miR-9下调CXCR4抑制鼻咽癌增殖、侵袭和转移的作用及机制研究
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摘要
目的和意义
鼻咽癌是我国南方地区高发的头颈部恶性肿瘤,易发生早期转移,确诊的鼻咽癌病例中有60%以上已有颈淋巴结转移或远处转移,而一旦发现转移病灶患者中位生存期不超过1年。因此,研究鼻咽癌发生和转移的分子机制,对提高鼻咽癌治疗疗效,延长患者生命有重要的临床指导意义。
microRNA分子是一类非编码小RNA,通过和靶基因3-UTR区结合抑制靶基因的转录或翻译,而其中的miR-9分子除参与胚胎神经发育、糖尿病和维护心肌功能外,还作为细胞增殖和分化的主控基因,参与Burkitt淋巴瘤、非霍奇金B细胞淋巴瘤、卵巢癌、胃癌、子宫内膜癌等恶性肿瘤的发生和转移,但miR-9究竟是抑癌基因还是癌基因因组织类型不同尚无定论,且在鼻咽癌甚至其他头颈部肿瘤miR-9的表达及功能研究尚无报道。
趋化因子受体-4(chemine receptor-4, CXCR4)是目前研究较为深入的趋化因子受体,文献报道CXCR4在多种恶性肿瘤的发生发展和转移中起重要作用,且与鼻咽癌的转移和预后密切相关。我们通过生物信息学方法分析发现miR-9分子存在能和CXCR4 3'-UTR相应序列结合的“种子序列”,提示两者之间可能存在直接的靶向调控关系,但仍需相关的实验证据去证实。
因此,本实验拟研究miR-9在慢性鼻咽炎组织和不同转移潜能的鼻咽癌组织、永生化鼻咽上皮细胞NP69和鼻咽癌细胞中的表达情况；通过5-AZA和TSA处理C666-1细胞后检测miR-9的表达变化和相应编码基因的甲基化状态变化,进而研究miR-9在鼻咽癌中表达失调是否与相应的编码基因启动子区CpG岛DNA甲基化相关,明确miR-9表达失调的分子机制。通过筛选miR-9过表达的C666-1细胞和体内外功能实验,明确miR-9对鼻咽癌细胞增殖、迁移、侵袭和转移的影响；通过构建和miR-9结合的CXCR4 3'-UTR双荧光报告质粒及上述体内外功能实验,探讨miR-9和CXCR4之间是否存在直接的靶向调控关系,miR-9能否部分逆转CXCR4在鼻咽癌中的生物学功能；
方法
1.临床标本收集和分组
收集2008年10月至2010年12月来我科就诊并活检的鼻咽癌组织和慢性鼻咽炎组织,共60例,包括40例鼻咽癌组织(其中无转移者20例,伴转移者20例)和20例慢性鼻咽炎组织(对照组),患者临床资料齐全,所有标本均行HE染色确诊,同时行miR-9原位杂交检测,并分析其表达与患者临床资料之间的相关性。
2.鼻咽癌细胞株的培养
鼻咽癌细胞C666-1、Sune-1、5-8F、6-10B、CNE-1、CNE-2和Hone-1以RP1640+10%FBS培养,正常的永生化鼻咽上皮细胞NP69以KMSF培养基培养,按常规条件消化传代。
3. Real time-PCR:抽提细胞内小分子RNA和总RNA,逆转录成cDNA后利用Taqman探针或引物进行PCR扩增,以2-ΔΔCt值表示基因相对表达强度。
4.原位杂交：石蜡组织和细胞爬片经组织脱蜡和水化(4%多聚甲醛固定)、胰酶消化(细胞透化)、预杂交、杂交、封闭、抗地高辛标记的一抗(1：1000)4℃孵育过夜、BCIP/NBT缓冲液洗涤、BCIP/NBT染色、核固红复染、封片等步骤后,显微镜下观察染色结果,并进行结果判读,判定标准同免疫组化结果的判读。
5.去甲基化药物5-AZA和去乙酰化药物TSA处理C666-1细胞
将鼻咽癌细胞C666-1以1×105的密度接种于6孔板中,生长过夜后加入终浓度为50uM的5-AZA,每隔24小时换含上述浓度的5-AZA的完全培养基一次,连续处理3天后收集细胞(5-AZA组),或在连续加入终浓度为50uM的5-AZA作用3天后继续加入终浓度为10nm的TSA维持作用24小时后收集细胞(5-AZA+TSA组),检测miR-9的表达变化和miR-9编码基因的甲基化状态变化。
6.hsa-miR-9-1, hsa-miR-9-2和hsa-miR-9-3启动子区的CpG岛预测：利用CpG Island Searcher软件对编码hsa-miR-9的基因hsa-miR-9-1, hsa-miR-9-2和hsa-miR-9-3启动子区的CpG岛进行分析,以相应1microRNA之cDNA的转录起始位点为+1,对基因上游1000bp到下游1000bp共2000bp的区段序列,以GC=55%, ObsCpG/ExpCpG=0.65, Length=300或200,Distance=100作为搜索参数进行分析。
7.重亚硫酸盐测序(bisulfite genomic sequencing, BSP):抽提细胞基因组DNA、
亚硫酸盐处理基因组DNA使得非甲基化胞嘧啶转化为尿嘧啶,同时使甲基化胞嘧啶保持不变,再以亚硫酸盐处理过的基因组DNA为模板,根据启动子CpG岛预测结果设计相应引物,利用高保真酶Platinum Taq Hifidility扩增目的片段BS-9-1、BS-9-2和BS-9-3,PCR产物凝胶回收后与pMD19-T载体连接,连接产物涂板并摇菌、质粒小抽后进行DNA测序检测甲基化情况。
8.载体的构建与鉴定
①pLVTHM/miR-9的构建及鉴定
合成pre-miR-9转录模板正义链和反义链的DNA Oligo序列,退火形成DNA双链后与pLVTHM载体同时进行MluI和ClaI双酶切,连接以构建载体pLVTHM/miR-9。
②psiCHECK-2-CXCR4 3'-UTR及其突变体的载体构建与鉴定
以正常人外周血基因组DNA为模板构建载体psiCHECK-2-CXCR4 3'-UTR(XhoI和NotI双酶切),同时运用重叠PCR技术对psiCHECK-2-CXCR4 3'-UTR的靶序列进行替换突变和缺失突变,构建载体psiCHECK-2-mt-s-CXCR4 3'-UTR(替换突变)和psiCHECK-2-mt-d-CXCR4 3'-UTR(缺失突变)。
③pReceiver-CXCR4-ORF和pReceiver-CXCR4-ORF+3'-UTR质粒的构建与鉴定
以正常人外周血基因组DNA为模板构建载体pReceiver-CXCR4-ORF和pReceiver-CXCR4-ORF+3'-UTR。
9.慢病毒的包装及滴度测定：病毒包装用293FT细胞生长融合度达70%—80%时用磷酸钙转染法进行病毒包装。转染6h后换液,48-72h后荧光显微镜下观察,发现大多数细胞发出绿色荧光时收集病毒上清,再以梯度稀释法测定慢病毒滴度。
10.筛选miR-9过表达的C666-1细胞
慢病毒悬液感染鼻咽癌C666-1细胞,荧光显微镜下观察表达绿色荧光的细胞比例超过30%时用流式细胞仪分选GFP+细胞,命名为C666-1/miR-9和C666-1/pLVTHM,荧光定量RT-PCR鉴定GFP+细胞中miR-9的表达,以western-blot法(或免疫荧光)检测CXCR4蛋白的表达。
11.Western blot:抽提细胞总蛋白、BCA蛋白定量法检测总蛋白的浓度后按昭SDS-PAGE电泳、转膜、免疫反应、显影和定影的步骤进行Western blot检测。
12.免疫荧光：细胞爬片以4%多聚甲醛固定30分钟,0.2%Triton X-100处理5分钟,10%正常山羊血清封闭30分钟后以CXCR4一抗(1：100稀释)4℃孵育过夜、荧光标记二抗(1：200)室温湿盒避光孵育1小时,DAPI室温湿盒避光孵育5分钟后镜检、拍照。
13.体内外功能实验：
①CCK-8检测细胞增殖能力：96孔板中每孔接种1000个细胞,每孔体积为200u1,每组5孔,同时设置空白对照,分别培养1-5天。每孔加入CCK8工作液20u1,37℃继续培养1h后终止培养,以空白对照孔调零,酶标仪上450nm测定各孔吸光度值(OD值),以相应OD比值表示细胞增殖能力大小。
②平板克隆形成实验：胰酶消化细胞后制成单细胞悬液,6孔板中每孔种入200个细胞,完全培养基中培养10-14天,待形成明显的细胞集落时4%多聚甲醛固定30min, PBS清洗后1%结晶紫染色10min,显微镜下计算含50个细胞以上的克隆数。
③细胞凋亡状态的检测：利用流式细胞仪检测、Caspase-3活性分析和Tunel原位凋亡检测细胞的凋亡状态。
④划痕实验：待细胞密度达到90%左右,吸干培养基,用200u1的加样枪头垂直在培养孔中部轻轻划过,制造多条互相平行的划痕,力度以刮落细胞而不在培养板上留痕为准。培养液冲去脱落细胞后继续培养,在制作划痕12h后对越过划痕边缘向空白处移动生长的细胞进行计数。
⑤Transwell迁移实验：胰酶消化细胞后以无血清培养基洗涤细胞并制作细胞悬液,均匀加入200u1至transwell小室上腔,加入600u1完全培基至transwell小室下腔,放置12小时后取出,PBS洗涤后4%多聚甲醛固定20min,1%结晶紫染色液染色10min,显微镜下计数穿膜细胞数。
⑥Transwell侵袭实验：细胞在进行Transwell侵袭实验前先血清饥饿24h,胰酶消化细胞后以0.5 ml无血清培养基重悬细胞(5×104)并加入transwell小室上腔的Matrigel基质胶表面,transwell小室下腔加入完全培养基作为趋化物。放置12小时后取出,PBS洗涤后4%多聚甲醛固定20min,1%结晶紫染色液染色10min,显微镜下计数穿透基质胶的细胞数。
⑦裸鼠皮下成瘤实验：选择4-6周龄BALB/C-nu/nu雄性裸鼠,体重20-24g进行裸鼠皮下成瘤实验。裸鼠背部皮下注射4×106细胞后定期观察肿瘤的生长情况,并以公式volume=1/2×xa×b2计算肿瘤的体积(a代表肿瘤的最长径,b代表肿瘤的最短径,单位为cm),当肿瘤体积接近500cm3时终止饲养。
⑧体内转移实验
选择4-6周龄的BALB/c nu/nu雄性裸鼠,体重18-24g,建立肝包膜下种植肺转移动物模型,待肝包膜下成瘤后利用动物整体成像系统观察体内肿瘤组织发出的绿色荧光,处死裸鼠HE染色观察肿瘤细胞在肝肺内转移情况。
14.miRNA靶标预测
通过常见的生物信息学软件PicTar(http://www.targetscan.org/)、miRBase(http://www.mirbase.org)、Targetscan(http://pictar.mdc-berlin.de/)来预测miR-9分子可能调控的靶基因,取3个软件预测结果的交集,确定我们感兴趣的靶基因。
15.双荧光素酶报告实验：采用双荧光素酶报告基因检测试剂盒进行双荧光素酶活性分析。转染前一天,将293FT细胞(2×105)接种到24孔板中,每孔加入500u1无抗生素的培养基,待细胞融合度达到95%时进行转染,转染48h后收集裂解细胞并检测双荧光素酶活性,以Firefly luciferase与Renilla luciferase的活性比值作为双荧光素酶的相对活性。
16.统计学分析
所有数据均采用SPSS13.0统计软件包进行统计学处理,P<0.05为差异有统计学意义。慢性鼻咽炎组织、无转移鼻咽癌组织、伴转移鼻咽癌组织中CXCR4蛋白表达水平的比较、hsa-miR-9表达水平的比较采用多个独立样本非参数检验(K independent samples test) (Kruskal-Wallis检验),CXCR4蛋白与hsa-miR-9表达水平的相关性分析采用Sperman等级相关。荧光定量PCR比较不同细胞株中miR-9的表达时采用单因素方差分析(One-way ANOVA),多重比较采用SNK(Student-Neuman-Keuls)法。方差不齐时采用基于方差不齐的多重比较方法Dunnett's T3法。平板克隆实验、划痕实验、(?)ranswell迁移实验、(?)ranswell侵袭实验等两样本均数的比较均采用两独立样本的t检验(independent-samples Ttest)；CCK8实验、皮下成瘤实验采用析因设计的方差分析；不同细胞间肝肺转移率的比较采用两个独立样本率比较的X2检验；C666-1、C666-1 5-AZA和C666-1 5-AZA+TSA三组之间miR-9表达水平的比较采用单因素方差分析(One-Way ANOVA), BSP结果的比较采用R×C表资料的X2检验进行统计学分析。
结果
l.hsa-miR-9在鼻咽癌组织和细胞中低表达
miR-9阳性表达于慢性鼻咽炎鼻咽上皮细胞和癌细胞的胞核和胞浆,以胞核为主,呈蓝褐色颗粒状。鼻咽癌组织中miR-9的表达水平(平均秩次22.25)显著低于慢性鼻咽炎组织(平均秩次47.00)(Z=-5.218,P=0.000)。在鼻咽癌不同T分期、N分期和临床分期miR-9的表达差异均有统计学意义(Z=20.477,P=0.000；X2=14.423,P=0.002；X22=17.561,P=0.001),随着T分期、N分期和临床分期的不断增高,miR-9的表达水平逐渐降低,其中T3-T4期明显低于T1-T2期,N3期明显低于N1-N2期,Ⅳ期明显低于Ⅰ-Ⅲ期,且NO期miR-9的表达水平(平均秩次30.56)显著高于N1-N3期(平均秩次17.58)(Z=-2.992,P=0.002),从而说明miR-9的表达与鼻咽癌的发生、转移及进展相关。以ΔCt值表示miR-9的相对表达水平,鼻咽癌细胞株中NP69细胞miR-9的表达水平(18.00±0.10)显著少于C666-1 (27.19±0.11)、Sune-1 (25.80±0.22)、5-8F (23.77±0.13)、6-10B(24.76±0.07)、CNE-1(24.16±0.12)、CNE-2(22.92±0.14)和Hone-1(26.77±0.21)细胞,差异有统计学意义(F=1212.097,P=0.000)。多重比较结果显示转移能力最强的C666-1细胞miR-9的表达水平均低于转移能力较弱的5-8F和CNE-2(P=0.000),均低于无转移能力的6-10B和CNE-1(P=0.000)。
2.miR-9表达下调与其编码基因hsa-miR-9-1, hsa-miR-9-2和hsa-miR-9-3启动子区CpG岛甲基化有关
①hsa-miR-9-1, hsa-miR-9-2和hsa-miR-9-3启动子区CpG岛分布：
我们利用CpG Island Searcher软件对hsa-miR-9-1、hsa-miR-9-2和hsa-miR-9-3启动子区的CpG岛进行分析,结果表明hsa-miR-9-1 165bp-1255bp之间共1091bp区域存在一个CpG岛,其中GC比例为62.1%,ObsCpG/ExpCpG为0.741,共含有18个CpG二核苷酸；hsa-miR-9-2 3bp-203bp之间共201bp区域存在一个CpG岛,其中GC比例为55.2%,ObsCpG/ExpCpG为0.756,共含有9个CpG二核苷酸；hsa-miR-9-3 87bp-1899bp之间共1813bp存在一个CpG岛,其中GC比例为65.4%, ObsCpG/ExpCpG为0.739,共有35个CpG二核苷酸。
②5-AZA和TSA药物干预鼻咽癌细胞C666-1后miR-9重新表达及编码基因部分去甲基化
5-AZA和TSA能诱导C666-1细胞miR-9基因重新表达,C666-1 5-AZA组、C666-15-AZA+TSA组同C666-1组相比,miR-9表达水平分别上调11.31倍和22.63倍,差异有统计学意义(F=780.280,P=0.000),组间比较发现C666-1 5-AZA组、C666-15-AZA+TSA组miR-9表达水平显著高于C666-1组(P=0.000),且C666-15-AZA+TSA组亦显著高于C666-1 5-AZA组(P=0.000)。BSP DNA测序结果显示C666-1、C666-1 5-AZA、C666-1 5-AZA+TSA三组has-miR-9-1的甲基化比率分别为78.8%(67/85)、32.9%(28/85)和14.1%(12/85),差异有统计学意义(Z=77.324,P=0.000); has-miR-9-2的甲基化比率分别为72.2%(39/54)、38.9%(21/54)和14.8%(8/54),差异有统计学意义(χ2=36.850, P=0.000); has-miR-9-3的甲基化比率分别为76%(133/175)、32%(56/175)和20.6%(36/175),差异有统计学意义(X2=122.407,P=0.000),C666-1细胞has-miR-9-1、has-miR-9-2和has-miR-9-3甲基化率最高,C666-1-AZA组次之,C666-1-AZA+TSA组最低,说明5-AZA和TSA能改变C666-1细胞miR-9编码基因的甲基化状态,换言之,启动子甲基化和去乙酰化是鼻咽癌细胞miR-9表达下调的主要原因。
3.miR-9过表达对鼻咽癌细胞C666-1生物学功能的影响
为证实miR-9对鼻咽癌细胞C666-1生物学功能的影响,我们构建了miR-9过表达载体,筛选出miR-9过表达的C666-1/miR-9细胞,并进行了一系列体内外功能实验：CCK8法结果示细胞种植后的第二天开始,C666-1/miR-9细胞的增殖速度显著慢于C666-1/PLVTHM细胞(F=696.168,P=0.000)；平板克隆形成实验示C666-1/miR-9细胞形成的平板克隆数(48.15±3.82)显著少于对照组C666-1/PLVTHM细胞(83.70±3.74)(t=-29.746,P=0.000),说明miR-9过表达可抑制鼻咽癌的增殖和平板克隆形成能力；C666/miR-9和对照组C666-1/PLVTHM细胞均未出现明显的细胞凋亡,说明miR-9过表达不影响鼻咽癌细胞的凋亡；划痕实验中C666-1/miR-9细胞穿越划痕的细胞数(47.167±2.552)较对照细胞C666-1/PLVTHM (95.917±3.118)明显减少(t=-41.912,P=0.000),穿越transwell膜的细胞数(36.500±4.542)较对照组C666-1/PLVTHM (93.417±5.195)亦明显减少(t=28.569,P=0.000),提示miR-9过表达可抑制鼻咽癌细胞体外迁移能力；铺板12小时后C666-1/miR-9细胞穿透基质胶的细胞数(32.667±3.257)较对照组C666-1/PLVTHM细胞(81.833±4.366)显著减少(t=31.270,P=0.000),说明miR-9过表达可抑制鼻咽癌细胞的体外侵袭能力。背部接种C666-1/PLVTHM和C666-1/miR-9的裸鼠,2周后皮下成瘤率100%,C666-1/PLVTHM组少数瘤体因生长过快出现瘤体的表面坏死,且C666-1/PLVTHM组瘤体的平均体积(208.699±151.858)显著高于C666-1/miR-9组(128.688±75.127)(F=1472.708,P=0.000)；肝包膜下接种C666-1/PLVTHM和C666-1/miR-9细胞的裸鼠,C666-1/PLVTHM组肝内转移率88.9%(8/9)显著高于C666-1/miR-9组肝内转移率44.4%(4/9)(χ2=4.000, P=0.046), C666-1/PLVTHM组肺内转移率77.8%(7/9)显著高于C666-1/miR-9组肺内转移率22.2%(2/9)(X2=5.556,P=0.018),说明miR-9过表达能抑制鼻咽癌细胞皮下成瘤和肝肺转移的能力。
4.miR-9抑制靶基因CXCR4的表达和功能
生物信息学方法预测miR-9可能通过其种子序列(CUUUGGU)与CXCR4mRNA 3'-UTR的7个碱基(ACCAAAG)完全互补结合,从而发挥作用。为证实上述推测,我们构建双荧光报告质粒psiCHECK-2-CXCR4 3'-UTR质粒及其缺失突变体psiCHECK-2-mt-d-CXCR4 3'-UTR和替换突变体psiCHECK-2-mt-s-CXCR4 3'-UTR,并将上述载体分别与miR-9 mimics和miR-9mimics NC, miR-9 inhibitor和miR-9 inhibitor NC共转染至293FT细胞,转染48h后收集裂解细胞检测荧光素酶活性,以Firefly luciferase与Renilla luciferase的活性比值作为双荧光素酶的相对活性进行不同样品间的比较,结果示psiCHECK-2-CXCR4 3'-UTR与miR-9mimic共转染可显著降低荧光素酶活性(P<0.001),而将该重组质粒与miR-9 inhibitor共转染后可显著增强荧光素酶活性(P=0.001),此外,缺失突变质粒psiCHECK-2-mt-d-CXCR4 3'-UTR和替换突变质粒psiCHECK-2-mt-s-CXCR4 3'-UTR与miR-9 mimic或者miR-9 inhibitor共转染均不能改变荧光素酶的活性(P>0.05),而miR-9mimics的突变体miR-9-mt与psiCHECK-2-CXCR4 3'-UTR共转染后亦不能改变荧光素酶的活性,间接说明miR-9可通过“种子序列”与CXCRmRNA 3'-UTR区特异性结合。
其次,我们构建了过表达CXCR4蛋白的真核载体pReceiver-CXCR4-ORF和pReceiver-CXCR4-ORF+3'-UTR,并将其转染至CXCR4表达水平极低的293FT细胞,48h后收集细胞并进行western blot检测,结果示两者CXCR4蛋白表达水平相当,说明CXCR4 3'-UTR作为非翻译区不影响蛋白的翻译；而niR-9 mimics与CXCR4 ORF+3'-UTR载体共转染后能显著抑制CXCR4蛋白的表达,但miR-9mimics mt(与CXCR4 3'-UTR区结合的“种子序列”发生突变)与CXCR4ORF+3'-UTR载体共转染后对CXCR4蛋白表达的抑制作用不明显,且miR-9mimics或miR-9 mimics mt与CXCR4 ORF载体共转染后均不能显著抑制CXCR4蛋白的表达,说明“丧失”CXCR4 3'-UTR区后,miR-9对CXCR4表达的调控便缺乏了关键的“桥梁”,无论miR-9分子序列是否发生突变,均不能影响CXCR4蛋白的表达,进一步证明miR-9通过与CXCR4 3'-UTR区靶向结合调控其表达;其次,我们通过慢病毒感染的方法获得稳定过表达miR9的细胞C666-1/miR-9和对照细胞C666-1/PLVTHM, Q-PCR和western-blot检测示两组细胞间CXCR4mRNA的表达水平并无显著差异(P>0.05),但C666-1/miR-9细胞CXCR4蛋白表达水平显著低于C666-1/PLVTHM细胞,而在C666-1/miR-9细胞中转染miR-9 inhibitor后CXCR4蛋白表达水平得到一定程度的“恢复”,说明miR-9通过抑制翻译而非影响转录来调控CXCR4蛋白的表达,免疫荧光结果与western-blot结果相吻合,进一步证实了上述观点。
最后,为探讨miR-9能否部分逆转CXCR4在鼻咽癌中促进细胞增殖、迁移、侵袭和转移的功能,我们筛选出C666-1/CXCR4+PLVTHM与C666-1/CXCR4+miR-9细胞,结果发现C666-1/CXCR4+miR-9的增殖速度显著慢于C666-1/CXCR4+PLVTHM (F=2392.535, P=0.000), C666-1/CXCR4+miR-9形成的平板克隆数(64.90±2.29)显著少于对照组C666-1/CXCR4+PLVTHM(115.20±2.44) (t=67.185, P=0.000),说明miR-9能部分逆转CXCR4促进鼻咽癌增殖和克隆形成的作用；C666-1/CXCR4+PLVTHM与C666-1/CXCR4+miR-9的细胞凋亡状态均无显著差别；C666-1/CXCR4+miR-9细胞穿越划痕的细胞数(53.000±7.520)较对照组C666-1/CXCR4+PLVTHM(236.083±10.423)显著减少(t=49.348,P=0.000),C666-1/CXCR4+miR-9穿越transwell膜的细胞数(56.667±3.229)较对照组C666-1/CXCR4+PLVTHM(115.833±5.524)亦显著减少(t=32.033,P= 0.000), C666-1/CXCR4+miR-9穿透基质胶的细胞数(45.250±3.745)较对照组C666-1/CXCR4+PLVTHM(106.500±3.802)显著减少(t=39.760,P=0.000),说明miR-9能部分逆转CXCR4促进鼻咽癌细胞体外迁移和侵袭能力的作用。背部和肝包膜下接种C666-1/CXCR4+PLVTHM与C666-1/CXCR4+miR-9细胞的裸鼠,2周后C666-1/CXCR4+PLVTHM组皮下瘤体的平均体积(249.404±143.100)显著大于C666-1/CXCR4+miR-9组(134.357±74.782)(F=659.513,P=0.000)；C666-1/CXCR4+PLVTHM组肝内转移率100%(9/9)显著高于C666-1/CXCR4+miR-9组肝内转移率55.6%(5/9)(X2=5.143,P=0.023),而C666-1/CXCR4+PLVTHM组肺内转移率88.9%(8/9)显著高于C666-1/CXCR4+miR-9组肺内转移率33.3%(3/9)(X2=5.844,P=0.016),说明miR-9能部分逆转CXCR4促进鼻咽癌细胞皮下成瘤和肝肺转移的能力。
结论
1.miR-9在鼻咽癌组织和细胞中表达下调。
2.鼻咽癌中miR-9表达下调与编码miR-9的基因miR-9-1, miR-9-2和miR-9-3启动子区CpG岛的甲基化有关。
3.miR-9过表达能抑制鼻咽癌细胞的增殖、侵袭和转移能力,miR-9在鼻咽癌中发挥抑癌作用。
4.在鼻咽癌中,CXCR4为miR-9的靶基因。
5.miR-9通过调控靶基因CXCR4的表达,部分逆转靶基因CXCR4在鼻咽癌中的促癌功能,从而抑制鼻咽癌细胞的增殖、侵袭和转移能力。
Objective
Nasopharyngeal carcinoma(NPC) is a kind of head and neck squamous cell carcinoma frequently occurred in south of China. It was difficult for early diagnosis in NPC in clinical and it was reported that more than 60% diagnosed NPC patients had metastasis of neck lymph node or distant organs.Furthermore, the middle survival was less than one year if metastasis was detected.Therefore, it is very important to study the formation and metastasis mechanisms of NPC for improving curative effects and prolonging overall survivals of patients.
miRNAs are small noncoding RNAs that serve as negative regulators of gene expression. Through interactions with the 3'-untranslated region (3'-UTR) of mRNA by partial sequence homology, miRNAs cause gene silencing either by mRNA degradation or translation repression. In addition to participation in embryonic neural development, diabetes and maintenance cardiac function, accumulating evidence suggests that miR-9 could be key players in regulation of tumor cell proliferation,invasion and metastasis in many kind of tumors,including Burkitt lymphoma,non-hodgkin's B cell lymphoma, ovarian cancer, gastric carcinoma, endometrial carcinoma. Increasing evidence indicates that miRNAs may function as either oncogenes or tumor suppressors in a cell type-specific manner,but there is no report about miR-9 expression and function in NPC or other head and neck carcinoma.
Chemine receptor-4(CXCR4) was deeply researched by many reachers. It was reported that CXCR4 played an important role in the process of progression and metastasis in many tumors, but rarely studied in NPC.
We found that miR-9 could bind with CXCR4 3'-UTR through its"seed sequence"by bioinformatics method,indicating the direct targeted regulation relationship between miR-9 and CXCR4 is to be confirmed.
Our study aimed to observe the expressions of miR-9 and CXCR4 in NPC tissues with different metastatic potential and chronic nasopharyngitis tissues, normal immortalization nasopharynx epithelial cell NP69 and nasopharyngeal carcinoma cell lines; to explicit the effect of miR-9 and CXCR4 on proliferation,migration,invasion and metastasis by screening C666-1 cell line of miR-9 overexpression and CXCR4 interference;to explore whether miR-9 and CXCR4 have direct targeted regulation relationship,whether miR-9 could partially reverse the stimulative effect of CXCR4 in NPC by constructing CXCR4 3'-UTR double fluorescence report plasmid including binding sites of miR-9 and function of CXCR4 by in vivo and vitro experiment;to discuss and explicit whether lower expression of miR-9 in NPC is related with DNA methylation in promoter of its encoding genes by detection of miR-9 expression and CpG island DNA methylation in miR-9 encoding genes after 5-AZA and TSA used in C666-1.
Methods
1.Clinical samples collecting and grouping
Forty patients with NPC (20 cases with metastasis and 20 cases without metastasis) and twenty chronic nasopharyngitis cases were recruited in this study from October 2008 to December 2010. All the specimen were confirmed by HE staining method and detected the expression level of miR-9 and CXCR4,analized their expression correlation between miR-9 and CXCR4.
2.Culture of NPC cell lines
NPC cell lines including C666-1,Sune-1,5-8F,6-10B,CNE-1,CNE-2 and Hone-1 were maintained in RMPI-1640 medium which was supplemented with 10% heated inactivated fetal calf serum, and normal immortalization nasopharynx epithelial cell NP69 was maintained in KMSF medium,all the cell lines were digested by common conditions.
3.Real Time-PCR
MicroRNA and total RNA were extracted from NPC cell lines and NP69,and then reversely transcripted into cDNA and PCR amplified using taqman probes and primers,relative expression strength of miR-9 and CXCR4 were expressed by 2-ΔΔCt method.
4.In situ hybridization
Paraffin organizations and cells climb pieces were dewaxed and hydrated(fixed by 4% paraformaldehyde),trypsin digested,prehybridizated,hybridizated,serum closed,incubated overnight by digoxin labeled antibody at 4℃,washed by BCIP/NBT buffer,stained by BCIP/NBT and nuclear fast red,and then staining results were observed.
5.IHC detection
Paraffin sections of tissues prepared for detection were stained by SP method, and the results of IHC were observed by light microscope and judged:Sections were observed in 10×objective lens and the staining intensities of all cells in 5 fields of vision were counted randomly, then scores were counted.
6.Vector construction and identification
①Construction and identification of pLVTHM/miR-9 and pLVTHM/ CXCR4-shRNA-miR (con) vector
pre-miR-9 transcription templates and shRNA-miR (con) DNA Oligo sequences were synthesized,annealed and double digested by MluI and Clal, connected to construct vectors:pLVTHM/miR-9 and pLVTHM/CXCR4-shRNA-miR (con)
②Construction and identification of psiCHECK-2-CXCR4 3'-UTR vector and its mutants
we double digested PCR product of CXCR4 3'-UTR and psiCHECK-2 by Xhol and NotI to construct vector psiCHECK-2-CXCR4 3'-UTR,meanwhile we constructed psiCHECK-2-mt-s-CXCR4 3'-UTR (replace mutation) and psiCHECK-2-mt-d-CXCR4 3'-UTR (missing mutation) by overlap PCR method.
③Construction and identification of pReceiver-CXCR4-ORF and pReceiver- CXCR4-ORF+3'-UTR vector
We construct pReceiver-CXCR4-ORF and pReceiver-CXCR4-ORF+3'-UTR vectors using peripheral genomic DNA of normal persons as template
7.Lentivirus Package
DNA-Cacl2 solution was used for lentivirus Package.
8.Screening miR-9 overexpression and CXCR4 interference cell lines.
We sorted C666-1 GFP+ cells by flow cytometric analysis after lentivirus infecting C666-1 cells, and named them as C666-1/miR-9 and C666-1/pLVTHM, C666-1/CXCR4-shRNA-miR and C666-1/CXCR4-shRNA-con,identificated them by RT-PCR and western-blot methods.
9.Western blot:Western blot analysis was performed according to the standard method with antibodies to CXCR4 and GAPDH after total protein were extracted and quantificated by BCA method.
10.Immunofluorescence assay. The expression of CXCR4 was determined by immunofluorescence assay. Cells were fixed by 4%paraformaldehyde and permeabilizated by 0.2%triton X-100 for 5mins, blocked by 10% normal goat serum for 30mins, inoculated with rabbit antihuman CXCR4 protein monoclonal antibody at 4℃for overnight and rinsed with phosphate buffered saline (PBS) with 0.2% tween 20 (PBST) three times for 10 min each. The mixture was then incubated with fluorescein isothiocyanate (FITC)-goat antimouse immunoglobulin G (IgG) antibody (1:200) at 37℃for 60 min and rinsed three times for 10 min each with PBST solution. Finally, inoculated with DAPI for 5mins.Cell pictures were obtained by fluorescence microscope in every sample and randomly selected to test cell fluorescence intensity.
11.Functional studies in vitro and in vivo:
①CCK-8 assay:CCK-8 assay was performed to assess the effect of miR-9 and CXCR4 on cell proliferation.Cells(1×103cell/well) were plated in a 96-well plate and maintained in RPMI1640 supplemented with 10% FBS.At 24,48,72,96 and 120h after seeding,culture medium was removed,cells were treated with 20ul CCK8 working solution for 1h at 37℃,and spectrometric absorbance at wavelength of 450nm was measured on a microplate reader.
②Colony formation assay:For colony formation assay,200 cells were seeded into 6-well plate. After being cultured for 1 10-14 days, surviving colonies (>50 cells/colony) were counted with 1% crystal violet staining. Triplicate independent experiments were performed.
③Cell apoptosis:Flow cytometry detection,caspase-3 activity analysis and Tunel in situ apoptosis detection were used to detect cell apoptosis status.
④wound healing assays:In vitro wound healing assay was carried out to determine the ability of cells to form membrane protrusion and cell migration. When the cell confluence reached 90%,a single wound was created in the center of cell monolayer by gentle removal of the attached cells with a sterile plastic pipette tip.The debris was removed by washing the cells with serum free medium.Migration of cells into the wound was then observed after 12h..
⑤Transwell migration assay:For cell migration assay, cells were starved with serum free medium for 24 hrs before the assay. Cells (5×104) were suspended in 0.5 ml serum-free medium and loaded on the upper compartment of transwell chamber. The lower compartment was filled with complete medium as chemoattractant. After 12 hrs, migration cells were fixed, stained, and counted under a microscope. Triplicate independent experiments were done.
⑥Transwell invasion assays
For invasion assay, cells were starved with serum free medium for 24 hrs before the assay.5×104 cells were suspended in 0.5 ml serum-free medium and loaded on the upper compartment of invasion chamber coated with Matrigel. The lower compartment was filled with complete medium as chemoattractant. After 12 hrs, invasive cells were fixed, stained, and counted under a microscope. Triplicate independent experiments were done.
⑦Tumor formation in nude mice:For in vivo experiment,cells (4×106) in 200μL serum-free RPMI1640 were injected s.c into the right flank of 4-6 weeks-old nude mice, respectively. The tumor volume was calculated by the formula V=0.5×L×W2(L:length;W:width),and when the volme of tumor was close to 500 cm3 nude mice were killed.
⑧In vivo metastasis experiment:Nude mice(BALB/C nu/nu,4-6 weeks old,18-24g in weight) were maintained under pathogen-free conditions(specific pathogen-free level).Primary tumors were established by direct injection of 2×106 cells into the liver as previously described.All the mice were sacrificed on day 21,and tissues were collected for pathologic analysis.GFP fluorescence images were observed under an in vivo fluorescence instrument.
12.MiRNA target site prediction. A search for predicted target miRNAs was performed usingthe databases TargetScan (http://www.targetscan.org/),miRbase (http://www.mirbase.org) and PicTar(http://pictar.mdc-berlin.de/).equence S conservation was examined using University of California Santa Cruz genome browser(http://genome.ucsc.edu/) and then we selected interesting target gene (http://genome.ucsc.edu/).
13.Dual-luciferase reporter gene assay:293FT cells, chosen based on their low endogenous expression of miRNAs (26), were grown to 80-90% confluence in white 96-well plates in DMEM supplemented with 10% FBS,1% nonessential amino acids, L-glutamine, and penicillin/straptamicin at 37℃under 5% CO2.Cells were transfected with 20 ng empty psiCHECK-2 vector or psiCHECK-2-CXCR4-3'-UTR reporter and miR- 9 mimics or its mutant-miR-9 mimics mt and negative control (miR control), miR-9 inhibitor or its negative control (miR- 9 inhibitor control)at concentrations of 20nM in reduced serum and antibiotics-free Opti-MEM with lipofectamine 2000. T he Firefly and Renilla luciferase were measured in cell lysates using a dual-luciferase reporter assay system on a fusion plate reader. Firefly luciferase activity was used for normalization and as an internal control for transfection efficiency.
14.5-AZA and TSA treatment of C666-1 cells. 1×105 C666-1 cells were split 12 to 24 h before treatment. Cells were then given one of the following treatments. (i) 50uM 5-Aza-2'-deoxycytidine (5-AZA) was used for 72 h. Medium containing 5-AZA was changed every 24 h;(ii) 50uM 5-AZA was used for 72 h followed by 10nm TSA for an additional 24 h. The dose of 5-AZA (50uM) and TSA(lOnm) was chosen based on preliminary studies showing optimal reactivation of miR-9 gene expression and DNA methylation in promoter.
15.hsa-miR-9-l,hsa-miR-9-2 and hsa-miR-9-3 CpG islands methylation analysis In an attempt to determine all CpG islands and all potential transcription start sites (TSS) for hsa-miR-9-1,hsa-miR-9-2 and hsa-miR-9-3, we first proceeded with the identification of the promoter sequence. The analyses were initiated using an identified RefSeq by GenBank accession number, after which we submitted the gene sequence to a Genome BLAT Search through the UCSC Genome bioinformatics website (http://genome.ucsc.edu). We selected 2000bps of sequence extending from the 5' upstream region to 1000 bps downstream of the region of the TSS. The BLAT program returned a sequence that was first submitted to the CpGPLOT program from the European Bioinformatics Institute website (http://www.ebi.ac.uk/emboss/cpgplot) This program defines a CpG island as 200 or 300bps of sequence with 50%C+G content and 0.65 CpG observed/CpG expected.
16.Bisulfite genomic sequencing:Genomic DNA was isolated from cell samples and 1000ng treated with sodium bisulfite using the DNA Methylation kit to convert unmethylated cytosine to uracil.Bisulfite-treated DNA was then amplified with specific primers for CpG islands surrounding miR-9-1,miR-9-2 and miR-9-3 genomic locations by PCR with platinum taq hifidility,finally PCR product and pMD19-T vector were connected and coated for DNA sequencing to detect DNA methylation status.
17.Statistical analysis:All analyses were carried out using the SPSS 13.0 software package.A P value less than 0.05 was considered statistically significant.Comparisiong of miR-9 and CXCR4 expression level in chronic nasopharyngitis tissues,nasopharyngeal carcinoma tissues without metastasis, nasopharyngeal carcinoma tissues with metastasis was analyzed with the Kruskal-Wallis test (K independent samples test).Sperman rank correlation was used for relationship between CXCR4 protein and miR-9 expression level.Comparison of mean in different cell lines about iR-9 and CXCR4 expession was analyzed by one-way ANOVA or indepent-samples T test and Student- Neuman-keuls methos for multiple comparison.Dunnetts T3 method was exployed for heterogeneity of variance.Theχ2 test for proportion was used to analyze the vivo metastasis characteristics of different groups.Factorial analysis was utilized to analyze the results of CCK8,invasion and wound healing assay.The difference in wound migration and invasion indices between C666-1 cell clones were analyzed by the independented t test.Comparison of mean in colony formation and wound healing assay in two sample was analyzed by independent-samples T test;CCK8 assay and tumor formation in nude mice was analyzed by one-way ANOVA;χ2 test was used for rates of liver and lung metastasis among different cell lines.miR-9 exprssion level in C666-1,C666-1 5-AZA and C666-1 5-AZA+TSA groups were analyzed by One-way ANOVA and R×Cχ2 t test for BSP results.
Results
1.miR-9 was lower expressed in NPC tissues and cell lines.
miR-9 was positively expressed in nuclei and membrane of nasophayngeal carcinoma cell and epithelial cells in chronic nasopharyngitis tissues.The expression levels of miR-9 detected by ISH in nasopharyngeal carcinoma tissues (mean rank 22.25) were significantly lower than that of chronic nasopharyngitis tissues (mean rank 47.00) (Z=-5.218, P=0.000), and the differences of miR-9 expression level among nasopharyngeal carcinoma tissues with different T stage,N stage and clinical stage were significant (χ2=20.477, P=0.000;χ2=14.423, P=0.002;χ2=17.561, P=0.001). miR-9 expression levels were down-regulated gradually as T stage,N stage and clinical stage were increased,and miR-9 expression levels in T3-T4 stage,N3 stage and IV stage NPC tissues were lower than those of T1-T2 stage, N1-N2 stage and I-III stage.Notely, miR-9 expression level in NO stage NPC tissues (mean rank 30.56) was significantly lower than that of N1-N3 stage NPC tissues (mean rank 17.58) (Z=-2.992, P=0.002),indicating that miR-9 is related with formation and metastasis of NPC. The expression level of miR-9 in NP69 (18.00±0.10) detected by Real time-PCR was significantly higher than those in C666-1 (27.19±0.11), Sune-1(25.80±0.22),5-8F(23.77±0.13),6-10B(24.76±0.07),CNE-1(24.16±0.12),CNE-2 (22.92±0.14) and Hone-1 (26.77±0.21) (F=1212.097, P=0.000), and the expreesion level of miR-9 in C666-1 was lower than those of 5-8F and CNE-2 (P=0.000),6-10B and CNE-1 (P=0.000)
2. miR-9 downregulation is related with CpG island DNA methylation in promoter of hsa-miR-9-1, hsa-miR-9-2 and hsa-miR-9-3.
①CpG island distribution in hsa-miR-9-1, hsa-miR-9-2 and hsa-miR-9-3 promoters:We used CpG island searcher software to analyze CpG islands in hsa-miR-9-1,hsa-miR-9-2 and hsa-miR-9-3 promoters,and found that there is a CpG island in hsa-miR-9-1 from 165bp to 1255bp(1091bp,including 18 CpG dinucleotides, GC%=62.1%, ObsCpG/ExpCpG=0.741),a CpG island in hsa-miR-9-2 from 3bp to 203bp(201bp,including 9 CpG dinucleotides,GC%=55.2%, ObsCpG/ExpCpG=0.756) and a CpG island in hsa-miR-9-3 from 87bp to 1899bp (1813bp,including 35 CpG dinucleotides, GC%=65.4%, ObsCpG/ExpCpG=0.739).
②5-AZA and TSA induce miR-9 re-expression and DNA demethylation in C666-1 cell.5-AZA and TSA could induce miR-9 re-expression in C666-1,miR-9 expression level in C666-1 5-AZA group and C666-1 5-AZA+TSA group increased 11.31 and 22.63 times (F=780.280,P=0.000),and miR-9 expression level in C666-1 5-AZA group and C666-1 5-AZA+TSA group were significantly higher than that of C666-1 group (P=0.000),and the same as C666-1 5-AZA+TSA group and C666-1 5-AZA group (P=0.000).Methylation ratios of hsa-miR-9-1 in C666-1、C666-1 5-AZA and C666-1 5-AZA+TSA groups were 78.8%(67/85)、32.9%(28/85) and 14.1%(12/85) respectively (χ2=77.324, P=0.000), methylation ratios of hsa-miR-9-2 were 72.2%(39/54)、38.9%(21/54) and 14.8%(8/54) respectively (χ2=36.850, P=0.000), methylation ratios of hsa-miR-9-3 were 76%(133/175)、32%(56/175) and 20.6%(36/175) (χ2=122.407, P=0.000) by BSP sequencing., methylation ratios of hsa-miR-9-1, hsa-miR-9-2 and hsa-miR-9-3 were highest in C666-1 group and lowest in C666-1-AZA+TSA group,indicating 5-AZA and TSA could change methylation status of miR-9 encoding genes in C666-1 cell, in other word,DNA methylation and acetylation in promoter were main reasons for miR-9 downregulation in NPC cell.
3. The effect of miR-9 on functions of NPC.
The proliferation rates of C666-1/miR-9 was significantly slow than that of C666-1/PLVTHM (F=696.168, P=0.000),The colony numbers formed by C666-1/miR-9(48.15±3.82) was significantly less than that by C666-1/PLVTHM (83.70±3.74) (t=29.746, P=0.000) indicating that miR-9 overexpression could inhibit proliferarion and colony formation ability of NPC cell;There was no obvious apoptosis induced by C666-1/miR-9 and C666-1/PLVTHM,indicating that miR-9 overexpression has no effect of NPC cell apotosis;In wound healing assay,The number of C666-1/miR-9 cells passed through the scratch (47.167±2.552) was less than that of C666-1/PLVTHM (95.917±3.118) (t=41.912, P= 0.000),and in transwell migration assay C666-1/miR-9(36.500±4.542)and C666-1/PLVTHM(93.417±5.195) (t=28.569, P= 0.000) were the same as in wound healing assay,indicating that miR-9 overexpression could inhibit migration ability of NPC cell.In transwell invasion assay,the number of C666-1/miR-9 cells penetrated Matrigel (32.667±3.257) was less than that of C666-1/PLVTHM (81.833±4.366) (t=31.270, P=0.000) indicating that miR-9 overexpression could inhibit invasion ability of NPC cell.In subcutaneous tumor formation assay,the average volume of tumor formed by C666-1/miR-9 (128.688±75.127) was less than that by C666-1/PLVTHM (208.699±151.858) (F=1472.708, P=0.000);After cells inoculated under liver capsule,the metastasis rate of intrahepatic spread in C666-1/miR-9(44.4%,4/9) was lower than that of C666-1/PLVTHM (88.9%,8/9) (χ2=4.000,P=0.046),and the metastasis rate of lung spread in C666-1/miR-9(22.2%,2/9) was lower than that of C666-1/PLVTHM (77.8%,7/9) (χ2=5.556, P=0.018) indicating that miR-9 overexpression could inhibit tumor formation and metastasis ability of NPC cell.
4. miR-9 inhibits expression and function of CXCR4
We predicted that miR-9 may play its role through combining its seed sequence(CUUUGGU) with 3'-UTR of CXCR4mRNA (ACCAAAG) by bioinformatics method.To confirm the hyposesis,we constructed plasmid psiCHECK-2-CXCR4 3'-UTR and its mutants psiCHECK-2-mt-d-CXCR4 3'-UTR or psiCHECK-2-mt-s-CXCR4 3'-UTR that to be transfected with miR-9 mimics,miR-9 NC,miR-9 inhibitor and miR-9 inhibitor NC into 293FT cell and then detected luciferase activity, finding that con-transfection of psiCHECK-2-CXCR4 3'-UTR and miR-9 mimics could significantly reduce luciferase activity (P<0.001), con-transfection of psiCHECK-2-CXCR4 3'-UTR and miR-9 inhibitor could significantly enhance luciferase activity(P=0.001).However,con-transfection of psiCHECK-2-mt-d-CXCR4 3'-UTR or psiCHECK-2-mt-s-CXCR4 3'-UTR with miR-9 mimics or miR-9 inhibitor even could not change luciferase activity (P>0.05),and con-transfection of mutant of miR-9mimics(miR-9-mt) and psiCHECK-2-CXCR4 3'-UTR also could not change luciferase activity,indicating that miR-9 could bind with 3'-UTR of CXCR4.
Next,we constructed pReceiver-CXCR4-ORF and vectors and transfected them into 293FT cells in which CXCR4 protein is lower expressed,founding that the expression level of CXCR4 protein was almost the same,indicating the 3'-UTR of CXCR4 has no effect on protein translation,but when we con-transfected the above vectors with miR-9 mimics and miR-9 mimics mt,we found miR-9 mimics rather than miR-9 mimics mt could significantly inhibit CXCR4 protein expression after being transfected with pReceiver-CXCR4-ORF+3'-UTR, and both of miR-9 mimics and miR-9 mimics mt could not significantly inhibit CXCR4 protein expression after being transfected with pReceiver-CXCR4-ORF,indicating miR-9 has no effect on CXCR4 protein expression no matter whether the sequence of miR-9 is mutanted when 3'-UTR of CXCR4 is lost;next,we detected miR-9 and CXCR4 expression in C666-1/miR-9 and C666-1/PLVTHM by Q-PCR and western-blot,and found that CXCR4 protein rather than CXCR4 mRNA was lower in C666-1/miR-9 than C666-1/PLVTHM,but CXCR4 protein expression could be recovered to some extent,indicating miR-9 could inhibit CXCR4 protein rather than CXCR4 mRNA expression,which was consistent with results by immune fluorescence.
Finally, in order to explore whether miR-9 could partial reverse the function of CXCR4 in cell proliferation,migration,invasion and metastasis,we screened C666-1/CXCR4+PLVTHM and C666-1/CXCR4+miR-9 cells,and found that the proliferation of C666-1/CXCR4+miR-9 was significantly slower than that of C666-1/CXCR4+PLVTHM (F=2392.535, P=0.000),and colony numbers formed by C666-1/CXCR4+miR-9(64.90±2.29) were significantly less than that of C666-1/CXCR4+PLVTHM(115.20±2.44) (t=67.185, P=0.000),indicating that miR-9 could partial reverse the function of CXCR4 in cell proliferation and colony formation;C666-1/CXCR4+PLVTHM and C666-1/CXCR4+miR-9 have no effect on cell apoptosis.The number of C666-1/CXCR4+miR-9 cells passed through scratches (53.000±7.520) was much less than that of C666-1/CXCR4+PLVTHM (236.083±10.423) (t=49.348, P= 0.000),and the number of C666-1/CXCR4+miR-9 cells penetrated transwell membrane (56.667±3.229) was also much less than that of C666-1/CXCR4+PLVTHM (115.833±5.524) (t= 32.033, P=0.000),the number of C666-1/CXCR4+miR-9 cells penetrated Matrigel (45.250±3.745) was also much less than that of C666-1/CXCR4+PLVTHM (106.500±3.802) (t=39.760,P=0.000), indicating that miR-9 could partial reverse the function of CXCR4 in cell migration and invasion.The average volumes of subcutaneous tumor in C666-1/CXCR4+PLVT HM(249.404±143.100) group were greater than that of C666-1/CXCR4+miR-9 group(134.357±74.782) (F= 659.513, P=0.000);The intrahepatic transfer rate in C666-1/CXCR4+PLVTHM group (100%,9/9) was higher than that of C666-1/CXCR4+miR-9 group (55.6%,5/9) (x2=5.143,P=0.023),and the pulmonary metastasis rate in C666-1/CXCR4+PLVTHM group (88.9%,8/9) was higher than that of C666-1/CXCR4+miR-9 group (33.3%,3/9) (χ2=5.844, P=0.016), indicating that miR-9 could partial reverse the function of CXCR4 in tumor formation and metastasis in vivo.
Conclusion
①miR-9 was down-regulated in nasopharyngeal carcinoma tissues and nasopharyngeal carcinoma cells.
②The lower expression of miR-9 in NPC is related with CpG island DNA methylation in promoter of miR-9 encoding genes hsa-miR-9-l,hsa-miR-9-2 and hsa-miR-9-3.
③miR-9 overexpression could inhibit the proliferation,colony formation, migration and invasion, subcutaneous tumor formation and liver, lung metastasis ability of C666-1 cell,indicating miR-9 acted as tumor suppressor gene in NPC.
④miR-9 inhibited the proliferation, migration and invasion ability of C666-1 cell by regulating expression of CXCR4 protein and partially reversing the function of CXCR4 in NPC.
鼻咽癌是我国南方地区高发的头颈部恶性肿瘤,易发生早期转移,确诊的鼻咽癌病例中有60%以上已有颈淋巴结转移或远处转移,而一旦发现转移病灶患者中位生存期不超过1年。因此,研究鼻咽癌发生和转移的分子机制,对提高鼻咽癌治疗疗效,延长患者生命有重要的临床指导意义。
microRNA分子是一类非编码小RNA,通过和靶基因3-UTR区结合抑制靶基因的转录或翻译,而其中的miR-9分子除参与胚胎神经发育、糖尿病和维护心肌功能外,还作为细胞增殖和分化的主控基因,参与Burkitt淋巴瘤、非霍奇金B细胞淋巴瘤、卵巢癌、胃癌、子宫内膜癌等恶性肿瘤的发生和转移,但miR-9究竟是抑癌基因还是癌基因因组织类型不同尚无定论,且在鼻咽癌甚至其他头颈部肿瘤miR-9的表达及功能研究尚无报道。
趋化因子受体-4(chemine receptor-4, CXCR4)是目前研究较为深入的趋化因子受体,文献报道CXCR4在多种恶性肿瘤的发生发展和转移中起重要作用,且与鼻咽癌的转移和预后密切相关。我们通过生物信息学方法分析发现miR-9分子存在能和CXCR4 3'-UTR相应序列结合的“种子序列”,提示两者之间可能存在直接的靶向调控关系,但仍需相关的实验证据去证实。
因此,本实验拟研究miR-9在慢性鼻咽炎组织和不同转移潜能的鼻咽癌组织、永生化鼻咽上皮细胞NP69和鼻咽癌细胞中的表达情况；通过5-AZA和TSA处理C666-1细胞后检测miR-9的表达变化和相应编码基因的甲基化状态变化,进而研究miR-9在鼻咽癌中表达失调是否与相应的编码基因启动子区CpG岛DNA甲基化相关,明确miR-9表达失调的分子机制。通过筛选miR-9过表达的C666-1细胞和体内外功能实验,明确miR-9对鼻咽癌细胞增殖、迁移、侵袭和转移的影响；通过构建和miR-9结合的CXCR4 3'-UTR双荧光报告质粒及上述体内外功能实验,探讨miR-9和CXCR4之间是否存在直接的靶向调控关系,miR-9能否部分逆转CXCR4在鼻咽癌中的生物学功能；
方法
1.临床标本收集和分组
收集2008年10月至2010年12月来我科就诊并活检的鼻咽癌组织和慢性鼻咽炎组织,共60例,包括40例鼻咽癌组织(其中无转移者20例,伴转移者20例)和20例慢性鼻咽炎组织(对照组),患者临床资料齐全,所有标本均行HE染色确诊,同时行miR-9原位杂交检测,并分析其表达与患者临床资料之间的相关性。
2.鼻咽癌细胞株的培养
鼻咽癌细胞C666-1、Sune-1、5-8F、6-10B、CNE-1、CNE-2和Hone-1以RP1640+10%FBS培养,正常的永生化鼻咽上皮细胞NP69以KMSF培养基培养,按常规条件消化传代。
3. Real time-PCR:抽提细胞内小分子RNA和总RNA,逆转录成cDNA后利用Taqman探针或引物进行PCR扩增,以2-ΔΔCt值表示基因相对表达强度。
4.原位杂交：石蜡组织和细胞爬片经组织脱蜡和水化(4%多聚甲醛固定)、胰酶消化(细胞透化)、预杂交、杂交、封闭、抗地高辛标记的一抗(1：1000)4℃孵育过夜、BCIP/NBT缓冲液洗涤、BCIP/NBT染色、核固红复染、封片等步骤后,显微镜下观察染色结果,并进行结果判读,判定标准同免疫组化结果的判读。
5.去甲基化药物5-AZA和去乙酰化药物TSA处理C666-1细胞
将鼻咽癌细胞C666-1以1×105的密度接种于6孔板中,生长过夜后加入终浓度为50uM的5-AZA,每隔24小时换含上述浓度的5-AZA的完全培养基一次,连续处理3天后收集细胞(5-AZA组),或在连续加入终浓度为50uM的5-AZA作用3天后继续加入终浓度为10nm的TSA维持作用24小时后收集细胞(5-AZA+TSA组),检测miR-9的表达变化和miR-9编码基因的甲基化状态变化。
6.hsa-miR-9-1, hsa-miR-9-2和hsa-miR-9-3启动子区的CpG岛预测：利用CpG Island Searcher软件对编码hsa-miR-9的基因hsa-miR-9-1, hsa-miR-9-2和hsa-miR-9-3启动子区的CpG岛进行分析,以相应1microRNA之cDNA的转录起始位点为+1,对基因上游1000bp到下游1000bp共2000bp的区段序列,以GC=55%, ObsCpG/ExpCpG=0.65, Length=300或200,Distance=100作为搜索参数进行分析。
7.重亚硫酸盐测序(bisulfite genomic sequencing, BSP):抽提细胞基因组DNA、
亚硫酸盐处理基因组DNA使得非甲基化胞嘧啶转化为尿嘧啶,同时使甲基化胞嘧啶保持不变,再以亚硫酸盐处理过的基因组DNA为模板,根据启动子CpG岛预测结果设计相应引物,利用高保真酶Platinum Taq Hifidility扩增目的片段BS-9-1、BS-9-2和BS-9-3,PCR产物凝胶回收后与pMD19-T载体连接,连接产物涂板并摇菌、质粒小抽后进行DNA测序检测甲基化情况。
8.载体的构建与鉴定
①pLVTHM/miR-9的构建及鉴定
合成pre-miR-9转录模板正义链和反义链的DNA Oligo序列,退火形成DNA双链后与pLVTHM载体同时进行MluI和ClaI双酶切,连接以构建载体pLVTHM/miR-9。
②psiCHECK-2-CXCR4 3'-UTR及其突变体的载体构建与鉴定
以正常人外周血基因组DNA为模板构建载体psiCHECK-2-CXCR4 3'-UTR(XhoI和NotI双酶切),同时运用重叠PCR技术对psiCHECK-2-CXCR4 3'-UTR的靶序列进行替换突变和缺失突变,构建载体psiCHECK-2-mt-s-CXCR4 3'-UTR(替换突变)和psiCHECK-2-mt-d-CXCR4 3'-UTR(缺失突变)。
③pReceiver-CXCR4-ORF和pReceiver-CXCR4-ORF+3'-UTR质粒的构建与鉴定
以正常人外周血基因组DNA为模板构建载体pReceiver-CXCR4-ORF和pReceiver-CXCR4-ORF+3'-UTR。
9.慢病毒的包装及滴度测定：病毒包装用293FT细胞生长融合度达70%—80%时用磷酸钙转染法进行病毒包装。转染6h后换液,48-72h后荧光显微镜下观察,发现大多数细胞发出绿色荧光时收集病毒上清,再以梯度稀释法测定慢病毒滴度。
10.筛选miR-9过表达的C666-1细胞
慢病毒悬液感染鼻咽癌C666-1细胞,荧光显微镜下观察表达绿色荧光的细胞比例超过30%时用流式细胞仪分选GFP+细胞,命名为C666-1/miR-9和C666-1/pLVTHM,荧光定量RT-PCR鉴定GFP+细胞中miR-9的表达,以western-blot法(或免疫荧光)检测CXCR4蛋白的表达。
11.Western blot:抽提细胞总蛋白、BCA蛋白定量法检测总蛋白的浓度后按昭SDS-PAGE电泳、转膜、免疫反应、显影和定影的步骤进行Western blot检测。
12.免疫荧光：细胞爬片以4%多聚甲醛固定30分钟,0.2%Triton X-100处理5分钟,10%正常山羊血清封闭30分钟后以CXCR4一抗(1：100稀释)4℃孵育过夜、荧光标记二抗(1：200)室温湿盒避光孵育1小时,DAPI室温湿盒避光孵育5分钟后镜检、拍照。
13.体内外功能实验：
①CCK-8检测细胞增殖能力：96孔板中每孔接种1000个细胞,每孔体积为200u1,每组5孔,同时设置空白对照,分别培养1-5天。每孔加入CCK8工作液20u1,37℃继续培养1h后终止培养,以空白对照孔调零,酶标仪上450nm测定各孔吸光度值(OD值),以相应OD比值表示细胞增殖能力大小。
②平板克隆形成实验：胰酶消化细胞后制成单细胞悬液,6孔板中每孔种入200个细胞,完全培养基中培养10-14天,待形成明显的细胞集落时4%多聚甲醛固定30min, PBS清洗后1%结晶紫染色10min,显微镜下计算含50个细胞以上的克隆数。
③细胞凋亡状态的检测：利用流式细胞仪检测、Caspase-3活性分析和Tunel原位凋亡检测细胞的凋亡状态。
④划痕实验：待细胞密度达到90%左右,吸干培养基,用200u1的加样枪头垂直在培养孔中部轻轻划过,制造多条互相平行的划痕,力度以刮落细胞而不在培养板上留痕为准。培养液冲去脱落细胞后继续培养,在制作划痕12h后对越过划痕边缘向空白处移动生长的细胞进行计数。
⑤Transwell迁移实验：胰酶消化细胞后以无血清培养基洗涤细胞并制作细胞悬液,均匀加入200u1至transwell小室上腔,加入600u1完全培基至transwell小室下腔,放置12小时后取出,PBS洗涤后4%多聚甲醛固定20min,1%结晶紫染色液染色10min,显微镜下计数穿膜细胞数。
⑥Transwell侵袭实验：细胞在进行Transwell侵袭实验前先血清饥饿24h,胰酶消化细胞后以0.5 ml无血清培养基重悬细胞(5×104)并加入transwell小室上腔的Matrigel基质胶表面,transwell小室下腔加入完全培养基作为趋化物。放置12小时后取出,PBS洗涤后4%多聚甲醛固定20min,1%结晶紫染色液染色10min,显微镜下计数穿透基质胶的细胞数。
⑦裸鼠皮下成瘤实验：选择4-6周龄BALB/C-nu/nu雄性裸鼠,体重20-24g进行裸鼠皮下成瘤实验。裸鼠背部皮下注射4×106细胞后定期观察肿瘤的生长情况,并以公式volume=1/2×xa×b2计算肿瘤的体积(a代表肿瘤的最长径,b代表肿瘤的最短径,单位为cm),当肿瘤体积接近500cm3时终止饲养。
⑧体内转移实验
选择4-6周龄的BALB/c nu/nu雄性裸鼠,体重18-24g,建立肝包膜下种植肺转移动物模型,待肝包膜下成瘤后利用动物整体成像系统观察体内肿瘤组织发出的绿色荧光,处死裸鼠HE染色观察肿瘤细胞在肝肺内转移情况。
14.miRNA靶标预测
通过常见的生物信息学软件PicTar(http://www.targetscan.org/)、miRBase(http://www.mirbase.org)、Targetscan(http://pictar.mdc-berlin.de/)来预测miR-9分子可能调控的靶基因,取3个软件预测结果的交集,确定我们感兴趣的靶基因。
15.双荧光素酶报告实验：采用双荧光素酶报告基因检测试剂盒进行双荧光素酶活性分析。转染前一天,将293FT细胞(2×105)接种到24孔板中,每孔加入500u1无抗生素的培养基,待细胞融合度达到95%时进行转染,转染48h后收集裂解细胞并检测双荧光素酶活性,以Firefly luciferase与Renilla luciferase的活性比值作为双荧光素酶的相对活性。
16.统计学分析
所有数据均采用SPSS13.0统计软件包进行统计学处理,P<0.05为差异有统计学意义。慢性鼻咽炎组织、无转移鼻咽癌组织、伴转移鼻咽癌组织中CXCR4蛋白表达水平的比较、hsa-miR-9表达水平的比较采用多个独立样本非参数检验(K independent samples test) (Kruskal-Wallis检验),CXCR4蛋白与hsa-miR-9表达水平的相关性分析采用Sperman等级相关。荧光定量PCR比较不同细胞株中miR-9的表达时采用单因素方差分析(One-way ANOVA),多重比较采用SNK(Student-Neuman-Keuls)法。方差不齐时采用基于方差不齐的多重比较方法Dunnett's T3法。平板克隆实验、划痕实验、(?)ranswell迁移实验、(?)ranswell侵袭实验等两样本均数的比较均采用两独立样本的t检验(independent-samples Ttest)；CCK8实验、皮下成瘤实验采用析因设计的方差分析；不同细胞间肝肺转移率的比较采用两个独立样本率比较的X2检验；C666-1、C666-1 5-AZA和C666-1 5-AZA+TSA三组之间miR-9表达水平的比较采用单因素方差分析(One-Way ANOVA), BSP结果的比较采用R×C表资料的X2检验进行统计学分析。
结果
l.hsa-miR-9在鼻咽癌组织和细胞中低表达
miR-9阳性表达于慢性鼻咽炎鼻咽上皮细胞和癌细胞的胞核和胞浆,以胞核为主,呈蓝褐色颗粒状。鼻咽癌组织中miR-9的表达水平(平均秩次22.25)显著低于慢性鼻咽炎组织(平均秩次47.00)(Z=-5.218,P=0.000)。在鼻咽癌不同T分期、N分期和临床分期miR-9的表达差异均有统计学意义(Z=20.477,P=0.000；X2=14.423,P=0.002；X22=17.561,P=0.001),随着T分期、N分期和临床分期的不断增高,miR-9的表达水平逐渐降低,其中T3-T4期明显低于T1-T2期,N3期明显低于N1-N2期,Ⅳ期明显低于Ⅰ-Ⅲ期,且NO期miR-9的表达水平(平均秩次30.56)显著高于N1-N3期(平均秩次17.58)(Z=-2.992,P=0.002),从而说明miR-9的表达与鼻咽癌的发生、转移及进展相关。以ΔCt值表示miR-9的相对表达水平,鼻咽癌细胞株中NP69细胞miR-9的表达水平(18.00±0.10)显著少于C666-1 (27.19±0.11)、Sune-1 (25.80±0.22)、5-8F (23.77±0.13)、6-10B(24.76±0.07)、CNE-1(24.16±0.12)、CNE-2(22.92±0.14)和Hone-1(26.77±0.21)细胞,差异有统计学意义(F=1212.097,P=0.000)。多重比较结果显示转移能力最强的C666-1细胞miR-9的表达水平均低于转移能力较弱的5-8F和CNE-2(P=0.000),均低于无转移能力的6-10B和CNE-1(P=0.000)。
2.miR-9表达下调与其编码基因hsa-miR-9-1, hsa-miR-9-2和hsa-miR-9-3启动子区CpG岛甲基化有关
①hsa-miR-9-1, hsa-miR-9-2和hsa-miR-9-3启动子区CpG岛分布：
我们利用CpG Island Searcher软件对hsa-miR-9-1、hsa-miR-9-2和hsa-miR-9-3启动子区的CpG岛进行分析,结果表明hsa-miR-9-1 165bp-1255bp之间共1091bp区域存在一个CpG岛,其中GC比例为62.1%,ObsCpG/ExpCpG为0.741,共含有18个CpG二核苷酸；hsa-miR-9-2 3bp-203bp之间共201bp区域存在一个CpG岛,其中GC比例为55.2%,ObsCpG/ExpCpG为0.756,共含有9个CpG二核苷酸；hsa-miR-9-3 87bp-1899bp之间共1813bp存在一个CpG岛,其中GC比例为65.4%, ObsCpG/ExpCpG为0.739,共有35个CpG二核苷酸。
②5-AZA和TSA药物干预鼻咽癌细胞C666-1后miR-9重新表达及编码基因部分去甲基化
5-AZA和TSA能诱导C666-1细胞miR-9基因重新表达,C666-1 5-AZA组、C666-15-AZA+TSA组同C666-1组相比,miR-9表达水平分别上调11.31倍和22.63倍,差异有统计学意义(F=780.280,P=0.000),组间比较发现C666-1 5-AZA组、C666-15-AZA+TSA组miR-9表达水平显著高于C666-1组(P=0.000),且C666-15-AZA+TSA组亦显著高于C666-1 5-AZA组(P=0.000)。BSP DNA测序结果显示C666-1、C666-1 5-AZA、C666-1 5-AZA+TSA三组has-miR-9-1的甲基化比率分别为78.8%(67/85)、32.9%(28/85)和14.1%(12/85),差异有统计学意义(Z=77.324,P=0.000); has-miR-9-2的甲基化比率分别为72.2%(39/54)、38.9%(21/54)和14.8%(8/54),差异有统计学意义(χ2=36.850, P=0.000); has-miR-9-3的甲基化比率分别为76%(133/175)、32%(56/175)和20.6%(36/175),差异有统计学意义(X2=122.407,P=0.000),C666-1细胞has-miR-9-1、has-miR-9-2和has-miR-9-3甲基化率最高,C666-1-AZA组次之,C666-1-AZA+TSA组最低,说明5-AZA和TSA能改变C666-1细胞miR-9编码基因的甲基化状态,换言之,启动子甲基化和去乙酰化是鼻咽癌细胞miR-9表达下调的主要原因。
3.miR-9过表达对鼻咽癌细胞C666-1生物学功能的影响
为证实miR-9对鼻咽癌细胞C666-1生物学功能的影响,我们构建了miR-9过表达载体,筛选出miR-9过表达的C666-1/miR-9细胞,并进行了一系列体内外功能实验：CCK8法结果示细胞种植后的第二天开始,C666-1/miR-9细胞的增殖速度显著慢于C666-1/PLVTHM细胞(F=696.168,P=0.000)；平板克隆形成实验示C666-1/miR-9细胞形成的平板克隆数(48.15±3.82)显著少于对照组C666-1/PLVTHM细胞(83.70±3.74)(t=-29.746,P=0.000),说明miR-9过表达可抑制鼻咽癌的增殖和平板克隆形成能力；C666/miR-9和对照组C666-1/PLVTHM细胞均未出现明显的细胞凋亡,说明miR-9过表达不影响鼻咽癌细胞的凋亡；划痕实验中C666-1/miR-9细胞穿越划痕的细胞数(47.167±2.552)较对照细胞C666-1/PLVTHM (95.917±3.118)明显减少(t=-41.912,P=0.000),穿越transwell膜的细胞数(36.500±4.542)较对照组C666-1/PLVTHM (93.417±5.195)亦明显减少(t=28.569,P=0.000),提示miR-9过表达可抑制鼻咽癌细胞体外迁移能力；铺板12小时后C666-1/miR-9细胞穿透基质胶的细胞数(32.667±3.257)较对照组C666-1/PLVTHM细胞(81.833±4.366)显著减少(t=31.270,P=0.000),说明miR-9过表达可抑制鼻咽癌细胞的体外侵袭能力。背部接种C666-1/PLVTHM和C666-1/miR-9的裸鼠,2周后皮下成瘤率100%,C666-1/PLVTHM组少数瘤体因生长过快出现瘤体的表面坏死,且C666-1/PLVTHM组瘤体的平均体积(208.699±151.858)显著高于C666-1/miR-9组(128.688±75.127)(F=1472.708,P=0.000)；肝包膜下接种C666-1/PLVTHM和C666-1/miR-9细胞的裸鼠,C666-1/PLVTHM组肝内转移率88.9%(8/9)显著高于C666-1/miR-9组肝内转移率44.4%(4/9)(χ2=4.000, P=0.046), C666-1/PLVTHM组肺内转移率77.8%(7/9)显著高于C666-1/miR-9组肺内转移率22.2%(2/9)(X2=5.556,P=0.018),说明miR-9过表达能抑制鼻咽癌细胞皮下成瘤和肝肺转移的能力。
4.miR-9抑制靶基因CXCR4的表达和功能
生物信息学方法预测miR-9可能通过其种子序列(CUUUGGU)与CXCR4mRNA 3'-UTR的7个碱基(ACCAAAG)完全互补结合,从而发挥作用。为证实上述推测,我们构建双荧光报告质粒psiCHECK-2-CXCR4 3'-UTR质粒及其缺失突变体psiCHECK-2-mt-d-CXCR4 3'-UTR和替换突变体psiCHECK-2-mt-s-CXCR4 3'-UTR,并将上述载体分别与miR-9 mimics和miR-9mimics NC, miR-9 inhibitor和miR-9 inhibitor NC共转染至293FT细胞,转染48h后收集裂解细胞检测荧光素酶活性,以Firefly luciferase与Renilla luciferase的活性比值作为双荧光素酶的相对活性进行不同样品间的比较,结果示psiCHECK-2-CXCR4 3'-UTR与miR-9mimic共转染可显著降低荧光素酶活性(P<0.001),而将该重组质粒与miR-9 inhibitor共转染后可显著增强荧光素酶活性(P=0.001),此外,缺失突变质粒psiCHECK-2-mt-d-CXCR4 3'-UTR和替换突变质粒psiCHECK-2-mt-s-CXCR4 3'-UTR与miR-9 mimic或者miR-9 inhibitor共转染均不能改变荧光素酶的活性(P>0.05),而miR-9mimics的突变体miR-9-mt与psiCHECK-2-CXCR4 3'-UTR共转染后亦不能改变荧光素酶的活性,间接说明miR-9可通过“种子序列”与CXCRmRNA 3'-UTR区特异性结合。
其次,我们构建了过表达CXCR4蛋白的真核载体pReceiver-CXCR4-ORF和pReceiver-CXCR4-ORF+3'-UTR,并将其转染至CXCR4表达水平极低的293FT细胞,48h后收集细胞并进行western blot检测,结果示两者CXCR4蛋白表达水平相当,说明CXCR4 3'-UTR作为非翻译区不影响蛋白的翻译；而niR-9 mimics与CXCR4 ORF+3'-UTR载体共转染后能显著抑制CXCR4蛋白的表达,但miR-9mimics mt(与CXCR4 3'-UTR区结合的“种子序列”发生突变)与CXCR4ORF+3'-UTR载体共转染后对CXCR4蛋白表达的抑制作用不明显,且miR-9mimics或miR-9 mimics mt与CXCR4 ORF载体共转染后均不能显著抑制CXCR4蛋白的表达,说明“丧失”CXCR4 3'-UTR区后,miR-9对CXCR4表达的调控便缺乏了关键的“桥梁”,无论miR-9分子序列是否发生突变,均不能影响CXCR4蛋白的表达,进一步证明miR-9通过与CXCR4 3'-UTR区靶向结合调控其表达;其次,我们通过慢病毒感染的方法获得稳定过表达miR9的细胞C666-1/miR-9和对照细胞C666-1/PLVTHM, Q-PCR和western-blot检测示两组细胞间CXCR4mRNA的表达水平并无显著差异(P>0.05),但C666-1/miR-9细胞CXCR4蛋白表达水平显著低于C666-1/PLVTHM细胞,而在C666-1/miR-9细胞中转染miR-9 inhibitor后CXCR4蛋白表达水平得到一定程度的“恢复”,说明miR-9通过抑制翻译而非影响转录来调控CXCR4蛋白的表达,免疫荧光结果与western-blot结果相吻合,进一步证实了上述观点。
最后,为探讨miR-9能否部分逆转CXCR4在鼻咽癌中促进细胞增殖、迁移、侵袭和转移的功能,我们筛选出C666-1/CXCR4+PLVTHM与C666-1/CXCR4+miR-9细胞,结果发现C666-1/CXCR4+miR-9的增殖速度显著慢于C666-1/CXCR4+PLVTHM (F=2392.535, P=0.000), C666-1/CXCR4+miR-9形成的平板克隆数(64.90±2.29)显著少于对照组C666-1/CXCR4+PLVTHM(115.20±2.44) (t=67.185, P=0.000),说明miR-9能部分逆转CXCR4促进鼻咽癌增殖和克隆形成的作用；C666-1/CXCR4+PLVTHM与C666-1/CXCR4+miR-9的细胞凋亡状态均无显著差别；C666-1/CXCR4+miR-9细胞穿越划痕的细胞数(53.000±7.520)较对照组C666-1/CXCR4+PLVTHM(236.083±10.423)显著减少(t=49.348,P=0.000),C666-1/CXCR4+miR-9穿越transwell膜的细胞数(56.667±3.229)较对照组C666-1/CXCR4+PLVTHM(115.833±5.524)亦显著减少(t=32.033,P= 0.000), C666-1/CXCR4+miR-9穿透基质胶的细胞数(45.250±3.745)较对照组C666-1/CXCR4+PLVTHM(106.500±3.802)显著减少(t=39.760,P=0.000),说明miR-9能部分逆转CXCR4促进鼻咽癌细胞体外迁移和侵袭能力的作用。背部和肝包膜下接种C666-1/CXCR4+PLVTHM与C666-1/CXCR4+miR-9细胞的裸鼠,2周后C666-1/CXCR4+PLVTHM组皮下瘤体的平均体积(249.404±143.100)显著大于C666-1/CXCR4+miR-9组(134.357±74.782)(F=659.513,P=0.000)；C666-1/CXCR4+PLVTHM组肝内转移率100%(9/9)显著高于C666-1/CXCR4+miR-9组肝内转移率55.6%(5/9)(X2=5.143,P=0.023),而C666-1/CXCR4+PLVTHM组肺内转移率88.9%(8/9)显著高于C666-1/CXCR4+miR-9组肺内转移率33.3%(3/9)(X2=5.844,P=0.016),说明miR-9能部分逆转CXCR4促进鼻咽癌细胞皮下成瘤和肝肺转移的能力。
结论
1.miR-9在鼻咽癌组织和细胞中表达下调。
2.鼻咽癌中miR-9表达下调与编码miR-9的基因miR-9-1, miR-9-2和miR-9-3启动子区CpG岛的甲基化有关。
3.miR-9过表达能抑制鼻咽癌细胞的增殖、侵袭和转移能力,miR-9在鼻咽癌中发挥抑癌作用。
4.在鼻咽癌中,CXCR4为miR-9的靶基因。
5.miR-9通过调控靶基因CXCR4的表达,部分逆转靶基因CXCR4在鼻咽癌中的促癌功能,从而抑制鼻咽癌细胞的增殖、侵袭和转移能力。
Objective
Nasopharyngeal carcinoma(NPC) is a kind of head and neck squamous cell carcinoma frequently occurred in south of China. It was difficult for early diagnosis in NPC in clinical and it was reported that more than 60% diagnosed NPC patients had metastasis of neck lymph node or distant organs.Furthermore, the middle survival was less than one year if metastasis was detected.Therefore, it is very important to study the formation and metastasis mechanisms of NPC for improving curative effects and prolonging overall survivals of patients.
miRNAs are small noncoding RNAs that serve as negative regulators of gene expression. Through interactions with the 3'-untranslated region (3'-UTR) of mRNA by partial sequence homology, miRNAs cause gene silencing either by mRNA degradation or translation repression. In addition to participation in embryonic neural development, diabetes and maintenance cardiac function, accumulating evidence suggests that miR-9 could be key players in regulation of tumor cell proliferation,invasion and metastasis in many kind of tumors,including Burkitt lymphoma,non-hodgkin's B cell lymphoma, ovarian cancer, gastric carcinoma, endometrial carcinoma. Increasing evidence indicates that miRNAs may function as either oncogenes or tumor suppressors in a cell type-specific manner,but there is no report about miR-9 expression and function in NPC or other head and neck carcinoma.
Chemine receptor-4(CXCR4) was deeply researched by many reachers. It was reported that CXCR4 played an important role in the process of progression and metastasis in many tumors, but rarely studied in NPC.
We found that miR-9 could bind with CXCR4 3'-UTR through its"seed sequence"by bioinformatics method,indicating the direct targeted regulation relationship between miR-9 and CXCR4 is to be confirmed.
Our study aimed to observe the expressions of miR-9 and CXCR4 in NPC tissues with different metastatic potential and chronic nasopharyngitis tissues, normal immortalization nasopharynx epithelial cell NP69 and nasopharyngeal carcinoma cell lines; to explicit the effect of miR-9 and CXCR4 on proliferation,migration,invasion and metastasis by screening C666-1 cell line of miR-9 overexpression and CXCR4 interference;to explore whether miR-9 and CXCR4 have direct targeted regulation relationship,whether miR-9 could partially reverse the stimulative effect of CXCR4 in NPC by constructing CXCR4 3'-UTR double fluorescence report plasmid including binding sites of miR-9 and function of CXCR4 by in vivo and vitro experiment;to discuss and explicit whether lower expression of miR-9 in NPC is related with DNA methylation in promoter of its encoding genes by detection of miR-9 expression and CpG island DNA methylation in miR-9 encoding genes after 5-AZA and TSA used in C666-1.
Methods
1.Clinical samples collecting and grouping
Forty patients with NPC (20 cases with metastasis and 20 cases without metastasis) and twenty chronic nasopharyngitis cases were recruited in this study from October 2008 to December 2010. All the specimen were confirmed by HE staining method and detected the expression level of miR-9 and CXCR4,analized their expression correlation between miR-9 and CXCR4.
2.Culture of NPC cell lines
NPC cell lines including C666-1,Sune-1,5-8F,6-10B,CNE-1,CNE-2 and Hone-1 were maintained in RMPI-1640 medium which was supplemented with 10% heated inactivated fetal calf serum, and normal immortalization nasopharynx epithelial cell NP69 was maintained in KMSF medium,all the cell lines were digested by common conditions.
3.Real Time-PCR
MicroRNA and total RNA were extracted from NPC cell lines and NP69,and then reversely transcripted into cDNA and PCR amplified using taqman probes and primers,relative expression strength of miR-9 and CXCR4 were expressed by 2-ΔΔCt method.
4.In situ hybridization
Paraffin organizations and cells climb pieces were dewaxed and hydrated(fixed by 4% paraformaldehyde),trypsin digested,prehybridizated,hybridizated,serum closed,incubated overnight by digoxin labeled antibody at 4℃,washed by BCIP/NBT buffer,stained by BCIP/NBT and nuclear fast red,and then staining results were observed.
5.IHC detection
Paraffin sections of tissues prepared for detection were stained by SP method, and the results of IHC were observed by light microscope and judged:Sections were observed in 10×objective lens and the staining intensities of all cells in 5 fields of vision were counted randomly, then scores were counted.
6.Vector construction and identification
①Construction and identification of pLVTHM/miR-9 and pLVTHM/ CXCR4-shRNA-miR (con) vector
pre-miR-9 transcription templates and shRNA-miR (con) DNA Oligo sequences were synthesized,annealed and double digested by MluI and Clal, connected to construct vectors:pLVTHM/miR-9 and pLVTHM/CXCR4-shRNA-miR (con)
②Construction and identification of psiCHECK-2-CXCR4 3'-UTR vector and its mutants
we double digested PCR product of CXCR4 3'-UTR and psiCHECK-2 by Xhol and NotI to construct vector psiCHECK-2-CXCR4 3'-UTR,meanwhile we constructed psiCHECK-2-mt-s-CXCR4 3'-UTR (replace mutation) and psiCHECK-2-mt-d-CXCR4 3'-UTR (missing mutation) by overlap PCR method.
③Construction and identification of pReceiver-CXCR4-ORF and pReceiver- CXCR4-ORF+3'-UTR vector
We construct pReceiver-CXCR4-ORF and pReceiver-CXCR4-ORF+3'-UTR vectors using peripheral genomic DNA of normal persons as template
7.Lentivirus Package
DNA-Cacl2 solution was used for lentivirus Package.
8.Screening miR-9 overexpression and CXCR4 interference cell lines.
We sorted C666-1 GFP+ cells by flow cytometric analysis after lentivirus infecting C666-1 cells, and named them as C666-1/miR-9 and C666-1/pLVTHM, C666-1/CXCR4-shRNA-miR and C666-1/CXCR4-shRNA-con,identificated them by RT-PCR and western-blot methods.
9.Western blot:Western blot analysis was performed according to the standard method with antibodies to CXCR4 and GAPDH after total protein were extracted and quantificated by BCA method.
10.Immunofluorescence assay. The expression of CXCR4 was determined by immunofluorescence assay. Cells were fixed by 4%paraformaldehyde and permeabilizated by 0.2%triton X-100 for 5mins, blocked by 10% normal goat serum for 30mins, inoculated with rabbit antihuman CXCR4 protein monoclonal antibody at 4℃for overnight and rinsed with phosphate buffered saline (PBS) with 0.2% tween 20 (PBST) three times for 10 min each. The mixture was then incubated with fluorescein isothiocyanate (FITC)-goat antimouse immunoglobulin G (IgG) antibody (1:200) at 37℃for 60 min and rinsed three times for 10 min each with PBST solution. Finally, inoculated with DAPI for 5mins.Cell pictures were obtained by fluorescence microscope in every sample and randomly selected to test cell fluorescence intensity.
11.Functional studies in vitro and in vivo:
①CCK-8 assay:CCK-8 assay was performed to assess the effect of miR-9 and CXCR4 on cell proliferation.Cells(1×103cell/well) were plated in a 96-well plate and maintained in RPMI1640 supplemented with 10% FBS.At 24,48,72,96 and 120h after seeding,culture medium was removed,cells were treated with 20ul CCK8 working solution for 1h at 37℃,and spectrometric absorbance at wavelength of 450nm was measured on a microplate reader.
②Colony formation assay:For colony formation assay,200 cells were seeded into 6-well plate. After being cultured for 1 10-14 days, surviving colonies (>50 cells/colony) were counted with 1% crystal violet staining. Triplicate independent experiments were performed.
③Cell apoptosis:Flow cytometry detection,caspase-3 activity analysis and Tunel in situ apoptosis detection were used to detect cell apoptosis status.
④wound healing assays:In vitro wound healing assay was carried out to determine the ability of cells to form membrane protrusion and cell migration. When the cell confluence reached 90%,a single wound was created in the center of cell monolayer by gentle removal of the attached cells with a sterile plastic pipette tip.The debris was removed by washing the cells with serum free medium.Migration of cells into the wound was then observed after 12h..
⑤Transwell migration assay:For cell migration assay, cells were starved with serum free medium for 24 hrs before the assay. Cells (5×104) were suspended in 0.5 ml serum-free medium and loaded on the upper compartment of transwell chamber. The lower compartment was filled with complete medium as chemoattractant. After 12 hrs, migration cells were fixed, stained, and counted under a microscope. Triplicate independent experiments were done.
⑥Transwell invasion assays
For invasion assay, cells were starved with serum free medium for 24 hrs before the assay.5×104 cells were suspended in 0.5 ml serum-free medium and loaded on the upper compartment of invasion chamber coated with Matrigel. The lower compartment was filled with complete medium as chemoattractant. After 12 hrs, invasive cells were fixed, stained, and counted under a microscope. Triplicate independent experiments were done.
⑦Tumor formation in nude mice:For in vivo experiment,cells (4×106) in 200μL serum-free RPMI1640 were injected s.c into the right flank of 4-6 weeks-old nude mice, respectively. The tumor volume was calculated by the formula V=0.5×L×W2(L:length;W:width),and when the volme of tumor was close to 500 cm3 nude mice were killed.
⑧In vivo metastasis experiment:Nude mice(BALB/C nu/nu,4-6 weeks old,18-24g in weight) were maintained under pathogen-free conditions(specific pathogen-free level).Primary tumors were established by direct injection of 2×106 cells into the liver as previously described.All the mice were sacrificed on day 21,and tissues were collected for pathologic analysis.GFP fluorescence images were observed under an in vivo fluorescence instrument.
12.MiRNA target site prediction. A search for predicted target miRNAs was performed usingthe databases TargetScan (http://www.targetscan.org/),miRbase (http://www.mirbase.org) and PicTar(http://pictar.mdc-berlin.de/).equence S conservation was examined using University of California Santa Cruz genome browser(http://genome.ucsc.edu/) and then we selected interesting target gene (http://genome.ucsc.edu/).
13.Dual-luciferase reporter gene assay:293FT cells, chosen based on their low endogenous expression of miRNAs (26), were grown to 80-90% confluence in white 96-well plates in DMEM supplemented with 10% FBS,1% nonessential amino acids, L-glutamine, and penicillin/straptamicin at 37℃under 5% CO2.Cells were transfected with 20 ng empty psiCHECK-2 vector or psiCHECK-2-CXCR4-3'-UTR reporter and miR- 9 mimics or its mutant-miR-9 mimics mt and negative control (miR control), miR-9 inhibitor or its negative control (miR- 9 inhibitor control)at concentrations of 20nM in reduced serum and antibiotics-free Opti-MEM with lipofectamine 2000. T he Firefly and Renilla luciferase were measured in cell lysates using a dual-luciferase reporter assay system on a fusion plate reader. Firefly luciferase activity was used for normalization and as an internal control for transfection efficiency.
14.5-AZA and TSA treatment of C666-1 cells. 1×105 C666-1 cells were split 12 to 24 h before treatment. Cells were then given one of the following treatments. (i) 50uM 5-Aza-2'-deoxycytidine (5-AZA) was used for 72 h. Medium containing 5-AZA was changed every 24 h;(ii) 50uM 5-AZA was used for 72 h followed by 10nm TSA for an additional 24 h. The dose of 5-AZA (50uM) and TSA(lOnm) was chosen based on preliminary studies showing optimal reactivation of miR-9 gene expression and DNA methylation in promoter.
15.hsa-miR-9-l,hsa-miR-9-2 and hsa-miR-9-3 CpG islands methylation analysis In an attempt to determine all CpG islands and all potential transcription start sites (TSS) for hsa-miR-9-1,hsa-miR-9-2 and hsa-miR-9-3, we first proceeded with the identification of the promoter sequence. The analyses were initiated using an identified RefSeq by GenBank accession number, after which we submitted the gene sequence to a Genome BLAT Search through the UCSC Genome bioinformatics website (http://genome.ucsc.edu). We selected 2000bps of sequence extending from the 5' upstream region to 1000 bps downstream of the region of the TSS. The BLAT program returned a sequence that was first submitted to the CpGPLOT program from the European Bioinformatics Institute website (http://www.ebi.ac.uk/emboss/cpgplot) This program defines a CpG island as 200 or 300bps of sequence with 50%C+G content and 0.65 CpG observed/CpG expected.
16.Bisulfite genomic sequencing:Genomic DNA was isolated from cell samples and 1000ng treated with sodium bisulfite using the DNA Methylation kit to convert unmethylated cytosine to uracil.Bisulfite-treated DNA was then amplified with specific primers for CpG islands surrounding miR-9-1,miR-9-2 and miR-9-3 genomic locations by PCR with platinum taq hifidility,finally PCR product and pMD19-T vector were connected and coated for DNA sequencing to detect DNA methylation status.
17.Statistical analysis:All analyses were carried out using the SPSS 13.0 software package.A P value less than 0.05 was considered statistically significant.Comparisiong of miR-9 and CXCR4 expression level in chronic nasopharyngitis tissues,nasopharyngeal carcinoma tissues without metastasis, nasopharyngeal carcinoma tissues with metastasis was analyzed with the Kruskal-Wallis test (K independent samples test).Sperman rank correlation was used for relationship between CXCR4 protein and miR-9 expression level.Comparison of mean in different cell lines about iR-9 and CXCR4 expession was analyzed by one-way ANOVA or indepent-samples T test and Student- Neuman-keuls methos for multiple comparison.Dunnetts T3 method was exployed for heterogeneity of variance.Theχ2 test for proportion was used to analyze the vivo metastasis characteristics of different groups.Factorial analysis was utilized to analyze the results of CCK8,invasion and wound healing assay.The difference in wound migration and invasion indices between C666-1 cell clones were analyzed by the independented t test.Comparison of mean in colony formation and wound healing assay in two sample was analyzed by independent-samples T test;CCK8 assay and tumor formation in nude mice was analyzed by one-way ANOVA;χ2 test was used for rates of liver and lung metastasis among different cell lines.miR-9 exprssion level in C666-1,C666-1 5-AZA and C666-1 5-AZA+TSA groups were analyzed by One-way ANOVA and R×Cχ2 t test for BSP results.
Results
1.miR-9 was lower expressed in NPC tissues and cell lines.
miR-9 was positively expressed in nuclei and membrane of nasophayngeal carcinoma cell and epithelial cells in chronic nasopharyngitis tissues.The expression levels of miR-9 detected by ISH in nasopharyngeal carcinoma tissues (mean rank 22.25) were significantly lower than that of chronic nasopharyngitis tissues (mean rank 47.00) (Z=-5.218, P=0.000), and the differences of miR-9 expression level among nasopharyngeal carcinoma tissues with different T stage,N stage and clinical stage were significant (χ2=20.477, P=0.000;χ2=14.423, P=0.002;χ2=17.561, P=0.001). miR-9 expression levels were down-regulated gradually as T stage,N stage and clinical stage were increased,and miR-9 expression levels in T3-T4 stage,N3 stage and IV stage NPC tissues were lower than those of T1-T2 stage, N1-N2 stage and I-III stage.Notely, miR-9 expression level in NO stage NPC tissues (mean rank 30.56) was significantly lower than that of N1-N3 stage NPC tissues (mean rank 17.58) (Z=-2.992, P=0.002),indicating that miR-9 is related with formation and metastasis of NPC. The expression level of miR-9 in NP69 (18.00±0.10) detected by Real time-PCR was significantly higher than those in C666-1 (27.19±0.11), Sune-1(25.80±0.22),5-8F(23.77±0.13),6-10B(24.76±0.07),CNE-1(24.16±0.12),CNE-2 (22.92±0.14) and Hone-1 (26.77±0.21) (F=1212.097, P=0.000), and the expreesion level of miR-9 in C666-1 was lower than those of 5-8F and CNE-2 (P=0.000),6-10B and CNE-1 (P=0.000)
2. miR-9 downregulation is related with CpG island DNA methylation in promoter of hsa-miR-9-1, hsa-miR-9-2 and hsa-miR-9-3.
①CpG island distribution in hsa-miR-9-1, hsa-miR-9-2 and hsa-miR-9-3 promoters:We used CpG island searcher software to analyze CpG islands in hsa-miR-9-1,hsa-miR-9-2 and hsa-miR-9-3 promoters,and found that there is a CpG island in hsa-miR-9-1 from 165bp to 1255bp(1091bp,including 18 CpG dinucleotides, GC%=62.1%, ObsCpG/ExpCpG=0.741),a CpG island in hsa-miR-9-2 from 3bp to 203bp(201bp,including 9 CpG dinucleotides,GC%=55.2%, ObsCpG/ExpCpG=0.756) and a CpG island in hsa-miR-9-3 from 87bp to 1899bp (1813bp,including 35 CpG dinucleotides, GC%=65.4%, ObsCpG/ExpCpG=0.739).
②5-AZA and TSA induce miR-9 re-expression and DNA demethylation in C666-1 cell.5-AZA and TSA could induce miR-9 re-expression in C666-1,miR-9 expression level in C666-1 5-AZA group and C666-1 5-AZA+TSA group increased 11.31 and 22.63 times (F=780.280,P=0.000),and miR-9 expression level in C666-1 5-AZA group and C666-1 5-AZA+TSA group were significantly higher than that of C666-1 group (P=0.000),and the same as C666-1 5-AZA+TSA group and C666-1 5-AZA group (P=0.000).Methylation ratios of hsa-miR-9-1 in C666-1、C666-1 5-AZA and C666-1 5-AZA+TSA groups were 78.8%(67/85)、32.9%(28/85) and 14.1%(12/85) respectively (χ2=77.324, P=0.000), methylation ratios of hsa-miR-9-2 were 72.2%(39/54)、38.9%(21/54) and 14.8%(8/54) respectively (χ2=36.850, P=0.000), methylation ratios of hsa-miR-9-3 were 76%(133/175)、32%(56/175) and 20.6%(36/175) (χ2=122.407, P=0.000) by BSP sequencing., methylation ratios of hsa-miR-9-1, hsa-miR-9-2 and hsa-miR-9-3 were highest in C666-1 group and lowest in C666-1-AZA+TSA group,indicating 5-AZA and TSA could change methylation status of miR-9 encoding genes in C666-1 cell, in other word,DNA methylation and acetylation in promoter were main reasons for miR-9 downregulation in NPC cell.
3. The effect of miR-9 on functions of NPC.
The proliferation rates of C666-1/miR-9 was significantly slow than that of C666-1/PLVTHM (F=696.168, P=0.000),The colony numbers formed by C666-1/miR-9(48.15±3.82) was significantly less than that by C666-1/PLVTHM (83.70±3.74) (t=29.746, P=0.000) indicating that miR-9 overexpression could inhibit proliferarion and colony formation ability of NPC cell;There was no obvious apoptosis induced by C666-1/miR-9 and C666-1/PLVTHM,indicating that miR-9 overexpression has no effect of NPC cell apotosis;In wound healing assay,The number of C666-1/miR-9 cells passed through the scratch (47.167±2.552) was less than that of C666-1/PLVTHM (95.917±3.118) (t=41.912, P= 0.000),and in transwell migration assay C666-1/miR-9(36.500±4.542)and C666-1/PLVTHM(93.417±5.195) (t=28.569, P= 0.000) were the same as in wound healing assay,indicating that miR-9 overexpression could inhibit migration ability of NPC cell.In transwell invasion assay,the number of C666-1/miR-9 cells penetrated Matrigel (32.667±3.257) was less than that of C666-1/PLVTHM (81.833±4.366) (t=31.270, P=0.000) indicating that miR-9 overexpression could inhibit invasion ability of NPC cell.In subcutaneous tumor formation assay,the average volume of tumor formed by C666-1/miR-9 (128.688±75.127) was less than that by C666-1/PLVTHM (208.699±151.858) (F=1472.708, P=0.000);After cells inoculated under liver capsule,the metastasis rate of intrahepatic spread in C666-1/miR-9(44.4%,4/9) was lower than that of C666-1/PLVTHM (88.9%,8/9) (χ2=4.000,P=0.046),and the metastasis rate of lung spread in C666-1/miR-9(22.2%,2/9) was lower than that of C666-1/PLVTHM (77.8%,7/9) (χ2=5.556, P=0.018) indicating that miR-9 overexpression could inhibit tumor formation and metastasis ability of NPC cell.
4. miR-9 inhibits expression and function of CXCR4
We predicted that miR-9 may play its role through combining its seed sequence(CUUUGGU) with 3'-UTR of CXCR4mRNA (ACCAAAG) by bioinformatics method.To confirm the hyposesis,we constructed plasmid psiCHECK-2-CXCR4 3'-UTR and its mutants psiCHECK-2-mt-d-CXCR4 3'-UTR or psiCHECK-2-mt-s-CXCR4 3'-UTR that to be transfected with miR-9 mimics,miR-9 NC,miR-9 inhibitor and miR-9 inhibitor NC into 293FT cell and then detected luciferase activity, finding that con-transfection of psiCHECK-2-CXCR4 3'-UTR and miR-9 mimics could significantly reduce luciferase activity (P<0.001), con-transfection of psiCHECK-2-CXCR4 3'-UTR and miR-9 inhibitor could significantly enhance luciferase activity(P=0.001).However,con-transfection of psiCHECK-2-mt-d-CXCR4 3'-UTR or psiCHECK-2-mt-s-CXCR4 3'-UTR with miR-9 mimics or miR-9 inhibitor even could not change luciferase activity (P>0.05),and con-transfection of mutant of miR-9mimics(miR-9-mt) and psiCHECK-2-CXCR4 3'-UTR also could not change luciferase activity,indicating that miR-9 could bind with 3'-UTR of CXCR4.
Next,we constructed pReceiver-CXCR4-ORF and vectors and transfected them into 293FT cells in which CXCR4 protein is lower expressed,founding that the expression level of CXCR4 protein was almost the same,indicating the 3'-UTR of CXCR4 has no effect on protein translation,but when we con-transfected the above vectors with miR-9 mimics and miR-9 mimics mt,we found miR-9 mimics rather than miR-9 mimics mt could significantly inhibit CXCR4 protein expression after being transfected with pReceiver-CXCR4-ORF+3'-UTR, and both of miR-9 mimics and miR-9 mimics mt could not significantly inhibit CXCR4 protein expression after being transfected with pReceiver-CXCR4-ORF,indicating miR-9 has no effect on CXCR4 protein expression no matter whether the sequence of miR-9 is mutanted when 3'-UTR of CXCR4 is lost;next,we detected miR-9 and CXCR4 expression in C666-1/miR-9 and C666-1/PLVTHM by Q-PCR and western-blot,and found that CXCR4 protein rather than CXCR4 mRNA was lower in C666-1/miR-9 than C666-1/PLVTHM,but CXCR4 protein expression could be recovered to some extent,indicating miR-9 could inhibit CXCR4 protein rather than CXCR4 mRNA expression,which was consistent with results by immune fluorescence.
Finally, in order to explore whether miR-9 could partial reverse the function of CXCR4 in cell proliferation,migration,invasion and metastasis,we screened C666-1/CXCR4+PLVTHM and C666-1/CXCR4+miR-9 cells,and found that the proliferation of C666-1/CXCR4+miR-9 was significantly slower than that of C666-1/CXCR4+PLVTHM (F=2392.535, P=0.000),and colony numbers formed by C666-1/CXCR4+miR-9(64.90±2.29) were significantly less than that of C666-1/CXCR4+PLVTHM(115.20±2.44) (t=67.185, P=0.000),indicating that miR-9 could partial reverse the function of CXCR4 in cell proliferation and colony formation;C666-1/CXCR4+PLVTHM and C666-1/CXCR4+miR-9 have no effect on cell apoptosis.The number of C666-1/CXCR4+miR-9 cells passed through scratches (53.000±7.520) was much less than that of C666-1/CXCR4+PLVTHM (236.083±10.423) (t=49.348, P= 0.000),and the number of C666-1/CXCR4+miR-9 cells penetrated transwell membrane (56.667±3.229) was also much less than that of C666-1/CXCR4+PLVTHM (115.833±5.524) (t= 32.033, P=0.000),the number of C666-1/CXCR4+miR-9 cells penetrated Matrigel (45.250±3.745) was also much less than that of C666-1/CXCR4+PLVTHM (106.500±3.802) (t=39.760,P=0.000), indicating that miR-9 could partial reverse the function of CXCR4 in cell migration and invasion.The average volumes of subcutaneous tumor in C666-1/CXCR4+PLVT HM(249.404±143.100) group were greater than that of C666-1/CXCR4+miR-9 group(134.357±74.782) (F= 659.513, P=0.000);The intrahepatic transfer rate in C666-1/CXCR4+PLVTHM group (100%,9/9) was higher than that of C666-1/CXCR4+miR-9 group (55.6%,5/9) (x2=5.143,P=0.023),and the pulmonary metastasis rate in C666-1/CXCR4+PLVTHM group (88.9%,8/9) was higher than that of C666-1/CXCR4+miR-9 group (33.3%,3/9) (χ2=5.844, P=0.016), indicating that miR-9 could partial reverse the function of CXCR4 in tumor formation and metastasis in vivo.
Conclusion
①miR-9 was down-regulated in nasopharyngeal carcinoma tissues and nasopharyngeal carcinoma cells.
②The lower expression of miR-9 in NPC is related with CpG island DNA methylation in promoter of miR-9 encoding genes hsa-miR-9-l,hsa-miR-9-2 and hsa-miR-9-3.
③miR-9 overexpression could inhibit the proliferation,colony formation, migration and invasion, subcutaneous tumor formation and liver, lung metastasis ability of C666-1 cell,indicating miR-9 acted as tumor suppressor gene in NPC.
④miR-9 inhibited the proliferation, migration and invasion ability of C666-1 cell by regulating expression of CXCR4 protein and partially reversing the function of CXCR4 in NPC.
引文
[1]易俊林,高黎,黄晓东,等.鼻咽癌的临床表现和预后[J].中国医学科学院学报,2006,28(3)：315-317.
[2]Bala S, Marcos M, Szabo G. Emerging role of microRNAs in liver diseases[J]. World J Gastroenterol,2009,15(45):5633-5640.
[3]Lee R C, Feinbaum R L, Ambros V. The C. elegahs heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14[J]. Cell,1993, 75(5):843-854.
[4]Reinhart B J, Slack F J, Basson M, et al. The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans[J]. Nature,2000,403(6772): 901-906.
[5]Winter J, Jung S, Keller S, et al. Many roads to maturity:microRNA biogenesis pathways and their regulation[J]. Nat Cell Biol,2009,11(3):228-234.
[6]Carthew R W, Sontheimer E J. Origins and Mechanisms of miRNAs and siRNAs[J]. Cell,2009,136(4):642-655.
[7]Inui M, Martello G, Piccolo S, et al. MicroRNA control of signal transduction[J]. Nat Rev Mol Cell Biol,2010,11(4):252-263.
[8]Zeng Y, Yi R, Cullen B R. MicroRNAs and small interfering RNAs can inhibit mRNA expression by similar mechanisms[J].Proc Natl Acad Sci USA,2003, 100(17):9779-9784.
[9]吴斌,俞洋.微小RNA与肿瘤研究进展[J].现代肿瘤医学,2009,17(2)：373-376.
[10]Lo AK, To KF, Lo KW, et al.Modulation of LMP1 protein expression by EBV-encoded microRNAs[J]. Proc Natl Acad Sci USA,2007,104(41): 16164-9.
[11]Choy EY, Siu KL, Kok KH, et al. An Epstein-Barr virus-encoded microRNA targets PUMA to promote host cell survival[J].J Exp Med,2008,205(11): 2551-60.
[12]Sengupta S, den Boon JA, Chen IH, et al.MicroRNA 29c is down-regulated in nasopharyngeal carcinomas, up-regulating mRNAs encoding extracellular matrix proteins[J].Proc Natl Acad Sci USA.2008,105(15):5874-8.
[13]Shi W, Alajez NM, Bastianutto C, et al. Significance of Plkl regulation by miR-100 in human nasopharyngeal cancer[J]. Int J Cancer,2010,126(9): 2036-48.
[14]Chen HC, Chen GH, Chen YH, et al. MicroRNA deregulation and pathway alterations in nasopharyngeal carcinoma[J]. Br J Cancer,2009,100(6):1002-11.
[15]Xia H, Ng SS, Jiang S, et al.miR-200a-mediated downregulation of ZEB2 and CTNNB1 differentially inhibits nasopharyngeal carcinoma cell growth, migration and invasion[J]. Biochem Biophys Res Commun,2010,391(1): 535-41.
[16]Wong TS, Man OY, Tsang CM, et al. MicroRNA let-7 suppresses nasopharyngeal carcinoma cells proliferation through downregulating c-Myc expression[J].J Cancer Res Clin Oncol,2011,137(3):415-22.
[17]Zhang L, Deng T, Li X, microRNA-141 is involved in a nasopharyngeal carcinoma-related genes network[J].Carcinogenesis,2010,31(4):559-66.
[18]Wan HY, Guo LM, Liu T, et al.Regulation of the transcription factor NF-kappaB1 by microRNA-9 in human gastric adenocarcinoma[J].Mol Cancer,2010,9 (16):1-10.
[19]Luo H, Zhang H, Zhang Z, et al.Down-regulated miR-9 and miR-433 in human gastric carcinoma[J]. J Exp Clin Cancer Res,2009,16 (28):82.
[20]Rotkrua P, Akiyama Y, Hashimoto Y, et al. MiR-9 down-regulates CDX2 expression in gastric cancer cells[J].Int J Cancer,2011, Jan 10.
[21]Guo LM, Pu Y, Han Z, et al.MicroRNA-9 inhibits ovarian cancer cell growth through regulation of NF-kappaBl [J].FEBS J,2009,276(19):5537-46.
[22]Tan HX, Wang Q, Chen LZ, et al.MicroRNA-9 reduces cell invasion and E-cadherin secretion in SK-Hep-1 cell[J].Med Oncol,2010,27 (3):654-60.
[23]Ma L, Young J, Prabhala H, et al. miR-9, a MYC/MYCN-activated microRNA, regulates E-cadherin and cancer metastasis[J]. Nat Cell Biol, 2010,12(3):247-56.
[24]晁腾飞,张瑜,颜兴起,等.MiR-9在人神经胶质瘤中调节CBX7的表达[J].中国医学科学院学报,2008,30(3)：268-274.
[25]Nie K, Gomez M, Landgraf P, et al.MicroRNA-mediated down-regulation of PRDM1/Blimp-1 in Hodgkin/Reed-Sternberg cells:apotential pathogenetic lesion in Hodgkin lymphomas[J].Am J Pathol,2008,173 (1):242-52.
[26]Myatt SS, Wang J, Monteiro LJ, et al.Defniition of microRNAs that repress expression of the tumor suppressor gene FOXO1 in endometrial cancer [J]. Cancer Res,2010,70(1):367-77.
[27]Tao Q, Chan AT.Nasopharyngeal carcinoma:molecular pathogenesis and therapeutic developments[J].Expert Rev Mol Med,2007,9 (12):1-24.
[28]Chow LS, Lo KW, Kwong J, et al.RASSF1A is a target tumor suppressor from 3p21.3 in nasopharyngeal carcinoma[J]. Int J Cancer,2004,109(6):839-47.
[29]Yau WL, Lung HL, Zabarovsky ER, et al.Functional studies of the chromosome 3p21.3 candidate tumor suppressor gene BLU/ZMYND10 in nasopharyngeal carcinoma[J]. Int J Cancer,2006,119(12):2821-6.
[30](30)Li L, Tao Q, Jin H, et al. The tumor suppressor UCHL1 forms a complex with p53/MDM2/ARF to promote p53 signaling and is frequently silenced in nasopharyngeal carcinoma [J]. Clin Cancer Res,2010,16(11):2949-58.
[31]Lung HL, Cheung AK, Xie D, et al.TSLC1 is a tumor suppressor gene associated with metastasis in nasopharyngeal carcinoma[J]. Cancer Res,2006, 6(19):9385-92.
[32]Wang Y, Li J, Cui Y, et al. CMTM3, located at the critical tumor suppressor locus 16q22.1, is silenced by CpG methylation in carcinomas and inhibits tumor cell growth through inducing apoptosis[J]. Cancer Res,2009,69(12): 5194-201.
[33]EstellerM.Epigenetics in cancer[J].N Engl J Med,2008,358:1148-1159.
[34]张恒,何成强,李云龙.哺乳动物DNA甲基化的功能和作用[J].生命的化学,2002,22：218-220.
[35]Ran Y, Wu S, You Y, et al.Demethylation of E-cadherin gene in nasopharyngeal carcinoma could serve as a potential therapeutic strategy[J]. J Biochem,2011, 149(1):49-54.
[36]Chen F, Mo Y, Ding H, et al. Frequent epigenetic inactivation of Myocardin in human nasopharyngeal carcinoma[J]. Head Neck,2011,33(1):54-9.
[37]Tong JH, Ng DC, Chau SL, et al.Putative tumour-suppressor gene DAB2 is frequently down regulated by promoter hypermethylation in nasopharyngeal carcinoma[J]. BMC Cancer,2010,10:253.
[38]Liu M, Peng Y, Wang X, et al.NGX6 gene mediated by promoter methylation as a potential molecular marker in colorectal cancer[J]. BMC Cancer,2010,10: 160.
[39]Li L, Tao Q, Jin H, et al.The tumor suppressor UCHL1 forms a complex with p53/MDM2/ARF to promote p53 signaling and is frequently silenced in nasopharyngeal carcinoma[J]. Clin Cancer Res,2010,16(11):2949-58.
[40]Wang T, Liu H, Chen Y, et al.Methylation associated inactivation of RASSF1A and its synergistic effect with activated K-Ras in nasopharyngeal carcinoma[J]. J Exp Clin Cancer Res,2009,28:160.
[41]Zhou L, Feng X, Shan W, et al.Epigenetic and genetic alterations of the EDNRB gene in nasopharyngeal carcinoma[J]. Oncology,2007,72(5-6): 357-63.
[42]Wang Y, Li J, Cui Y, et al.CMTM3, located at the critical tumor suppressor locus 16q22.1, is silenced by CpG methylation in carcinomas and inhibits tumor cell growth through inducing apoptosis[J]. Cancer Res,2009,69(12): 5194-201.
[43]Shao L, Cui Y, Li H, et al.CMTM5 exhibits tumor suppressor activities and is frequently silenced by methylation in carcinoma cell lines[J].Clin Cancer Res, 2007,13(19):5756-62.
[44]Tan SH, Ida H, Goh BC, et al.Analyses of promoter hypermethylation for RUNX3 and other tumor suppressor genes in nasopharyngeal carcinoma[J]. Anticancer Res,2006,26(6B):4287-92.
[45]Trankenschuh W, Puls F, Christgen M, et al.Frequent and distinct aberrations of DNA methylation patterns in fibrolamellar carcinoma of the liver[J].PLoS One,2010,5(10):e13688.
[46]Hildebrandt MA, Gu J, Lin J, et al.Hsa-miR-9 methylation status is associated with cancer development and metastatic recurrence in patients with clear cell renal cell carcinoma[J].Oncogene,2010,29(42):5724-8.
[47]Bandres E, Agirre X, Bitarte N, et al. Epigenetic regulation of microRNA expression in colorectal cancer[J].Int J Cancer,2009,125(11):2737-43.
[48]Lehmann U, Hasemeier B, Christgen M, et al. Epigenetic inactivation of microRNA gene hsa-mir-9-1 in human breast cancer[J]. J Pathol,2008,214(1): 17-20
[49]Ding YL, Zhang JL, Tang SF, et al. Effect of chemokine stromal cell derived factor-1 and its receptor CXCR4 on the peritoneal carcinometastasis of gastric cancer[J].Zhonghua Yi Xue Za Zhi,2008,88(3):202-5.
[50]Chinni SR, Yamamoto H, Dong Z, et al.CXCL12/CXCR4 Transactivates HER2 in Lipid Rafts of Prostate Cancer Cells and Promotes Growth of Metastatic Deposits in Bone[J].Mol Cancer Res,2008,6(3):446-57.
[51]Nigris F, Rossiello R, Schiano C, et al. Deletion of Yin Yang 1 protein in osteosarcoma cells on cell invasion and CXCR4/angiogenesis and metastasis[J]. Cancer Res,2008,68(6):1797-808.
[52]Kim J, Mori T, Chen SL, et al. Chemokine receptor CXCR4 expression in patients with melanoma and colorectal cancer liver metastases and the association with disease outcome[J].Ann Surg,2006,244(1):113-20.
[53]Holland JD, Kochetkova M, Akekawatchai C, et al.Differential functional activation of chemokine receptor CXCR4 is mediated by G proteins in breast cancer cells[J].Cancer Res,2006,66(8):4117-24.
[54]Engl T, Relja B, Marian D, et al.CXCR4 chemokine receptor mediates prostate tumor cell adhesion through alpha5 and beta3 integrins[J]. Neoplasia,2006, 8(4):290-301.
[55]Jones J, Marian D, Weich E, et al.CXCR4 chemokine receptor engagement modifies integrin dependent adhesion of renal carcinoma cells[J]. Exp Cell Res,2007,313(19):4051-65.
[56]Fukunaga S, Maeda K, Noda E, et al. Association between expression of vascular endothelial growth factor C, chemokine receptor CXCR4 and lymph node metastasis in colorectal cancer[J]. Oncology,2006,71(3-4):204-11.
[57]Yoon Y, Liang Z, Zhang X, et al.CXC chemokine receptor-4 antagonist blocks both growth of primary and metastasis of head and neck cancer in xenograft mouse models[J].Cancer Res,2007,67(15):7518-24.
[58]de Nigris F, Crudele V, Giovane A, et al. CXCR4/YY1 inhibition impairs VEGF network and angiogenesis during malignancy[J].Proc Natl Acad Sci USA,2010,107(32):14484-9.
[59]Lee A, Frischer J, Serur A, et al.Inhibition of cyclooxygenase-2 disrupts tumor vascular mural cell recruitment and survival signaling[J]. Cancer Res,2006, 6(8):4378-84.
[60]Tang CH, Tan TW, Fu WM, et al. Involvement of matrix metalloproteinase-9 in stromal cell-derived factor-1/CXCR4 pathway of lung cancer metastasis[J]. Carcinogenesis,2008,29(1):35-43.
[61]Guo Z, Cai S, Fang R, et al.The synergistic effects of CXCR4 and EGFR on promoting EGF-mediated metastasis in ovarian cancer cells[J].Colloids SurfB Biointerfaces,2007,60(1):1-6.
[62]Hu J, Deng X, Bian X,et al. The expression of functional chemokine receptor CXCR4 is associated with the metastatic potential of human nasopharyngeal carcinoma.Clin Cancer Res.2005 Jul 1;11(13):4658-65.
[63]Ou DL, Chen CL, Lin SB, et al.Chemokine receptor expression profiles in nasopharyngeal carcinoma and their association with metastasis and radiotherapy. J Pathol.2006 Nov;210(3):363-73.
[64]Wang N, Wu QL, Fang Y,et al. Expression of chemokine receptor CXCR4 in nasopharyngeal carcinoma:pattern of expression and correlation with clinical outcome. J Transl Med.2005 Jun 26;3:26.
[65]Segawa Y, Oda Y, Yamamoto H,et al. Close correlation between CXCR4 and VEGF expression and their prognostic implications in nasopharyngeal carcinoma.Oncol Rep.2009 May;21(5):1197-202
[66]罗花南,李湘平,刘雄等.CXCR4/SDF-1α与鼻咽癌器官特异性转移的相关性研究[J].中华肿瘤杂志,2009,31(4)：260-264.
[67]Labbaye C, Spinello I, Quaranta MT, et al.A three-step pathway comprising PLZF/miR-146a/CXCR4 controls megakaryopoiesis[J]. Nat Cell Biol,2008, 10(7):788-801.
[1]Wong TS, Man OY, Tsang CM,et al. MicroRNA let-7 suppresses nasopharyngeal carcinoma cells proliferation through downregulating c-Myc expression. J Cancer Res Clin Oncol.2011 Mar;137(3):415-22.
[2]Lu J, He ML, Wang L,et al. MiR-26a inhibits cell growth and tumorigenesis of nasopharyngeal carcinoma through repression of EZH2. Cancer Res.2011 Jan 1;71(1):225-33.
[3]Sengupta S, den Boon JA, Chen IH,et al. MicroRNA 29c is down-regulated in nasopharyngeal carcinomas, up-regulating mRNAs encoding extracellular matrix proteins. Cancer Res.2011 Mar 15;71(6):2381-91.
[4]Xia H, Ng SS, Jiang S,et al. miR-200a-mediated downregulation of ZEB2 and CTNNB1 differentially inhibits nasopharyngeal carcinoma cell growth, migration and invasion. Biochem Biophys Res Commun.2010 Jan 1;391(1):535-41.
[5]Alajez NM, Lenarduzzi M, Ito E,et al. MiR-218 suppresses nasopharyngeal cancer progression through downregulation of survivin and the SLIT2-ROBO1 pathway. Cancer Res.2011 Mar 15;71(6):2381-91.
[6]Li Q Wu Z, Peng Y,et al. MicroRNA-10b induced by Epstein-Barr virus-encoded latent membrane protein-1 promotes the metastasis of human nasopharyngeal carcinoma cells. Cancer Lett.2010 Dec 18;299(1):29-36.
[7]Zhang L, Deng T, Li X,et al. microRNA-141 is involved in a nasophayngeal carcinoma-related genes network. Carcinogenesis.2010 Apr;31(4):559-66.
[8]Guo LM, Pu Y, Han Z,et al. MicroRNA-9 inhibits ovarian cancer cell growth through regulation of NF-kappaB1. Nat Cell Biol.2010 Mar;12(3):247-56.
[9]Wan HY, Guo LM, Liu T,et al. Regulation of the transcription factor NF-kappaB1 by microRNA-9 in human gastric adenocarcinoma. FEBS J.2009 Oct;276(19):5537-46.
[10]Ma L, Young J, Prabhala H,. miR-9, a MYC/MYCN-activated microRNA, regulates E-cadherin and cancer metastasis. Nat Cell Biol.2010 Mar;12(3):247-56.
[11]Li W, Ruan K. MicroRNA detection by microarray[J]. Anal Bioanal Chem, 2009,394(4):1117-24.
[12]Mestdagh P, Feys T, Bernard N, et al.High-throughput stem-loop RT-qPCR miRNA expression profiling using minute amounts of input RNA[J].Nucleic Acids Res,2008,36(21):e143.
[13]Sempere LF, Preis M, Yezefski T, et al.Fluorescence-based codetection with protein markers reveals distinct cellular compartments for altered MicroRNA expression in solid tumors[J].Clin Cancer Res,2010,16(16):4246-55.
[14]Haramati S, Chapnik E, Sztainberg Y, et al.miRNA malfunction causes spinal i. motor neuron disease.Proc Natl Acad Sci USA,2010,107(29):13111-6.
[15]Saunders LR, Sharma AD, Tawney J, et al. miRNAs regulate SIRT1 expression during mouse embryonic stem cell differentiation and in adult mouse tissues. Aging (Albany NY),2010,2(7):415-31.
[16]Zhong X, Li N, Liang S, et al. Identification of microRNAs regulating reprogramming factor LIN28 in embryonic stem cells and cancer cells.J Biol Chem,2010,285(53):41961-71.
[17]Otaegi G, Pollock A, Hong J, et al. MicroRNA miR-9 modifies motor neuron columns by a tuning regulation of FoxPl levels in developing spinal cords. J Neurosci,2011,31(3):809-18.
[18]Bonev B, Pisco A, Papalopulu N.MicroRNA-9 reveals regional diversity of neural progenitors along the anterior-posterior axis. Dev Cell,2011,20(1): 19-32.
[19]Kong L, Zhu J, Han W, et al.Significance of serum microRNAs in pre-diabetes and newly diagnosed type 2 diabetes:a clinical study. Acta Diabetol,2010 Sep 21.
[20]Plaisance V, Abderrahmani A, Perret-Menoud V, et al.MicroRNA-9 controls the expression of Granuphilin/Slp4 and the secretory response of insulin-producing cells.J Biol Chem.J Biol Chem,2006,281(37):26932-42.
[21]Wang K, Long B, Zhou J, et al. miR-9 and NFATc3 regulate myocardin in cardiac hypertrophy.Biol Chem,2010,285(16):11903-12.
[22]Onnis A, De Falco G, Antonicelli G, et al.Alteration of microRNAs regulated by c-Myc in Burkitt lymphoma. PLoS one,2010,5(9):e12960.
[23]Lin J, Lwin T, Zhao JJ, et al.Follicular dendritic cell-induced microRNA-mediated upregulation of PRDM1 and downregulation of BCL-6 in non-Hodgkin's B-cell lymphomas.Leukemia,2011,25(1):145-52.
[24]Guo R, Wu Q, Liu F, et al.Description of the CD 133+ subpopulation of the human ovarian cancer cell line OVCAR3.Oncol Rep,2011,25(1):141-6.
[25]Rotkrua P, Akiyama Y, Hashimoto Y, et al.MiR-9 down-regulates CDX2 expression in gastric cancer cells.Int J Cancer,2011 Jan 10.
[26]Myatt SS, Wang J, Monteiro LJ, et al.Definition of microRNAs that repress expression of the tumor suppressor gene FOXO1 in endometrial cancer. Cancer Res,2010,70(1):367-77.
[27]Guo LM, Pu Y, Han Z, et al. MicroRNA-9 inhibits ovarian cancer cell growth through regulation of NF-kappaB1. FEBS J.2009 Oct;276(19):5537-46.)
[28]Lund E, Dahlberg JE.Substrate selectivity of exportin 5 and Dicer in the biogenesis of microRNAs.Cold Spring Harb Symp Quant Biol,2006,71:59-66.
[29]Saini S, Majid S, Yamamura S, Regulatory role of miR-203 in prostate cancer progression and metastasis.Clin Cancer Res.2010 Dec 15.
[30]Nass D, Rosenwald S, Meiri E, et al。MiR-92b and miR-9/9* are specifically expressed in brain primary tumors and can be used to differentiate primary from metastatic brain tumors. Brain Pathol.2009 Jul;19(3):375-83.
[1]Herman JG, Graff JR, Myohanen S, et al.Methylation-specific PCR:a novel PCR assay for methylation status of CpG islands [J].Proc Natl Acad Sci U S A, 1996,93(18):9821-6.
[2]Hu SL, Kong XY, Cheng ZD, et al.Promoter methylation of p16, Runx3, DAPK and CHFR genes is frequent in gastric carcinoma[J].Tumori,2010, 96(5):726-33.
[3]Chang Sun, Laura L Reimers, Robert D Burk.Methylation of HPV16 genome CpG sites is associated with cervix precancer and cancer[J].Gynecol Oncol, 2011 Feb 7.
[4][4]Youwei Zhang, Rui Wang, Haizhu Song, et al.Methylation of multiple genes as a candidate biomarker in non-small cell lung cancer[J].Cancer Lett,2011 Jan 19.
[5]Ran Y, Wu S, You Y. Demethylation of E-cadherin gene in nasopharyngeal carcinoma could serve as a potential therapeutic strategy [J]. J Biochem,2011, 149(1):49-54.
[6]Tong JH.Putative tumour-suppressor gene DAB2 is frequently down regulated by promoter hypermethylation in nasopharyngeal carcinoma [J]. BMC Cancer, 2010,3 (0):253.
[7]Seo SY, Kim EO, Jang KL, et al.Epstein-Barr virus latent membrane protein 1 suppresses the growth-inhibitory effect of retinoic acid by inhibiting retinoic acid receptor-beta2 expression via DNA methylation [J].Cancer Lett,2008, 270(1):66-76.
[8]Langevin SM, Stone RA, Bunker CH, et al. microRNA-137 promoter methylation is associated with poorer overall survival in patients with squamous cell carcinoma of the head and neck[J]. Cancer,2010, Nov 8.
[9]Balaguer F, Link A, Lozano JJ, et al.Epigenetic silencing of miR-137 is an early event in colorectal carcinogenesis[J]. Cancer Res,2010,70(16):6609-18.
[10]Suzuki H, Yamamoto E, Nojima M, et al. Methylation -associated silencing of microRNA-34b/c in gastric cancer and its involvement in an epigenetic field defect[J]. Carcinogenesis.2010,31(12):2066-73.
[11]Ceppi P, Mudduluru G, Kumarswamy R, et al.Loss of miR-200c expression induces an aggressive, invasive and chemoresistant phenotype in non-small cell lung cancer[J].Mol Cancer Res,2010,8(9):1207-16.
[12]Hanoun N, Delpu Y, Suriawinata AA, et al.The silencing of microRNA 148a production by DNA hypermethylation is an early event in pancreatic carcinogenesis[J].Clin Chem,2010,56(7):1107-18.
[13]U Lehmann, B Hasemeier, M Christgen, et al.Epigenetic inactivation of microRNA gene hsa-mir-9-1 in human breast cancer[J]. J Pathol,2008,214(1): 17-24.
[14]Bandres E, Agirre X, Bitarte N, et al. Epigenetic regulation of microRNA expression in colorectal cancer[J].Int J Cancer,2009,125(11):2737-43.
[15]Trankenschuh W, Puls F, Christgen M, et al.Frequent and distinct aberrations of DNA methylation patterns in fibrolamellar carcinoma of the liver[J]. PLoS One,2010,5(10):e13688.
[16]Amaia Lujambio, George A Calinc, Alberto Villanueva, et al. A microRNA DNA methylation signature for human cancer metastasis[J].Proc Nat Acad Sci U S A,2008,105(36):13556-61.
[17]MAT Hildebrandt, J Gu, J Lin, et al.Hsa-miR-9 methylation status is associated with cancer development and metastatic recurrence in patients with clear cell renal cell carcinoma[J]. Oncogene,2010,29(42):5724-8.
[18]Pei-Yin Hsu, Daniel E Deatherage, Benjamin AT Rodriguez, et al.Xenoestrogen-Induced Epigenetic Repression of microRNA-9-3 in Breast Epithelial Cells[J]. Cancer Res,2009,69(14):5936-5945.
[19]Dannenberg LO, Edenberg HJ. Epigenetics of gene expression in human hepatoma cells:expression profiling the response to inhibition of DNA methylation and histone deacetylation[J]. BMC Genomics,2006,19(7):181.
[20]Gui CY, Ngo L, Xu WS, et al.Histone deacetylase(HDAC)inhibitor activation of p21WAF1 involves changes in promoter-associated proteins, including HDAC1[J].Proc Natl Acad Sci U S A,2004,101(5):1241-6.
[21]Pero R, Peluso S, Angrisano T, et al.Chromatin and DNA methylation dynamics of Helicobacter pylori-induced COX-2 activation[J].Int J Med Microbiol,2011, 301(2):140-9.
[22]Sharma G, Mirza S, Prasad CP, et al.Promoter hypermethylation of p16INK4A, p14ARF, CyclinD2 and Slit2 in serum and tumor DNA from breast cancer patients[J]. Life Sci,2007,80(20):1873-81.
[23]Jones PL, Veenstra GJ, Wade PA.Methylated DNA and MeCP2 recruit histone deacetylase to repress transcription[J]. Nat Genet,1998,9(2):187-91.
[24]Tabe Y, Konopleva M, Kondo Y, et al. PML-RARalpha and AML1-ETO translocations are rarely associated with methylation of the RAR02 promoter[J].Ann Hematol,2006,85(10):689-704.
[1]Shi W, Alajez NM, Bastianutto C, et al. Significance of Plkl regulation by miR-100 in human nasopharyngeal cancer.Int J Cancer,2010,126(9):2036-48.
[2]Xia H, Ng SS, Jiang S, et al. miR-200a-mediated downregulation of ZEB2 and CTNNB1 differentially inhibits nasopharyngeal carcinoma cell growth,migration and invasion. Biochem Biophys Res Commun,2010,91(1): 535-41.
[3]Wong TS, Man OY, Tsang CM, et al. MicroRNA let-7 suppresses nasopharyngeal carcinoma cells proliferation through downregulating c-Myc expression. J Cancer Res Clin Oncol.2011,137(3):415-22.
[4]Li G, Wu Z, Peng Y, et al. MicroRNA-10b induced by Epstein-Barr virus-encoded latent membrane protein-1 promotes the metastasis of human nasopharyngeal carcinoma cells. Cancer Lett,2010,299(1):29-36.
[5]Lu J, He ML, Wang L, et al. MiR-26a inhibits cell growth and tumorigenesis of nasopharyngeal carcinoma through repression of EZH2. Cancer Res,2011, 71(1):225-33.
[6]Shibata M, Nakao H, Kiyonari H, et al.MicroRNA-9 regulates neurogenesis in mouse telencephalon by targeting multiple transcription factors.J Neurosci.2011 Mar 2;31(9):3407-22.
[7]Otaegi G, Pollock A, Hong J, et al. MicroRNA miR-9 modifies motor neuron columns by a tuning regulation of FoxPl levels in developing spinal cords.J Neurosci.2011 Jan 19;31(3):809-18.)
[8]Bonev B, Pisco A, Papalopulu N. MicroRNA-9 reveals regional diversity of neural progenitors along the anterior-posterior axis. Dev Cell.2011 Jan 18;20(1):19-32.
[9]Tan HX, Wang Q, Chen LZ, et al.MicroRNA-9 reduces cell invasion and E-cadherin secretion in SK-Hep-1 cell[J].Med Oncol,2010,27(3):654-60.
[10]Ma L, Young J, Prabhala H, et al. miR-9, a MYC/MYCN-activated microRNA, regulates E-cadherin and cancer metastasis [J].Nat Cell Biol,2010,12(3): 247-56.
[11]Rotkrua P, Akiyama Y, Hashimoto Y, et al.MiR-9 down-regulates CDX2 expression in gastric cancer cells.Int J Cancer,2011 Jan 10.
[12]Guo LM, Pu Y, Han Z, et al. MicroRNA-9 inhibits ovarian cancer cell growth through regulation of NF-kappaB1. FEBS J,2009,276(19):5537-46.
[13]Levine BL, Hum eau LM, Boyer J, et al. Gene transfer in humans using a conditionally replicating lentiviral vector[J].Proc Natl Acad Sci USA,2006, 103(46):17372-17377.
[14]Baka K, Goto-Koshino Y, Mizukoshi F, et al. Inhibition of the replication of feline immunodeficiency virus by lentiviral vector mediated RNA interference in feline cell lines [J].J Vet Med Sci,2008,70(8):777-783.
[15]Bauer G, Dao MA, Case SS, et al. In vivo biosafety model to assess the risk of adverse events from retroviral and lentiviral vectors[J]. Mol Ther,2008,16(7): 1308-1315.
[16]Ferretti E,De Smaele E,Po A,et al.MicroRNA profiling in human medulloblastoma. Int J Cancer.2009,124(3):568-77.
[17]Liu L, Chen L, Xu Y, et al. microRNA-195 promotes apoptosis and suppresses tumorigenicity of human colorectal cancer cells.Biochem Biophys Res Commun,2010,400(2):236-40.
[18]Yoshitomi T, Kawakami K, Enokida H, et al.Restoration of miR-517a expression induces cell apoptosis in bladder cancer cell lines.Oncol Rep,2011, 25(6):1661-8.
[19]Feng S, Cong S, Zhang X, et al.MicroRNA-192 targeting retinoblastoma 1 inhibits cell proliferation and induces cell apoptosis in lung cancer cells. Nucleic Acids Res,2011 Apr 21.
[20]J. Kota, R.R. Chivukula, K.A. O'Donnell, et al. Therapeutic microRNA delivery suppresses tumorigenesis in a murine liver cancer model.Cell,2009 (137):1005-1017.
[21]B.Zhang, X.X.Liu, J.R. He, et al.Pathologically decreased miR-26a antagonizes apoptosis and facilitates carcinogenesis by targeting MTDH and EZH2 in breast cancer.Carcinogenesis,2011(32):2-9.
[22]Liu XX, Li XJ, Zhang B, et al. MicroRNA-26b is underexpressed in human breast cancer and induces cell apoptosis by targeting SLC7A11. FEBS Lett, 2011 Apr 14.
[23]Shi L, Chen J, Yang J, et al. MiR-21 protected human glioblastoma U87MG cells from chemotherapeutic drug temozolomide induced apoptosis by decreasing Bax/Bcl-2 ratio and caspase-3 activity. Brain Res,2010,1352: 255-64.
[24]Li JH, Xiao X, Zhang YN, et al. MicroRNA miR-886-5p inhibits apoptosis by down-regulating Bax expression in human cervical carcinoma cells. Gynecol Oncol,2011,120(1):145-51.
[25]曾益新.肿瘤学(第2版).北京：人民卫生出版社,2003；97.
[1]Ding YL, Zhang JL, Tang SF, et al. Effect of chemokine stromal cell derived factor-1 and its receptor CXCR4 on the peritoneal carcinometastasis of gastric cancer[J].Zhonghua Yi Xue Za Zhi,2008,88(3):202-5.
[2]Chinni SR, Yamamoto H, Dong Z, et al.CXCL 12/CXCR4 Transactivates HER2 in Lipid Rafts of Prostate Cancer Cells and Promotes Growth of Metastatic Deposits in Bone[J].Mol Cancer Res,2008,6(3):446-57.
[3]Nigris F, Rossiello R, Schiano C, et al. Deletion of Yin Yang 1 protein in osteosarcoma cells on cell invasion and CXCR4/angiogenesis and metastasis[J]. Cancer Res,2008,68(6):1797-808.
[4]Kim J, Mori T, Chen SL, et al. Chemokine receptor CXCR4 expression in patients with melanoma and colorectal cancer liver metastases and the association with disease outcome[J].Ann Surg,2006,244(1):113-20.
[5]Holland JD,Kochetkova M,Akekawatchai C,et al.Differential functional activation of chemokine receptor CXCR4 is mediated by G proteins in breast cancer cells[J].Cancer Res,2006,66(8):4117-24.
[6]Hu J, Deng X, Bian X,et al. The expression of functional chemokine receptor CXCR4 is associated with the metastatic potential of human nasopharyngeal carcinoma.Clin Cancer Res.2005 Jul 1;11(13):4658-65.
[7]Ou DL, Chen CL, Lin SB, et al.Chemokine receptor expression profiles in nasopharyngeal carcinoma and their association with metastasis and radiotherapy. J Pathol.2006 Nov;210(3):363-73.
[8]Wang N, Wu QL, Fang Y,et al. Expression of chemokine receptor CXCR4 in nasopharyngeal carcinoma:pattern of expression and correlation with clinical outcome. J Transl Med.2005 Jun 26;3:26.
[9]Segawa Y, Oda Y, Yamamoto H,et al. Close correlation between CXCR4 and VEGF expression and their prognostic implications in nasopharyngeal carcinoma.Oncol Rep.2009 May;21 (5):1197-202
[10]罗花南,李湘平,刘雄等.CXCR4/SDF-1α与鼻咽癌器官特异性转移的相关性研究[J].中华肿瘤杂志,2009,31(4)：260-264.
[11]Ott CE, Grunhagen J, Jager M, et al.MicroRNAs Differentially Expressed in Postnatal Aortic Development Downregulate Elastin via 3' UTR and Coding-Sequence Binding Sites[J]. PLoS One,2011,6(1):e16250.
[12]Wu E, Thivierge C, Flamand M, et al. Pervasive and cooperative deadenylation of 3'UTRs by embryonic microRNA families[J]. Mol Cell,2010,40(4):558-70.
[13]Bagga S, Bracht J, Hunter S, et al.Regulation by let-7 and lin-4 miRNAs results in target mRNA degradation[J]. Cell,2005,122(4):553-63.
[14]Wada R, Akiyama Y, Hashimoto Y, et al.miR-212 is downregulated and suppresses methyl-CpG-binding protein MeCP2 in human gastric cancer[J].Int J Cancer,2010,27(5):1106-14.
[15]Zhao JJ, Lin J, Yang H, et al.MicroRNA-221/222 negatively regulates estrogen receptor alpha and is associated with tamoxifen resistance in breast cancer[J].J Biol Chem,2008,283(45):31079-86.
[16]Shin KH, Bae SD, Hong HS, et al.miR-181a shows tumor suppressive effect against oral squamous cell carcinoma cells by downregulating K-ras[J].Biochem Biophys Res commun,2011,404(4):896-902.
[17]Lo WL, Yu CC, Chiou GY, et al. MicroRNA-200c attenuates tumor growth and metastasis of presumptive head and neck squamous cell carcinoma stem cells squamous cell carcinoma stem cells[J].J Pathol,2011,223 (4):482-95.
[18]Guo H, Ingolia NT, Weissman JS, et al. Mammalian microRNAs predominantly act to decrease target mRNA levels[J].Nature,2010,466(7308):835-40.
[19]Wan HY, Guo LM, Liu T, et al.Regulation of the transcription factor NF-kappaB1 by microRNA-9 in human gastric adenocarcinoma[J]. Mol Cancer,2010,9 (16):1-10.
[20]Luo H, Zhang H, Zhang Z, et al.Down-regulated miR-9 and miR-433 in human gastric carcinoma[J]. J Exp Clin Cancer Res,2009,16 (28):82.
[21]Rotkrua P, Akiyama Y, Hashimoto Y, et al. MiR-9 down-regulates CDX2 expression in gastric cancer cells[J].Int J Cancer,2011, Jan 10.
[22]Guo LM, Pu Y, Han Z, et al.MicroRNA-9 inhibits ovarian cancer cell growth through regulation of NF-kappaB1[J].FEBS J,2009,276(19):5537-46.
[23]Tan HX, Wang Q, Chen LZ, et al.MicroRNA-9 reduces cell invasion and E-cadherin secretion in SK-Hep-1 cell[J].Med Oncol,2010,27(3):654-60.
[24]Nie K, Gomez M, Landgraf P, et al.MicroRNA-mediated down-regulation of PRDM1/Blimp-1 in Hodgkin/Reed-Sternberg cells:apotential pathogenetic lesion in Hodgkin lymphomas[J].Am J Pathol,2008,173 (1):242-52.
[25]Guo LM, Pu Y, Han Z, et al.MicroRNA-9 inhibits ovarian cancer cell growth through regulation of NF-kappaB1.FEBS J,2009,276(19):5537-46.
[26]Myatt SS, Wang J, Monteiro LJ,et al. Definition of microRNAs that repress expression of the tumor suppressor gene FOXO1 in endometrial cancer. Cancer Res,2010,70(1):367-77.
[27]Liu M, Lang N, Chen X,et al. miR-185 targets RhoA and Cdc42 expression and inhibits the proliferation potential of human colorectal cells. Cancer Lett, 2011,301(2):151-60.
[2]Bala S, Marcos M, Szabo G. Emerging role of microRNAs in liver diseases[J]. World J Gastroenterol,2009,15(45):5633-5640.
[3]Lee R C, Feinbaum R L, Ambros V. The C. elegahs heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14[J]. Cell,1993, 75(5):843-854.
[4]Reinhart B J, Slack F J, Basson M, et al. The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans[J]. Nature,2000,403(6772): 901-906.
[5]Winter J, Jung S, Keller S, et al. Many roads to maturity:microRNA biogenesis pathways and their regulation[J]. Nat Cell Biol,2009,11(3):228-234.
[6]Carthew R W, Sontheimer E J. Origins and Mechanisms of miRNAs and siRNAs[J]. Cell,2009,136(4):642-655.
[7]Inui M, Martello G, Piccolo S, et al. MicroRNA control of signal transduction[J]. Nat Rev Mol Cell Biol,2010,11(4):252-263.
[8]Zeng Y, Yi R, Cullen B R. MicroRNAs and small interfering RNAs can inhibit mRNA expression by similar mechanisms[J].Proc Natl Acad Sci USA,2003, 100(17):9779-9784.
[9]吴斌,俞洋.微小RNA与肿瘤研究进展[J].现代肿瘤医学,2009,17(2)：373-376.
[10]Lo AK, To KF, Lo KW, et al.Modulation of LMP1 protein expression by EBV-encoded microRNAs[J]. Proc Natl Acad Sci USA,2007,104(41): 16164-9.
[11]Choy EY, Siu KL, Kok KH, et al. An Epstein-Barr virus-encoded microRNA targets PUMA to promote host cell survival[J].J Exp Med,2008,205(11): 2551-60.
[12]Sengupta S, den Boon JA, Chen IH, et al.MicroRNA 29c is down-regulated in nasopharyngeal carcinomas, up-regulating mRNAs encoding extracellular matrix proteins[J].Proc Natl Acad Sci USA.2008,105(15):5874-8.
[13]Shi W, Alajez NM, Bastianutto C, et al. Significance of Plkl regulation by miR-100 in human nasopharyngeal cancer[J]. Int J Cancer,2010,126(9): 2036-48.
[14]Chen HC, Chen GH, Chen YH, et al. MicroRNA deregulation and pathway alterations in nasopharyngeal carcinoma[J]. Br J Cancer,2009,100(6):1002-11.
[15]Xia H, Ng SS, Jiang S, et al.miR-200a-mediated downregulation of ZEB2 and CTNNB1 differentially inhibits nasopharyngeal carcinoma cell growth, migration and invasion[J]. Biochem Biophys Res Commun,2010,391(1): 535-41.
[16]Wong TS, Man OY, Tsang CM, et al. MicroRNA let-7 suppresses nasopharyngeal carcinoma cells proliferation through downregulating c-Myc expression[J].J Cancer Res Clin Oncol,2011,137(3):415-22.
[17]Zhang L, Deng T, Li X, microRNA-141 is involved in a nasopharyngeal carcinoma-related genes network[J].Carcinogenesis,2010,31(4):559-66.
[18]Wan HY, Guo LM, Liu T, et al.Regulation of the transcription factor NF-kappaB1 by microRNA-9 in human gastric adenocarcinoma[J].Mol Cancer,2010,9 (16):1-10.
[19]Luo H, Zhang H, Zhang Z, et al.Down-regulated miR-9 and miR-433 in human gastric carcinoma[J]. J Exp Clin Cancer Res,2009,16 (28):82.
[20]Rotkrua P, Akiyama Y, Hashimoto Y, et al. MiR-9 down-regulates CDX2 expression in gastric cancer cells[J].Int J Cancer,2011, Jan 10.
[21]Guo LM, Pu Y, Han Z, et al.MicroRNA-9 inhibits ovarian cancer cell growth through regulation of NF-kappaBl [J].FEBS J,2009,276(19):5537-46.
[22]Tan HX, Wang Q, Chen LZ, et al.MicroRNA-9 reduces cell invasion and E-cadherin secretion in SK-Hep-1 cell[J].Med Oncol,2010,27 (3):654-60.
[23]Ma L, Young J, Prabhala H, et al. miR-9, a MYC/MYCN-activated microRNA, regulates E-cadherin and cancer metastasis[J]. Nat Cell Biol, 2010,12(3):247-56.
[24]晁腾飞,张瑜,颜兴起,等.MiR-9在人神经胶质瘤中调节CBX7的表达[J].中国医学科学院学报,2008,30(3)：268-274.
[25]Nie K, Gomez M, Landgraf P, et al.MicroRNA-mediated down-regulation of PRDM1/Blimp-1 in Hodgkin/Reed-Sternberg cells:apotential pathogenetic lesion in Hodgkin lymphomas[J].Am J Pathol,2008,173 (1):242-52.
[26]Myatt SS, Wang J, Monteiro LJ, et al.Defniition of microRNAs that repress expression of the tumor suppressor gene FOXO1 in endometrial cancer [J]. Cancer Res,2010,70(1):367-77.
[27]Tao Q, Chan AT.Nasopharyngeal carcinoma:molecular pathogenesis and therapeutic developments[J].Expert Rev Mol Med,2007,9 (12):1-24.
[28]Chow LS, Lo KW, Kwong J, et al.RASSF1A is a target tumor suppressor from 3p21.3 in nasopharyngeal carcinoma[J]. Int J Cancer,2004,109(6):839-47.
[29]Yau WL, Lung HL, Zabarovsky ER, et al.Functional studies of the chromosome 3p21.3 candidate tumor suppressor gene BLU/ZMYND10 in nasopharyngeal carcinoma[J]. Int J Cancer,2006,119(12):2821-6.
[30](30)Li L, Tao Q, Jin H, et al. The tumor suppressor UCHL1 forms a complex with p53/MDM2/ARF to promote p53 signaling and is frequently silenced in nasopharyngeal carcinoma [J]. Clin Cancer Res,2010,16(11):2949-58.
[31]Lung HL, Cheung AK, Xie D, et al.TSLC1 is a tumor suppressor gene associated with metastasis in nasopharyngeal carcinoma[J]. Cancer Res,2006, 6(19):9385-92.
[32]Wang Y, Li J, Cui Y, et al. CMTM3, located at the critical tumor suppressor locus 16q22.1, is silenced by CpG methylation in carcinomas and inhibits tumor cell growth through inducing apoptosis[J]. Cancer Res,2009,69(12): 5194-201.
[33]EstellerM.Epigenetics in cancer[J].N Engl J Med,2008,358:1148-1159.
[34]张恒,何成强,李云龙.哺乳动物DNA甲基化的功能和作用[J].生命的化学,2002,22：218-220.
[35]Ran Y, Wu S, You Y, et al.Demethylation of E-cadherin gene in nasopharyngeal carcinoma could serve as a potential therapeutic strategy[J]. J Biochem,2011, 149(1):49-54.
[36]Chen F, Mo Y, Ding H, et al. Frequent epigenetic inactivation of Myocardin in human nasopharyngeal carcinoma[J]. Head Neck,2011,33(1):54-9.
[37]Tong JH, Ng DC, Chau SL, et al.Putative tumour-suppressor gene DAB2 is frequently down regulated by promoter hypermethylation in nasopharyngeal carcinoma[J]. BMC Cancer,2010,10:253.
[38]Liu M, Peng Y, Wang X, et al.NGX6 gene mediated by promoter methylation as a potential molecular marker in colorectal cancer[J]. BMC Cancer,2010,10: 160.
[39]Li L, Tao Q, Jin H, et al.The tumor suppressor UCHL1 forms a complex with p53/MDM2/ARF to promote p53 signaling and is frequently silenced in nasopharyngeal carcinoma[J]. Clin Cancer Res,2010,16(11):2949-58.
[40]Wang T, Liu H, Chen Y, et al.Methylation associated inactivation of RASSF1A and its synergistic effect with activated K-Ras in nasopharyngeal carcinoma[J]. J Exp Clin Cancer Res,2009,28:160.
[41]Zhou L, Feng X, Shan W, et al.Epigenetic and genetic alterations of the EDNRB gene in nasopharyngeal carcinoma[J]. Oncology,2007,72(5-6): 357-63.
[42]Wang Y, Li J, Cui Y, et al.CMTM3, located at the critical tumor suppressor locus 16q22.1, is silenced by CpG methylation in carcinomas and inhibits tumor cell growth through inducing apoptosis[J]. Cancer Res,2009,69(12): 5194-201.
[43]Shao L, Cui Y, Li H, et al.CMTM5 exhibits tumor suppressor activities and is frequently silenced by methylation in carcinoma cell lines[J].Clin Cancer Res, 2007,13(19):5756-62.
[44]Tan SH, Ida H, Goh BC, et al.Analyses of promoter hypermethylation for RUNX3 and other tumor suppressor genes in nasopharyngeal carcinoma[J]. Anticancer Res,2006,26(6B):4287-92.
[45]Trankenschuh W, Puls F, Christgen M, et al.Frequent and distinct aberrations of DNA methylation patterns in fibrolamellar carcinoma of the liver[J].PLoS One,2010,5(10):e13688.
[46]Hildebrandt MA, Gu J, Lin J, et al.Hsa-miR-9 methylation status is associated with cancer development and metastatic recurrence in patients with clear cell renal cell carcinoma[J].Oncogene,2010,29(42):5724-8.
[47]Bandres E, Agirre X, Bitarte N, et al. Epigenetic regulation of microRNA expression in colorectal cancer[J].Int J Cancer,2009,125(11):2737-43.
[48]Lehmann U, Hasemeier B, Christgen M, et al. Epigenetic inactivation of microRNA gene hsa-mir-9-1 in human breast cancer[J]. J Pathol,2008,214(1): 17-20
[49]Ding YL, Zhang JL, Tang SF, et al. Effect of chemokine stromal cell derived factor-1 and its receptor CXCR4 on the peritoneal carcinometastasis of gastric cancer[J].Zhonghua Yi Xue Za Zhi,2008,88(3):202-5.
[50]Chinni SR, Yamamoto H, Dong Z, et al.CXCL12/CXCR4 Transactivates HER2 in Lipid Rafts of Prostate Cancer Cells and Promotes Growth of Metastatic Deposits in Bone[J].Mol Cancer Res,2008,6(3):446-57.
[51]Nigris F, Rossiello R, Schiano C, et al. Deletion of Yin Yang 1 protein in osteosarcoma cells on cell invasion and CXCR4/angiogenesis and metastasis[J]. Cancer Res,2008,68(6):1797-808.
[52]Kim J, Mori T, Chen SL, et al. Chemokine receptor CXCR4 expression in patients with melanoma and colorectal cancer liver metastases and the association with disease outcome[J].Ann Surg,2006,244(1):113-20.
[53]Holland JD, Kochetkova M, Akekawatchai C, et al.Differential functional activation of chemokine receptor CXCR4 is mediated by G proteins in breast cancer cells[J].Cancer Res,2006,66(8):4117-24.
[54]Engl T, Relja B, Marian D, et al.CXCR4 chemokine receptor mediates prostate tumor cell adhesion through alpha5 and beta3 integrins[J]. Neoplasia,2006, 8(4):290-301.
[55]Jones J, Marian D, Weich E, et al.CXCR4 chemokine receptor engagement modifies integrin dependent adhesion of renal carcinoma cells[J]. Exp Cell Res,2007,313(19):4051-65.
[56]Fukunaga S, Maeda K, Noda E, et al. Association between expression of vascular endothelial growth factor C, chemokine receptor CXCR4 and lymph node metastasis in colorectal cancer[J]. Oncology,2006,71(3-4):204-11.
[57]Yoon Y, Liang Z, Zhang X, et al.CXC chemokine receptor-4 antagonist blocks both growth of primary and metastasis of head and neck cancer in xenograft mouse models[J].Cancer Res,2007,67(15):7518-24.
[58]de Nigris F, Crudele V, Giovane A, et al. CXCR4/YY1 inhibition impairs VEGF network and angiogenesis during malignancy[J].Proc Natl Acad Sci USA,2010,107(32):14484-9.
[59]Lee A, Frischer J, Serur A, et al.Inhibition of cyclooxygenase-2 disrupts tumor vascular mural cell recruitment and survival signaling[J]. Cancer Res,2006, 6(8):4378-84.
[60]Tang CH, Tan TW, Fu WM, et al. Involvement of matrix metalloproteinase-9 in stromal cell-derived factor-1/CXCR4 pathway of lung cancer metastasis[J]. Carcinogenesis,2008,29(1):35-43.
[61]Guo Z, Cai S, Fang R, et al.The synergistic effects of CXCR4 and EGFR on promoting EGF-mediated metastasis in ovarian cancer cells[J].Colloids SurfB Biointerfaces,2007,60(1):1-6.
[62]Hu J, Deng X, Bian X,et al. The expression of functional chemokine receptor CXCR4 is associated with the metastatic potential of human nasopharyngeal carcinoma.Clin Cancer Res.2005 Jul 1;11(13):4658-65.
[63]Ou DL, Chen CL, Lin SB, et al.Chemokine receptor expression profiles in nasopharyngeal carcinoma and their association with metastasis and radiotherapy. J Pathol.2006 Nov;210(3):363-73.
[64]Wang N, Wu QL, Fang Y,et al. Expression of chemokine receptor CXCR4 in nasopharyngeal carcinoma:pattern of expression and correlation with clinical outcome. J Transl Med.2005 Jun 26;3:26.
[65]Segawa Y, Oda Y, Yamamoto H,et al. Close correlation between CXCR4 and VEGF expression and their prognostic implications in nasopharyngeal carcinoma.Oncol Rep.2009 May;21(5):1197-202
[66]罗花南,李湘平,刘雄等.CXCR4/SDF-1α与鼻咽癌器官特异性转移的相关性研究[J].中华肿瘤杂志,2009,31(4)：260-264.
[67]Labbaye C, Spinello I, Quaranta MT, et al.A three-step pathway comprising PLZF/miR-146a/CXCR4 controls megakaryopoiesis[J]. Nat Cell Biol,2008, 10(7):788-801.
[1]Wong TS, Man OY, Tsang CM,et al. MicroRNA let-7 suppresses nasopharyngeal carcinoma cells proliferation through downregulating c-Myc expression. J Cancer Res Clin Oncol.2011 Mar;137(3):415-22.
[2]Lu J, He ML, Wang L,et al. MiR-26a inhibits cell growth and tumorigenesis of nasopharyngeal carcinoma through repression of EZH2. Cancer Res.2011 Jan 1;71(1):225-33.
[3]Sengupta S, den Boon JA, Chen IH,et al. MicroRNA 29c is down-regulated in nasopharyngeal carcinomas, up-regulating mRNAs encoding extracellular matrix proteins. Cancer Res.2011 Mar 15;71(6):2381-91.
[4]Xia H, Ng SS, Jiang S,et al. miR-200a-mediated downregulation of ZEB2 and CTNNB1 differentially inhibits nasopharyngeal carcinoma cell growth, migration and invasion. Biochem Biophys Res Commun.2010 Jan 1;391(1):535-41.
[5]Alajez NM, Lenarduzzi M, Ito E,et al. MiR-218 suppresses nasopharyngeal cancer progression through downregulation of survivin and the SLIT2-ROBO1 pathway. Cancer Res.2011 Mar 15;71(6):2381-91.
[6]Li Q Wu Z, Peng Y,et al. MicroRNA-10b induced by Epstein-Barr virus-encoded latent membrane protein-1 promotes the metastasis of human nasopharyngeal carcinoma cells. Cancer Lett.2010 Dec 18;299(1):29-36.
[7]Zhang L, Deng T, Li X,et al. microRNA-141 is involved in a nasophayngeal carcinoma-related genes network. Carcinogenesis.2010 Apr;31(4):559-66.
[8]Guo LM, Pu Y, Han Z,et al. MicroRNA-9 inhibits ovarian cancer cell growth through regulation of NF-kappaB1. Nat Cell Biol.2010 Mar;12(3):247-56.
[9]Wan HY, Guo LM, Liu T,et al. Regulation of the transcription factor NF-kappaB1 by microRNA-9 in human gastric adenocarcinoma. FEBS J.2009 Oct;276(19):5537-46.
[10]Ma L, Young J, Prabhala H,. miR-9, a MYC/MYCN-activated microRNA, regulates E-cadherin and cancer metastasis. Nat Cell Biol.2010 Mar;12(3):247-56.
[11]Li W, Ruan K. MicroRNA detection by microarray[J]. Anal Bioanal Chem, 2009,394(4):1117-24.
[12]Mestdagh P, Feys T, Bernard N, et al.High-throughput stem-loop RT-qPCR miRNA expression profiling using minute amounts of input RNA[J].Nucleic Acids Res,2008,36(21):e143.
[13]Sempere LF, Preis M, Yezefski T, et al.Fluorescence-based codetection with protein markers reveals distinct cellular compartments for altered MicroRNA expression in solid tumors[J].Clin Cancer Res,2010,16(16):4246-55.
[14]Haramati S, Chapnik E, Sztainberg Y, et al.miRNA malfunction causes spinal i. motor neuron disease.Proc Natl Acad Sci USA,2010,107(29):13111-6.
[15]Saunders LR, Sharma AD, Tawney J, et al. miRNAs regulate SIRT1 expression during mouse embryonic stem cell differentiation and in adult mouse tissues. Aging (Albany NY),2010,2(7):415-31.
[16]Zhong X, Li N, Liang S, et al. Identification of microRNAs regulating reprogramming factor LIN28 in embryonic stem cells and cancer cells.J Biol Chem,2010,285(53):41961-71.
[17]Otaegi G, Pollock A, Hong J, et al. MicroRNA miR-9 modifies motor neuron columns by a tuning regulation of FoxPl levels in developing spinal cords. J Neurosci,2011,31(3):809-18.
[18]Bonev B, Pisco A, Papalopulu N.MicroRNA-9 reveals regional diversity of neural progenitors along the anterior-posterior axis. Dev Cell,2011,20(1): 19-32.
[19]Kong L, Zhu J, Han W, et al.Significance of serum microRNAs in pre-diabetes and newly diagnosed type 2 diabetes:a clinical study. Acta Diabetol,2010 Sep 21.
[20]Plaisance V, Abderrahmani A, Perret-Menoud V, et al.MicroRNA-9 controls the expression of Granuphilin/Slp4 and the secretory response of insulin-producing cells.J Biol Chem.J Biol Chem,2006,281(37):26932-42.
[21]Wang K, Long B, Zhou J, et al. miR-9 and NFATc3 regulate myocardin in cardiac hypertrophy.Biol Chem,2010,285(16):11903-12.
[22]Onnis A, De Falco G, Antonicelli G, et al.Alteration of microRNAs regulated by c-Myc in Burkitt lymphoma. PLoS one,2010,5(9):e12960.
[23]Lin J, Lwin T, Zhao JJ, et al.Follicular dendritic cell-induced microRNA-mediated upregulation of PRDM1 and downregulation of BCL-6 in non-Hodgkin's B-cell lymphomas.Leukemia,2011,25(1):145-52.
[24]Guo R, Wu Q, Liu F, et al.Description of the CD 133+ subpopulation of the human ovarian cancer cell line OVCAR3.Oncol Rep,2011,25(1):141-6.
[25]Rotkrua P, Akiyama Y, Hashimoto Y, et al.MiR-9 down-regulates CDX2 expression in gastric cancer cells.Int J Cancer,2011 Jan 10.
[26]Myatt SS, Wang J, Monteiro LJ, et al.Definition of microRNAs that repress expression of the tumor suppressor gene FOXO1 in endometrial cancer. Cancer Res,2010,70(1):367-77.
[27]Guo LM, Pu Y, Han Z, et al. MicroRNA-9 inhibits ovarian cancer cell growth through regulation of NF-kappaB1. FEBS J.2009 Oct;276(19):5537-46.)
[28]Lund E, Dahlberg JE.Substrate selectivity of exportin 5 and Dicer in the biogenesis of microRNAs.Cold Spring Harb Symp Quant Biol,2006,71:59-66.
[29]Saini S, Majid S, Yamamura S, Regulatory role of miR-203 in prostate cancer progression and metastasis.Clin Cancer Res.2010 Dec 15.
[30]Nass D, Rosenwald S, Meiri E, et al。MiR-92b and miR-9/9* are specifically expressed in brain primary tumors and can be used to differentiate primary from metastatic brain tumors. Brain Pathol.2009 Jul;19(3):375-83.
[1]Herman JG, Graff JR, Myohanen S, et al.Methylation-specific PCR:a novel PCR assay for methylation status of CpG islands [J].Proc Natl Acad Sci U S A, 1996,93(18):9821-6.
[2]Hu SL, Kong XY, Cheng ZD, et al.Promoter methylation of p16, Runx3, DAPK and CHFR genes is frequent in gastric carcinoma[J].Tumori,2010, 96(5):726-33.
[3]Chang Sun, Laura L Reimers, Robert D Burk.Methylation of HPV16 genome CpG sites is associated with cervix precancer and cancer[J].Gynecol Oncol, 2011 Feb 7.
[4][4]Youwei Zhang, Rui Wang, Haizhu Song, et al.Methylation of multiple genes as a candidate biomarker in non-small cell lung cancer[J].Cancer Lett,2011 Jan 19.
[5]Ran Y, Wu S, You Y. Demethylation of E-cadherin gene in nasopharyngeal carcinoma could serve as a potential therapeutic strategy [J]. J Biochem,2011, 149(1):49-54.
[6]Tong JH.Putative tumour-suppressor gene DAB2 is frequently down regulated by promoter hypermethylation in nasopharyngeal carcinoma [J]. BMC Cancer, 2010,3 (0):253.
[7]Seo SY, Kim EO, Jang KL, et al.Epstein-Barr virus latent membrane protein 1 suppresses the growth-inhibitory effect of retinoic acid by inhibiting retinoic acid receptor-beta2 expression via DNA methylation [J].Cancer Lett,2008, 270(1):66-76.
[8]Langevin SM, Stone RA, Bunker CH, et al. microRNA-137 promoter methylation is associated with poorer overall survival in patients with squamous cell carcinoma of the head and neck[J]. Cancer,2010, Nov 8.
[9]Balaguer F, Link A, Lozano JJ, et al.Epigenetic silencing of miR-137 is an early event in colorectal carcinogenesis[J]. Cancer Res,2010,70(16):6609-18.
[10]Suzuki H, Yamamoto E, Nojima M, et al. Methylation -associated silencing of microRNA-34b/c in gastric cancer and its involvement in an epigenetic field defect[J]. Carcinogenesis.2010,31(12):2066-73.
[11]Ceppi P, Mudduluru G, Kumarswamy R, et al.Loss of miR-200c expression induces an aggressive, invasive and chemoresistant phenotype in non-small cell lung cancer[J].Mol Cancer Res,2010,8(9):1207-16.
[12]Hanoun N, Delpu Y, Suriawinata AA, et al.The silencing of microRNA 148a production by DNA hypermethylation is an early event in pancreatic carcinogenesis[J].Clin Chem,2010,56(7):1107-18.
[13]U Lehmann, B Hasemeier, M Christgen, et al.Epigenetic inactivation of microRNA gene hsa-mir-9-1 in human breast cancer[J]. J Pathol,2008,214(1): 17-24.
[14]Bandres E, Agirre X, Bitarte N, et al. Epigenetic regulation of microRNA expression in colorectal cancer[J].Int J Cancer,2009,125(11):2737-43.
[15]Trankenschuh W, Puls F, Christgen M, et al.Frequent and distinct aberrations of DNA methylation patterns in fibrolamellar carcinoma of the liver[J]. PLoS One,2010,5(10):e13688.
[16]Amaia Lujambio, George A Calinc, Alberto Villanueva, et al. A microRNA DNA methylation signature for human cancer metastasis[J].Proc Nat Acad Sci U S A,2008,105(36):13556-61.
[17]MAT Hildebrandt, J Gu, J Lin, et al.Hsa-miR-9 methylation status is associated with cancer development and metastatic recurrence in patients with clear cell renal cell carcinoma[J]. Oncogene,2010,29(42):5724-8.
[18]Pei-Yin Hsu, Daniel E Deatherage, Benjamin AT Rodriguez, et al.Xenoestrogen-Induced Epigenetic Repression of microRNA-9-3 in Breast Epithelial Cells[J]. Cancer Res,2009,69(14):5936-5945.
[19]Dannenberg LO, Edenberg HJ. Epigenetics of gene expression in human hepatoma cells:expression profiling the response to inhibition of DNA methylation and histone deacetylation[J]. BMC Genomics,2006,19(7):181.
[20]Gui CY, Ngo L, Xu WS, et al.Histone deacetylase(HDAC)inhibitor activation of p21WAF1 involves changes in promoter-associated proteins, including HDAC1[J].Proc Natl Acad Sci U S A,2004,101(5):1241-6.
[21]Pero R, Peluso S, Angrisano T, et al.Chromatin and DNA methylation dynamics of Helicobacter pylori-induced COX-2 activation[J].Int J Med Microbiol,2011, 301(2):140-9.
[22]Sharma G, Mirza S, Prasad CP, et al.Promoter hypermethylation of p16INK4A, p14ARF, CyclinD2 and Slit2 in serum and tumor DNA from breast cancer patients[J]. Life Sci,2007,80(20):1873-81.
[23]Jones PL, Veenstra GJ, Wade PA.Methylated DNA and MeCP2 recruit histone deacetylase to repress transcription[J]. Nat Genet,1998,9(2):187-91.
[24]Tabe Y, Konopleva M, Kondo Y, et al. PML-RARalpha and AML1-ETO translocations are rarely associated with methylation of the RAR02 promoter[J].Ann Hematol,2006,85(10):689-704.
[1]Shi W, Alajez NM, Bastianutto C, et al. Significance of Plkl regulation by miR-100 in human nasopharyngeal cancer.Int J Cancer,2010,126(9):2036-48.
[2]Xia H, Ng SS, Jiang S, et al. miR-200a-mediated downregulation of ZEB2 and CTNNB1 differentially inhibits nasopharyngeal carcinoma cell growth,migration and invasion. Biochem Biophys Res Commun,2010,91(1): 535-41.
[3]Wong TS, Man OY, Tsang CM, et al. MicroRNA let-7 suppresses nasopharyngeal carcinoma cells proliferation through downregulating c-Myc expression. J Cancer Res Clin Oncol.2011,137(3):415-22.
[4]Li G, Wu Z, Peng Y, et al. MicroRNA-10b induced by Epstein-Barr virus-encoded latent membrane protein-1 promotes the metastasis of human nasopharyngeal carcinoma cells. Cancer Lett,2010,299(1):29-36.
[5]Lu J, He ML, Wang L, et al. MiR-26a inhibits cell growth and tumorigenesis of nasopharyngeal carcinoma through repression of EZH2. Cancer Res,2011, 71(1):225-33.
[6]Shibata M, Nakao H, Kiyonari H, et al.MicroRNA-9 regulates neurogenesis in mouse telencephalon by targeting multiple transcription factors.J Neurosci.2011 Mar 2;31(9):3407-22.
[7]Otaegi G, Pollock A, Hong J, et al. MicroRNA miR-9 modifies motor neuron columns by a tuning regulation of FoxPl levels in developing spinal cords.J Neurosci.2011 Jan 19;31(3):809-18.)
[8]Bonev B, Pisco A, Papalopulu N. MicroRNA-9 reveals regional diversity of neural progenitors along the anterior-posterior axis. Dev Cell.2011 Jan 18;20(1):19-32.
[9]Tan HX, Wang Q, Chen LZ, et al.MicroRNA-9 reduces cell invasion and E-cadherin secretion in SK-Hep-1 cell[J].Med Oncol,2010,27(3):654-60.
[10]Ma L, Young J, Prabhala H, et al. miR-9, a MYC/MYCN-activated microRNA, regulates E-cadherin and cancer metastasis [J].Nat Cell Biol,2010,12(3): 247-56.
[11]Rotkrua P, Akiyama Y, Hashimoto Y, et al.MiR-9 down-regulates CDX2 expression in gastric cancer cells.Int J Cancer,2011 Jan 10.
[12]Guo LM, Pu Y, Han Z, et al. MicroRNA-9 inhibits ovarian cancer cell growth through regulation of NF-kappaB1. FEBS J,2009,276(19):5537-46.
[13]Levine BL, Hum eau LM, Boyer J, et al. Gene transfer in humans using a conditionally replicating lentiviral vector[J].Proc Natl Acad Sci USA,2006, 103(46):17372-17377.
[14]Baka K, Goto-Koshino Y, Mizukoshi F, et al. Inhibition of the replication of feline immunodeficiency virus by lentiviral vector mediated RNA interference in feline cell lines [J].J Vet Med Sci,2008,70(8):777-783.
[15]Bauer G, Dao MA, Case SS, et al. In vivo biosafety model to assess the risk of adverse events from retroviral and lentiviral vectors[J]. Mol Ther,2008,16(7): 1308-1315.
[16]Ferretti E,De Smaele E,Po A,et al.MicroRNA profiling in human medulloblastoma. Int J Cancer.2009,124(3):568-77.
[17]Liu L, Chen L, Xu Y, et al. microRNA-195 promotes apoptosis and suppresses tumorigenicity of human colorectal cancer cells.Biochem Biophys Res Commun,2010,400(2):236-40.
[18]Yoshitomi T, Kawakami K, Enokida H, et al.Restoration of miR-517a expression induces cell apoptosis in bladder cancer cell lines.Oncol Rep,2011, 25(6):1661-8.
[19]Feng S, Cong S, Zhang X, et al.MicroRNA-192 targeting retinoblastoma 1 inhibits cell proliferation and induces cell apoptosis in lung cancer cells. Nucleic Acids Res,2011 Apr 21.
[20]J. Kota, R.R. Chivukula, K.A. O'Donnell, et al. Therapeutic microRNA delivery suppresses tumorigenesis in a murine liver cancer model.Cell,2009 (137):1005-1017.
[21]B.Zhang, X.X.Liu, J.R. He, et al.Pathologically decreased miR-26a antagonizes apoptosis and facilitates carcinogenesis by targeting MTDH and EZH2 in breast cancer.Carcinogenesis,2011(32):2-9.
[22]Liu XX, Li XJ, Zhang B, et al. MicroRNA-26b is underexpressed in human breast cancer and induces cell apoptosis by targeting SLC7A11. FEBS Lett, 2011 Apr 14.
[23]Shi L, Chen J, Yang J, et al. MiR-21 protected human glioblastoma U87MG cells from chemotherapeutic drug temozolomide induced apoptosis by decreasing Bax/Bcl-2 ratio and caspase-3 activity. Brain Res,2010,1352: 255-64.
[24]Li JH, Xiao X, Zhang YN, et al. MicroRNA miR-886-5p inhibits apoptosis by down-regulating Bax expression in human cervical carcinoma cells. Gynecol Oncol,2011,120(1):145-51.
[25]曾益新.肿瘤学(第2版).北京：人民卫生出版社,2003；97.
[1]Ding YL, Zhang JL, Tang SF, et al. Effect of chemokine stromal cell derived factor-1 and its receptor CXCR4 on the peritoneal carcinometastasis of gastric cancer[J].Zhonghua Yi Xue Za Zhi,2008,88(3):202-5.
[2]Chinni SR, Yamamoto H, Dong Z, et al.CXCL 12/CXCR4 Transactivates HER2 in Lipid Rafts of Prostate Cancer Cells and Promotes Growth of Metastatic Deposits in Bone[J].Mol Cancer Res,2008,6(3):446-57.
[3]Nigris F, Rossiello R, Schiano C, et al. Deletion of Yin Yang 1 protein in osteosarcoma cells on cell invasion and CXCR4/angiogenesis and metastasis[J]. Cancer Res,2008,68(6):1797-808.
[4]Kim J, Mori T, Chen SL, et al. Chemokine receptor CXCR4 expression in patients with melanoma and colorectal cancer liver metastases and the association with disease outcome[J].Ann Surg,2006,244(1):113-20.
[5]Holland JD,Kochetkova M,Akekawatchai C,et al.Differential functional activation of chemokine receptor CXCR4 is mediated by G proteins in breast cancer cells[J].Cancer Res,2006,66(8):4117-24.
[6]Hu J, Deng X, Bian X,et al. The expression of functional chemokine receptor CXCR4 is associated with the metastatic potential of human nasopharyngeal carcinoma.Clin Cancer Res.2005 Jul 1;11(13):4658-65.
[7]Ou DL, Chen CL, Lin SB, et al.Chemokine receptor expression profiles in nasopharyngeal carcinoma and their association with metastasis and radiotherapy. J Pathol.2006 Nov;210(3):363-73.
[8]Wang N, Wu QL, Fang Y,et al. Expression of chemokine receptor CXCR4 in nasopharyngeal carcinoma:pattern of expression and correlation with clinical outcome. J Transl Med.2005 Jun 26;3:26.
[9]Segawa Y, Oda Y, Yamamoto H,et al. Close correlation between CXCR4 and VEGF expression and their prognostic implications in nasopharyngeal carcinoma.Oncol Rep.2009 May;21 (5):1197-202
[10]罗花南,李湘平,刘雄等.CXCR4/SDF-1α与鼻咽癌器官特异性转移的相关性研究[J].中华肿瘤杂志,2009,31(4)：260-264.
[11]Ott CE, Grunhagen J, Jager M, et al.MicroRNAs Differentially Expressed in Postnatal Aortic Development Downregulate Elastin via 3' UTR and Coding-Sequence Binding Sites[J]. PLoS One,2011,6(1):e16250.
[12]Wu E, Thivierge C, Flamand M, et al. Pervasive and cooperative deadenylation of 3'UTRs by embryonic microRNA families[J]. Mol Cell,2010,40(4):558-70.
[13]Bagga S, Bracht J, Hunter S, et al.Regulation by let-7 and lin-4 miRNAs results in target mRNA degradation[J]. Cell,2005,122(4):553-63.
[14]Wada R, Akiyama Y, Hashimoto Y, et al.miR-212 is downregulated and suppresses methyl-CpG-binding protein MeCP2 in human gastric cancer[J].Int J Cancer,2010,27(5):1106-14.
[15]Zhao JJ, Lin J, Yang H, et al.MicroRNA-221/222 negatively regulates estrogen receptor alpha and is associated with tamoxifen resistance in breast cancer[J].J Biol Chem,2008,283(45):31079-86.
[16]Shin KH, Bae SD, Hong HS, et al.miR-181a shows tumor suppressive effect against oral squamous cell carcinoma cells by downregulating K-ras[J].Biochem Biophys Res commun,2011,404(4):896-902.
[17]Lo WL, Yu CC, Chiou GY, et al. MicroRNA-200c attenuates tumor growth and metastasis of presumptive head and neck squamous cell carcinoma stem cells squamous cell carcinoma stem cells[J].J Pathol,2011,223 (4):482-95.
[18]Guo H, Ingolia NT, Weissman JS, et al. Mammalian microRNAs predominantly act to decrease target mRNA levels[J].Nature,2010,466(7308):835-40.
[19]Wan HY, Guo LM, Liu T, et al.Regulation of the transcription factor NF-kappaB1 by microRNA-9 in human gastric adenocarcinoma[J]. Mol Cancer,2010,9 (16):1-10.
[20]Luo H, Zhang H, Zhang Z, et al.Down-regulated miR-9 and miR-433 in human gastric carcinoma[J]. J Exp Clin Cancer Res,2009,16 (28):82.
[21]Rotkrua P, Akiyama Y, Hashimoto Y, et al. MiR-9 down-regulates CDX2 expression in gastric cancer cells[J].Int J Cancer,2011, Jan 10.
[22]Guo LM, Pu Y, Han Z, et al.MicroRNA-9 inhibits ovarian cancer cell growth through regulation of NF-kappaB1[J].FEBS J,2009,276(19):5537-46.
[23]Tan HX, Wang Q, Chen LZ, et al.MicroRNA-9 reduces cell invasion and E-cadherin secretion in SK-Hep-1 cell[J].Med Oncol,2010,27(3):654-60.
[24]Nie K, Gomez M, Landgraf P, et al.MicroRNA-mediated down-regulation of PRDM1/Blimp-1 in Hodgkin/Reed-Sternberg cells:apotential pathogenetic lesion in Hodgkin lymphomas[J].Am J Pathol,2008,173 (1):242-52.
[25]Guo LM, Pu Y, Han Z, et al.MicroRNA-9 inhibits ovarian cancer cell growth through regulation of NF-kappaB1.FEBS J,2009,276(19):5537-46.
[26]Myatt SS, Wang J, Monteiro LJ,et al. Definition of microRNAs that repress expression of the tumor suppressor gene FOXO1 in endometrial cancer. Cancer Res,2010,70(1):367-77.
[27]Liu M, Lang N, Chen X,et al. miR-185 targets RhoA and Cdc42 expression and inhibits the proliferation potential of human colorectal cells. Cancer Lett, 2011,301(2):151-60.