miR-10b在鼻咽癌转移中的功能和调控机制研究
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摘要
研究背景和目的
鼻咽癌是中国南方和东南亚地区最常见的恶性肿瘤之一,以明显的区域分布特征、与EB病毒的密切关系以及强烈的转移倾向显著区别于其他头颈部肿瘤。转移是鼻咽癌治疗失败的主要原因,尽管诊疗技术不断进步,但由于60-85%的患者确诊时已发生临床转移,鼻咽癌的5年生存率提升缓慢,探究鼻咽癌转移的分子机制,寻找新的治疗靶点,减少转移的发生,是我们面临的巨大挑战。
LMP-1是鼻咽癌最重要的外源性癌基因,也是鼻咽癌最重要的促转移基因。在鼻咽癌中,LMP-1发挥类似肿瘤坏死因子受体(tumor necrosis factorreceptor,TNFR)的功能,通过激活NF-κB,AP-1,ets-1,MAPKs,JAK/STAT,PI3K/Akt等不同信号通路,对E-cadherin,MMPs,c-Met,VEGF,EGFR,COX-2等多个转移相关基因的调控,影响着鼻咽癌转移的几乎每个环节。沉默LMP-1能够减少鼻咽癌细胞转移。
miRNA(microRNA)是广泛存在于生物体内的19-25nt的非编码RNA。转录完成后,miRNA与靶mRNA的3'UTR的互补序列结合,通过抑制mRNA的翻译或直接降解mRNA调控基因表达。人类基因组编码的miRNA多达1000个,参加了包括胚胎形成、发育、代谢和重大疾病在内的几乎所有生命活动。一些miRNA能够影响细胞凋亡、增殖与分化,扮演着癌基因的角色,被称为oncomirs。肿瘤转移是一个及其复杂的生物学过程,包括上皮细胞去极化,细胞间黏附改变,运动性增强,基底膜穿透,血管生成等一系列变化,涉及了大量信号通路激活和多个基因的表达变化。这一过程同样离不开miRNA对基因表达的精准调控。我们对oncomirs在肿瘤转移中的功能和作用机制还知之甚少,最新研究发现,miR-17-92家族,miR-200家族,miR-205,miR-29C,miR-21,Let-7等oncomirs在肿瘤转移过程中,也发挥着重要作用。
miR-10b促进乳腺癌转移的发现更是备受瞩目。Ma和同事等发现miR-10b仅在高转移的乳腺癌细胞中表达,阻断miR-10b能够使MDA-MB-231细胞侵袭能力下降90%以上,而不具备转移能力的SUM149细胞过表达miR-10b后,在裸鼠体内发生远处转移。临床资料也表明,发生转移的乳腺癌患者,miR-10b表达高于非转移组。这一研究还发现。乳腺癌细胞中miR-10b的表达,正是由转录因子Twist启动的。
Twist属于碱性的螺旋-环-螺旋(Helix-loop-helix)蛋白家族中高度保守的转录因子,可以调节胚胎发育过程中的组织重建,并赋予细胞迁移的能力,也是促进多种肿瘤的转移与侵袭的关键基因,在鼻咽癌的转移中也发挥重要作用。Horikawa T.等发现LMP-1能够诱导转录因子Twist,促进鼻咽癌转移和侵袭。据此我们提出研究假说:miR-10b受LMP-1/Twist信号通路调控,在鼻咽癌进展过程中发挥促进转移的功能。
为了证实这一假说,我们首先检测miR-10b在鼻咽癌组织和不同的鼻咽癌细胞中的表达水平,分析miR-10b表达与细胞转移能力的关系,并观察LMP-1和Twist的表达变化对miR-10b的影响。利用pre-miR-10b慢病毒表达载体,在鼻咽癌细胞中过表达miR-10b,或转染miR-10b抑制剂阻断miR-10b,分析其表达变化对细胞周期、增殖、凋亡,移动、侵袭和体内转移能力的影响,以初步探讨鼻咽癌转移中miR-10b的功能和作用机制。
方法
1.鼻咽癌组织和细胞中miR-10b的检测
收集鼻咽癌组织标本45例(其中转移26例,未转移19例),慢性鼻咽炎组织16例作对照。所有新鲜鼻咽癌组织标本分为2份,一份经10%甲醛固定,包埋、切片免疫组化染色检测LMP-1表达情况。另一份标本液氮冻存,制作冰冻切片,用原位杂交检测miR-10b表达。
2.鼻咽癌细胞中miR-10b的检测
长期携带EB病毒基因的人类低分化鼻咽癌细胞株C666-1,高分化鼻咽癌细胞CNE1,低分化鼻咽癌细胞CNE2、HONE1、HNE-1细胞,以及SUNE-1细胞的高转移亚株5-8F和不转移亚株6-10B,永生化鼻咽上皮细胞株NP69细胞,8株细胞分别采用荧光定量PCR和原位杂交检测miR-10b表达。
3.沉默及过表达LMP-1表达
合成能够转录出LMP-1干扰模板的双链DNA片段,插入shRNA的表达载体pAVU6+27,获得pAVU6+27/shLMP-1质粒。以空pAVU6+27载体为对照,分别转染C666-1细胞后,通过G418筛选和巢式PCR检测获得LMP-1阴性的亚株C666/shLMP-1。含有N-LMP-1 cDNA的质粒pCR~(?)Ⅱ-TOPO/LMP-1,用Lipofectamine-2000转染细胞,48h后RT-PCR检测LMP-1mRNA水平。
4.miRNA提取和Real time PCR检测
按照mirVana miRNA isolation kit说明书进行操作,富集分子量小于200nt的小分子RNA。按照TaqMan MicRNA RT Kit说明书操作,采用TaqMan MicRNAAssays中的逆转录引物,以cDNA为模板,进行逆转录反应,U6 RNA作为内对照。
5.制备慢病毒载体稳定过表达miR-10b
根据miR-10b前体pre-mir-10b(MI000681)的序列,在5'端和3'端引入Mlu I和Cla I的酶切位点,合成全长102bp的pre-mir-10b转录模板,连接转化,挑取重组阳性克隆,经PCR、Mlu I和Xba I双酶切鉴定,鉴定正确的质粒送DNA测序。采用脂质体法将慢病毒包装系统中3种质粒按比例共转染293FT细胞,收集上清液,离心过滤后,得到慢病毒悬液。病毒悬液连续稀释法计算病毒滴度。慢病毒pLVTHM/miR-10b和空病毒载体pLVTHM感染鼻咽癌细胞。采用流式细胞仪分选出GFP(+)细胞扩大培养。Real PCR检测miR-10b表达水平。
6.siRNA及anti-miR转染
细胞融合度达到40%左右,更换无血清培养基。将anti-miR充分溶解于不含有血清的转染专用培养基Opti-MEM中。阳离子脂质体Lipofectamine2000用Opti-MEM稀释后,与siRNA或anti-miR溶液混合,轻轻振荡后,置于室温20min,加入细胞培养板。37℃孵育4h后,加入30%胎牛血清FBS,继续培养24h,48h,72h后,荧光定量PCR方法检测miR-10b表达水平的变化。
7.miR-10b报告基因质粒的构建和双荧光报告实验
以基因组DNA为模板扩增出将含有miR-10b种子区互补序列的片段作为miR-10b结合位点,将片段插入psiCHECK-2质粒海肾荧光素Renilla下游XhoI和NotI位点之间。用Lipofectamine-2000将psiCHECK-2/miR-10b-binding质粒anti-miR,同时转染细胞,24h后按照Dual-Luciferase Reporter Assay System的说明进行样品的Luciferase活性检测,根据荧光素比值:Luciferaseactivity(Renilla/Firefly)变化分析miRNA inhibitor抑制miR-10b的效果。
8.细胞增殖实验
细胞以每孔1×10~3个细胞接种于96孔培养板中,每孔体积200μl,每组5孔,同时设空白对照(仅加培养基),分别培养1-5天。每孔加入5mg/ml的MTT20μl,37℃继续培养4h后终止培养,加入DMSO,酶标仪上570nm测定各孔吸光度值(OD值),以相对应OD比值表示细胞增殖能力大小。各组取5孔平均值,绘制增殖曲线。
9.流式细胞实验
C666/shLMP-1和C666/shLMP-1/miR-10b细胞,0.25%胰酶消化,PBS洗涤3次后调整细胞浓度为5×10~5/ml的细胞悬液,离心后,按照BD公司荧光染色试剂盒说明书,进行荧光标记,流式细胞仪进行细胞周期检测。
10.划痕实验
在6孔细胞培养板中,每组细胞作3个复孔。待细胞密度达到90%左右,吸干培养基,用同样大小的加样枪头,垂直在培养孔中部轻轻划过,制造多条相互平行的划痕,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。每个孔随即选取9个有划痕穿过的视野,在不同时间点,对越过划痕边缘向空白出移动生长的细胞进行计数,独立实验重复3次。
11.侵袭实验
采用美国BD公司的肿瘤细胞基底膜穿透实验系统Biocoat MatrigelInvasion Chambers,按照说明书操作,C666-1细胞胰酶消化后,以无血清培养基种植与小室内5.0×10~3/孔,24孔细胞培养板,每孔中预先加入含有10%胎牛血清的培养基0.5ml,然后将培养杯置入孔中培养。不同时间后取出小室,棉签拭去基底膜正面的细胞,将部分小室基底膜沿杯底剪下翻转,用甲苯胺蓝对能够穿透基底膜的细胞进行染色,显微镜下计数。
12.体内转移实验
建立肝被膜下种植肺转移动物模型。4～6周龄BALB/c nu/nu裸鼠48只,鼻咽癌细胞调制成2×10~6/ml细胞悬液,用1ml注射器于裸鼠肝包膜下缓慢注入0.1ml细胞悬液,全层缝合腹壁。其中36只裸鼠随机分成2组,分别接种C666/shLMP-1和C666/shLMP-1/miR-10b细胞,每组18只,其中每组随机取9只用于观察抗肿瘤效果,其余9只用于生存期观察。另外12只裸鼠分成2组,每组6只,分别接种过表达miR-10b的HNE1和HNE1/miR-10b细胞。
用于生存期观察的18只裸鼠,观察活体动物体内荧光分布,能够了解肿瘤细胞在体内扩散的情况。并继续饲养至自然死亡,记录生存期。用于观察抗肿瘤效果的18只裸鼠第三周末断髓处死,立即取荷瘤裸鼠肝脏及肺脏组织,PBC冲洗2次,用动物整体成像系统下观察带荧光的肿瘤组织分布情况,并拍照。荧光照相后的肝脏及肺脏组织,用10%甲醛固定,常规石蜡包埋HE染色,显微镜下观察肿瘤细胞在肝脏内成瘤情况,以及肺、腹膜等器官肿瘤转移情况。
13.统计学处理
采用SPSS 13.0软件对数据进行分析,均以P＜0.05表示差异有显著性意义。免疫组化和原位杂交结果采用X~2检验以及两独立样本非参数秩和检验Mann-Whitney U法进行统计学分析。荧光定量PCR和细胞周期实验,样本均数比较采用,两独立样本的t检验(independent-samples T test)或单因素方差分析(One-way ANOVA),多重比较采用SNK法(Student-Neuman-Keuls)。方差不齐者采用基于方差不齐的多重比较方法Dunnett's T3法进行。MTT实验、体外侵袭实验、划痕实验采用析因设计的方差分析。动物生存时间分析采用Kaplan-Meier法,肺转移、肝内转移和腹水情况的比较采用X~2检验。
结果
1.miR-10b的表达与鼻咽癌转移以及LMP-1的关系
①miR-10b表达与鼻咽癌发病
在全部45例鼻咽癌组织中,miR-10b表达率为80%(36/45),主要定位于细胞核,鼻咽慢性炎症组织中表达率为56.25%(9/16),二者无显著差异(X~2=3.441,P=0.097)。
②miR-10b表达与鼻咽癌转移
将45例鼻咽癌组织按照确诊时有无淋巴结或远处转移分为转移组(26例)和未转移组(19例),鼻咽癌转移组miR-10b的表达较强(Z=-3.243,P=0.001)。
③miR-10b表达与LMP-1
45例鼻咽癌组织中,LMP-1阳性率为37.78%(17/45),LMP-1(+)组miR-10b的表达较强(Z=-2.013,P=0.044)。在LMP-1阳性组的中转移者miR-10b表达高于未转移者(Z=-2.504,P=0.012),而LMP-1阴性组中转移和未转移者的miR-10b表达没有统计学差异(Z=-1.857,P=0.063)。
2.miR-10b表达水平与细胞转移能力和LMP-1表达的相关性
①.miR-10b在转移的鼻咽癌细胞中高表达
7种鼻咽癌细胞株与永生化人鼻咽上皮细胞NP69相比较,miR-10b在8株细胞中的表达存在显著差异(F=96.395,P=0.000),具备转移能力的C666-1和5-8F细胞表达水平高于其他5种不转移的鼻咽癌细胞。原位杂交显示C666-1和5-8F细胞miR-10b表达为阳性,6-10B和HONE-1细胞为阴性,与荧光定量PCR检测结果一致。
②miR-10b表达水平与LMP-1一致
RT-PCR结果显示,转染pCR~(?)Ⅱ-TOPO/LMP-1 C666/LMP-1细胞LMP-1mRNA较C666-1明显提高。转染C666/shLMP-1细胞中LMP-1稳定沉默。LMP-1沉默的C666/shLMP-1细胞miR-10b表达明显下降,而过表达LMP-1的C666/LMP-1的miR-10b表达提高明显提高,LMP-1含量与miR-10b表达呈一致变化。
3.miR-10b过表达鼻咽癌细胞株的建立
由于LMP-1是鼻咽癌主要的外源性癌基因,为了排除LMP-1基因表达变化对细胞的影响,我们利用LMP-1沉默的C666/shLMP-1细胞进行miR-10b的功能研究。首先利用慢病毒载体系统在中过表达miR-10b,制备了miR-10b稳定过表达的慢病毒载体pLVTHM/pre-miR-10b,感染C666/shLMP-1细胞48-72h后,阳性C666-1细胞发出绿色荧光,经流式细胞仪分选出带荧光的细胞经反复传代后,miR-10b水平比感染pLVTHM空载体C666/shLMP-1最多提高45倍,成为稳定过表达的亚克隆细胞C666/shLMP/miR-10b。
4.miR-10b过表达对C666-1细胞增殖和细胞周期的影响
MTT结果和流式细胞实验表明,C666/shLMP-1和C666/shLMP-1/miR-10b细胞的体外增殖能力及细胞周期均无显著性差异。
5.miR-10b过表达对鼻咽癌细胞体外侵袭和移动能力的影响
基底膜穿透实验和划痕实验表明过表达miR-10b的C666/shLMP-1/miR-10b细胞侵袭和移动能力较对照组增强。miR-10b过表达提高鼻咽癌细胞体外侵袭和移动能力
6.miR-10b过表达对鼻咽癌细胞体内侵袭、移动和生存时间的影响
接种C666/shLMP-1和C666/shLMP-1/miR-10b鼻咽癌细胞的裸鼠,3周后肝脏成瘤率达100%,其中C666/shLMP-1/miR-10b组动物全部出现种植瘤肝内广泛转移腹水出现时间较早(X~2=5.556,P=0.018),肺转移发生率均高于C666/shLMP-1组(X~2=5.844,P=0.016),生存时间较C666/shLMP-1组缩短(X~2=5.526,P=0.019)。而接种HNE1/miR-10b和HNE1组也可在局部成瘤,但均未发现肝内播散和肺转移。说明miR-10b过表达提高鼻咽癌细胞体内侵袭和移动能力。
7.miR-10b抑制物阻断miR-10b表达对鼻咽癌细胞转移能力的影响
荧光报告检测结果表明,miR-10b抑制物(anti-miR-10b)与转染后,只有C666/shLMP/miR-10b侵袭能力增强(t=-5.509,P=0.000),而C666/shLMP-1和C666/control侵袭能力无明显变化(图-6)。说明anti-miR-10b能够逆转由于miR-10b过表达造成的侵袭能力提高,而对于miR-10b表达很低的C666/shLMP-1没有影响,同样对于LMP-1阳性C666/control,单独抑制miR-10b也不足以改变其转移能力。
8.LMP-1通过Twist基因诱导miR-10b表达
利用RNA干扰抑制Twist的表达,分析LMP-1是否通过Twist启动miR-10b转录。Twist siRNA转染C666-1细胞72小时后,Twist mRNA含量下降超过87.4%。LMP-1(+)的C666/control(t=7.060,P=0.002)和C666/LMP细胞(t=7.564,P=0.002)中miR-10b均下降,而LMP-1(-)的C666/shLMP(t=-0.579,P=0.594)和C666/shLMP/miR-10b细胞(t=-0.090,P=0.932)miR-10b的含量没有明显变化。说明LMP-1诱导miR-10b的表达,很可能是通过转录因子Twist实现的。
结论:
1.miR-10b在转移的鼻咽癌组织和细胞中高表达,表达水平与EB病毒LMP-1基因相关。
2.miR-10b的过表达能够促进鼻咽癌细胞体外和体内转移能力,但对细胞增殖、凋亡及细胞周期没有影响。
3.LMP-1基因通过转录因子Twist调控miR-10b表达,鼻咽癌中可能存在LMP-1/Twist/miR-10b促转移信号传导通路。
4.miR-10b在鼻咽癌转移分子机制研究中有重要意义,可以作为判断预后指导治疗的生物学标记,但能否作为基因治疗靶点不能定论。
BACKGROUND AND OBJECTIVE
The incidence of nasopharyngeal carcinoma(NPC)is most prevalent in Southeast Asia,particularly in Southern China.NPC is distinctive among the head and neck carcinomas for its marked tendency to metastasis and invasion.It has been shown that at the time of diagnosis,60-85%of NPC patients already have clinically detectable aggressive metastasis in the regional lymph nodes,in distant organs such as the lungs and in bone.So far there is still no effective treatment for NPC at the stage of metastasis.As a result,prognosis of NPC is poor and the 5-year survival rate is less than 50%.Meanwhile,the mechanisms that control NPC metastasis remain poorly understood.
Epstein-Barr virus(EBV)encoding latent membrane protein-1(LMP-1)is a primary oncoprotein in NPC.It has been suggested to contribute to the highly metastatic nature of NPC.LMP-1 functions as a constitutively active tumor necrosis factor receptor(TNFR)and contributes to multiple aspects of NPC,mainly through activating a number of signaling pathways,including NF-kB,AP-1,ets-1,MAPKs, and JAK/STAT,and controlling the expression of metastasis related gene,such as E-cadherin,matrix metalloproteinase(MMPs),c-Met,VEGF,EGFR and COX-2. Knock down of LMP-1 reduces the metastasis abilities of NPC cells.
MicroRNAs(miRNAs)are 19-25 nt regulatory RNAs that participate in the regulation of various biological functions in numerous eukaryotic lineages,including plants,insects,vertebrate,and mammals.miRNAs can have pleiotropic effects on cell proliferation,apoptosis,and cell differentiation.They are generally believed to act by binding to imperfectly complementary sequences in the 3' untranslated region of the target genes,resulting in decreased translation or degradation of the target transcript. Bioinformatic and experimental evidence indicate that each miRNA may target several dozen to several hundred gene transcripts,thus allowing for coordinated and combinatorial regulation of multiple genes by miRNAs.More than 1000 miRNAs have been identified in humans so far,and many of the genes in the human genome are predicted to be subject to miRNA regulation.The expression of many miRNAs is usually specific to a tissue or developmental stage,and the miRNA expression pattern is altered during the development of many diseases miRNAs are integral to gene regulation,apoptosis,hematopoietic development,and the maintenance of cell differentiation.There are a growing number of examples of particular miRNAs whose expression is increased in cancer(i.e.,mir-21,the mir-17-92b cluster,mir-155,and mir-372/373)whereas the expression of other miRNAs is decreased in tumor versus normal cells.miRNAs that have been experimentally shown to directly induce tumor have been termed“oncomirs”,with the first example being the mir-17-92b cluster. Metastasis is a central problem in cancer,yet the underlying mechanisms remain poorly understood.The involvement of miRNAs in the development of metastases has come under intense scrutiny in recent years.Members of miR-17-92 family, miR-200 family,miR-205,miR-29C,miR-21 and Let-7 were proved to be involved in cancer metastasis.
Ma and coworkers from Robert Weinberg's group found that miR-10b initiated breast cancer invasion and metastasis.They also found that Twist,a metastasis promoting transcription factor,could induce miR-10b expression and demonstrated that miR-10b was an essential element in the Twist-induced metastasis program. Others' work showed that induction of Twist by an Epstein-Barr virus (EBV)-encoded human viral oncoprotein latent membrane protein-1(LMP-1)directly contributes to the metastatic nature of NPC.
Therefore,here we explored whether miR-10b could be induced by EBV oncogene LMP-1,and its association with Twist as well.We took advantage of an established pre-miR-10b lentiviral vector and RNA interference technology to determine the relationship between EBV LMP-1 and miR-10b,and how induction of miR-10b by LMP-1 might contribute to the highly metastatic feature of NPC.We first showed that LMP-1 could activate miR-10b,and possibly via Twist,and miR-10b stimulated cell migration and invasion in vitro.We showed next that in cell culture,blocking of miR-10b could directly inhibit the invasion behavior of miR-10b over-expressed NPC,but we did not find such effect in control cells without miR-10b over-expression.We established a NPC metastasis model in mice by transplanting miR-10b over-expressed EBV-positive NPC cells,C666/shLMP-l/miR-10b,in the livers of nude mice and observed lung metastasis.Metastasis was detected in lung and liver,while no metastasis was found when miR-10b over-expressed EBV-negative non-metastatic NPC cells were transplanted into mice.
This report suggested that miR-10b facilitates the metastasis of EBV related NPC cells,but miR-10b gene might temporarily not be considered as a therapeutic target for NPC metastasis.
MATERIALS AND METHODS
1.Expression characteristics of miR-10b in NPC tissues
This study was conducted on a total of 45 NPC samples(26 metastatic NPC and 19 non-metastatic)and 16 chronic nasopharyngitis samples.All the samples was divided into two.One part of the specimen was made into frozen-section for miR-10b in situ hybridization,the other for paraffin section and immunohistochemistry analysis of LMP-1.
2.Expression characteristics of miR-10b in NPC cells
Quantitive real-time PCR was used to detect the expression of miR-10b in 7 NPC-derived cell lines(HNE1,HONE1,CNE1,CNE2,5-8F,6-10B,C666-1,)and immortalized nasopharyngeal epithelial cells NP69.
3.miRNA isolation and Real-time PCR
Cells were collected and miRNAs were prepared using MirVana miRNA isolation kit and RT reactions were performed using TaqMan MicRNA RT Kit(ABI, USA).Real-time PCR was performed with TaqMan Universal Master Mix(ABI) using Mx3000P real-time PCR instrument(Stratagene,USA).U6 RNA was used as an endogenous control for miRNA detection.The data were analyzed using the standard curve analysis.
4.Knockdown and overexpression of LMP-1
To stably knockdown of LMP-1,C666-1 cells were transfected with shRNA vector pAVU6+27/shLMP-l targeting LMP-1(CATAGGCCTTGCTCTCCTTCTCC) using the lipofectamine 2000 reagent(Invitrogen)following the protocol provided by the manufacturer.Forty-eight hours later,cells were harvested and plated on 10 cm tissue culture plate,and clones stably expressing shRNA(C666/shLMP-l)were selected using 400μg/ml G418.To overexpression of LMP-1,C666-1 cells were transfected with N-LMP-1 cDNA containing vector pCR~?Ⅱ-TOPO/LMP-l(A kindly gift of professor Marie C.Lin from the Department of Chemistry,The University of Hong Kong,China)using the lipofectamine 2000 reagent following the protocol provided by the manufacturer.Forty-eight hours later,cells were harvested and LMP-1 mRNA was detected by RT-PCR.
5.Lentiviral vector mediated overexpression of miR-10b
To generate a miR-10b expression vector,miR-10b precursor pre-miR-10b was amplified by PCR and cloned into pLVTHM vector(GFP-encoding)and transfected with psPAX2 and pMD2.G into 293FT using lipofectamine 2000 to package lentivirus pLVTHM/miR-10b.Cells were infected.After 3 days,cells were sorted by BD FACSAriaⅡcell sorter(Becton-Dickinson Labware)to selectively amplify GFP-positive cells.miR-10b mRNA expression after infection of lentiviral vector was revealed by real-time PCR.
6.Oligonucleotide transfection
Twist siRNA(AAGCTGAGCAAGATTCAGACC),its control siRNA and anti-miR-10b miRNA inhibitor were transfected using Lipofectamine 2000 (Invitrogen)following the protocol provided by the manufacturer.
7.MTT assay
MTT assay was performed to assess the effect of miR-10b on cell proliferation. Cells(1×10~3cells/well)were plated in a 96-well plate and maintained in RPMI-1640 supplemented with 10%FBS.At 24,48,72,96 and 120h after seeding,culture medium was removed,cells were treated with 20μl sterile MTT dye(5mg/ml,Sigma, USA)for 4h at 37℃,and then 200μl of DMSO was added and thoroughly mixed for 30min.Spectrometric absorbance at wavelength of 570nm was measured on a microplate reader.
8.Flow cytometry
Cell cycle profiles are analyzed by flow cytometry.Cells(5×l0~5cells/well) were plated in a 6-well plate and were transfected.After 48 h,cells were collected and washed three times in PBS.After resuspension,solution A,B and C from fluorescence labeling kit were added to label DNA.Sample analysis was performed by flow cytometry(BD FACSAriaⅡcell sorter,Becton-Dickinson Labware).The cell cycle phase distribution was calculated from the resultant DNA histogram using Multicycle AV software(Phoenix Flow System,San Diego,CA,USA).
9.Wound healing assays
In vitro wound healing assay was carried out to determine the ability of cells to form membrane protrusion and cell migration.Equal numbers of cells(1×10~5)were seeded into six-well cell culture plates.When the confluence reached 90%,a single wound was created in the center of the 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 at different time points.A total of nine areas were selected randomly in each well under a 40×objective and cells in three wells of each group were quantified in each experiment.
10.In vitro Matrigel invasion assay
Cell invasiveness was determined in vitro by the ability of the cell to transmigrate a layer of extracellular matrix(ECM)in Matrigel in Biocoat Matrigel Invasion Chambers(Becton-Dickinson Labware,Bedford,MA).Cells were plated at a density of 5.0×10~3 cells/insert,respectively.Medium with 10%FBS was added to the lower chamber as a chemoattractant.After 24 h incubation,cells on the upper surface of the membrane were removed.Invasive cells,which were able to breach the 8μm pores and grow on the lower surface,were fixed in 100%methanol,stained with 1%Toluidine(Sigma),and counted under an inverted microscope(Leica,German). The cells were counted in three random optical fields(200×magnification)from duplicate experiments.
11.Construction of miR-10b reporter plasmids and luciferase assays
Complement fragment of miR-10b(ACAAATTCGGTTCTACAGGGTA)was cloned into psiCHECKTM-2 Vector(Promega,Madison,WI)downstream of the Renilla luciferase cDNA.Luciferase activity was assayed using Dual-Luciferase Reporter Assay System(Promega,USA)following the protocol provided by the manufacturer.
12.In vivo metastasis experiment
Nude mice(BALB/c nu/nu,4-6 weeks old,18-24 g in weight)were purchased from Charles River Labs(Wilmington,WA)and were maintained under pathogen-free conditions(specific pathogen-free level).Mice were divided into two treatment groups(n=18/group),primary tumors were established by direct injection of 2×10~6 C666/shLMP or C666/shLMP-l/miR-10b into the liver as previously described.Nine mice from each group were randomly chosen for long-term survival study on day 10.The remaining nine mice were sacrificed on day 21,and tissues were collected for pathologic analysis.GFP fluorescence images were observed under an in vivo fluorescence instrument.
13.Statistical analysis
All analyses were carried out using the SPSS 13.0 software package.Expression of miR-10b in relation to clinical data was analyzed with the Mann-Whitney U test. Comparison of mean was analyzed by one-way ANOVA and Student-Neuman-Keuls method for multiple comparison.Dunnett's T3 method was employed for heterogeneity of variance.Survival curves were plotted by the Kaplan-Meier method and compared by the log-rank test.The X~2 test for proportion was used to analyze the in vivo metastasis characteristics of different groups.Factorial analysis was utilized to analyze the results MTT,invasion and wound healing assays.The differences in wound migration and invasion indices between C666-1 cell clones were analyzed by the independented t test.A P value less than 0.05 was considered statistically significant.
RESULTS
1.miR-10b was over-expressed in metastatic NPC samples and was correlate with LMP-1 expression
In situ hybridization results showed that,although there is no difference of miR-10b expression between NPC(36/45)and chronic nasopharyngitis(9/16) samples(X~2=3.441,P=0.097),miR-10b was positively associated with NPC metastasis(Z=-3.243,P=0.001).Furthermore,expression of miR-10b was correlated with LMP-1 expressing(Z=-2.013,P=0.044).In LMP-1(+)samples,metastatic NPC present higher miR-10b rate than non-metastatic ones(Z=-2.504,P=0.012). However,the same thing didn't happened in LMP-l(-)cases(Z=-1.857,P=0.063).
2.miR-10b was over-expressed in metastatic NPC cells and its expression was correlate with LMP-1 expression
Using real-time PCR,miR-10b expression in 7 NPC-derived cell lines(HNE1, HONE1,CNE1,CNE2,5-8F,6-10B,C666-1,)and immortalized nasopharyngeal epithelial cells NP69 were examined.The results showed that the expression of miR-10b in 8 cell lines was significantly different from each other(F=96.395, P=0.000).And expression of miR-10b was significantly increased in the metastatic C666-1 and 5-8F cell comparing with the non-metastatic NPC cells.
In situ hybridization showed negative miR-10b expression in 6-10B,HONE and strongly positive in C666-1 and 5-8F which supported the real-time PCR results.
To test whether miR-10b was regulated by LMP-1,LMP-1 expression in C666-1 cells was down-regulated by RNAi and isolation of clones C666/shLMP stably expressing shRNA targeting LMP-1 or up-regulated by transfecting the expression plasmid pCR?Ⅱ-TOPO/LMP-l.Real-time PCR showed that when LMP-1 expression was down-regulated,the expression of miR-10b was correspondingly decreased(C666-1 vs C666/shLMP-l);when LMP-1 expression was up-regulated,the expression of miR-10b was also increased(C666-1 vs C666/LMP-1).These results correlated miR-10b expression with LMP-1 expression and aggressive tumor phenotypes.
3.Establishment of miR-10b over-expression clone
C666/shLMP was infected by pLVTHM/pre-miR-10b lentiviouas vector and were sorted by BD FACSAriaⅡcell sorter.the selected GFP~+ clone was named C666/shLMP/miR-10b,and miR-10b level of which is about 45 folds that of C666/shLMP.
4.Over-expression of miR-10b had no effect on cell proliferation and cell cycle
To determine whether over-expression of miR-10b could increased cell proliferation,we compared the growth rates of miR-10b and vector-transduced cells. We found no statistically significant differences between them by MTT proliferation assays.Similarly,no change in cell cycle was observed by flow cytometry analysis in miR-10b over-expressed cell.
5.Over-expression of miR-10b increased cell mobility and invasion
Active cell motility is a rate-limiting step of rumor cell invasion.The mobility of NPC cells was measured using a well-established in vitro wound-healing assay.The miR-10b expressing C666/shLMP-l cells displayed a significantly higher cell invasion activity as compared to the control C666/shLMP-l cells.At 8 h after wound formation,C666/shLMP-l/miR-10b cells had fully migrated towards the open wound.This result suggested that over-expression of miR-10b by lentiviral vector led to a significant increase in the migration of the cells.These results indicated that over-expression of miR-10b increased cell invasion,supporting a role for miR-10b in the metastasis of EBV positive NPCs.
Invasion of basement membranes by tumor cell is best approximated in vitro by evaluating the transmigration of a biologically active matrix such as Matrigel. Therefore,we measured the ability of these cells to transmigrate through the Matrigel membrane.We found that the miR-10b transfected C666/shLMP-l cells displayed a significantly higher transmembrane migration activity as compared to the mock transfected c666/shLMP-l cells.
6.miR-10b over-expression promoted NPC metastasis in vivo
To determine whether miR-10b over-expression could shorten the lifespan of the nude mice,the nude mice were divided into two treatment groups(n=18/group) with the primary tumors being established by either direct injection of 2×10~6 C666/shLMP-l or C666/shLMP-l/miR-10b under the liver capsule.Nine mice from each group were randomly chosen for long-term survival study.The average survival period of the miR-10b over-expressed group was significantly shorter than the C666/shLMP-l inoculation control group(X~2=5.526,P=0.019).
Liver morphologies from these two groups of nude mice in NPC metastasis study were then evaluated 21 days after tumor inoculation.In the nude mice study(n =9),livers from 77.8%of C666/shLMP-l/miR-10b and 33.3%of C666/shLMP-l treatment mice displayed multiple metastasized tumors of different sizes on the surface of the liver.Most of the control C666/shLMP-treated mice showed normal lung morphology with no signs of tumors,whereas the C666/shLMP/miR-10b treated mice clearly showed multiple lung tumors(X~2=5.844,P=0.016).In addition, C666/shLMP-l/miR-10b treatment also significantly enhanced the development of ascites fluid accumulations(X~2=5.556,P=0.018).Moreover,in miR-10b over-expressed non-metastatic HNE-1-treated mice,no indication of lung and liver metastasis could be found in these mice.
7.Inhibition of miR-10b expression
To determine the effect of lowering the expression of miR-10b,we transfected antisense oligonucleotides anti-miR-10b into NPC cell lines.Effective miR-10b silencing,which was demonstrated by luciferase assay,strongly suppressed the invasive behavior of miR-10b-highly-expressed C666/shLMP-l/miR-10b cells in ECM invasion assays.These results suggested that over-expression of miR-10b increased cell invasion,supporting a role of miR-10b in the metastasis of EBV positive NPCs.
8.Twist induced miR-10b expression in metastatic NPC cells
Transcription factor Twist has been newly implicated in miR-10b related dissemination of breast carcinoma cells.We explored whether Twist could affect miR-10b expression in NPC cells.Twist siRNA was used to knockdown the expression of Twist.After 72 h of transfection,miR-10b was decreased by 87.4%in C666-1 cells.Similar result was also observed in C666/LMP-1 cells.While in miR-10b over-expressed C666/shLMP/miR-10b cells,the expression of miR-10b did not change with Twist RNAi.These results demonstrated that Twist was involved in miR-10b expression in metastatic NPC cells.Because Twist has been demonstrated to be induced by LMP-1,LMP-1 might modulate miR-10b expression via Twist.
CONCLUSIONS
1.Expression of miR-10b is up-regulated in the metastasis NPC tissues and cells
2.Elevated miR-10b expression promotes NPC aggressiveness,likely by increasing the metastatic cells activities.
3.The LMP-1/Twist signaling pathways would promote metastasis partly by up-regulating miR-10b.
4.miR-10b could prove to be useful prognostic markers,but further investigation is needed to confirm whether it would be a therapeutic target for NPC metastasis needs.
鼻咽癌是中国南方和东南亚地区最常见的恶性肿瘤之一,以明显的区域分布特征、与EB病毒的密切关系以及强烈的转移倾向显著区别于其他头颈部肿瘤。转移是鼻咽癌治疗失败的主要原因,尽管诊疗技术不断进步,但由于60-85%的患者确诊时已发生临床转移,鼻咽癌的5年生存率提升缓慢,探究鼻咽癌转移的分子机制,寻找新的治疗靶点,减少转移的发生,是我们面临的巨大挑战。
LMP-1是鼻咽癌最重要的外源性癌基因,也是鼻咽癌最重要的促转移基因。在鼻咽癌中,LMP-1发挥类似肿瘤坏死因子受体(tumor necrosis factorreceptor,TNFR)的功能,通过激活NF-κB,AP-1,ets-1,MAPKs,JAK/STAT,PI3K/Akt等不同信号通路,对E-cadherin,MMPs,c-Met,VEGF,EGFR,COX-2等多个转移相关基因的调控,影响着鼻咽癌转移的几乎每个环节。沉默LMP-1能够减少鼻咽癌细胞转移。
miRNA(microRNA)是广泛存在于生物体内的19-25nt的非编码RNA。转录完成后,miRNA与靶mRNA的3'UTR的互补序列结合,通过抑制mRNA的翻译或直接降解mRNA调控基因表达。人类基因组编码的miRNA多达1000个,参加了包括胚胎形成、发育、代谢和重大疾病在内的几乎所有生命活动。一些miRNA能够影响细胞凋亡、增殖与分化,扮演着癌基因的角色,被称为oncomirs。肿瘤转移是一个及其复杂的生物学过程,包括上皮细胞去极化,细胞间黏附改变,运动性增强,基底膜穿透,血管生成等一系列变化,涉及了大量信号通路激活和多个基因的表达变化。这一过程同样离不开miRNA对基因表达的精准调控。我们对oncomirs在肿瘤转移中的功能和作用机制还知之甚少,最新研究发现,miR-17-92家族,miR-200家族,miR-205,miR-29C,miR-21,Let-7等oncomirs在肿瘤转移过程中,也发挥着重要作用。
miR-10b促进乳腺癌转移的发现更是备受瞩目。Ma和同事等发现miR-10b仅在高转移的乳腺癌细胞中表达,阻断miR-10b能够使MDA-MB-231细胞侵袭能力下降90%以上,而不具备转移能力的SUM149细胞过表达miR-10b后,在裸鼠体内发生远处转移。临床资料也表明,发生转移的乳腺癌患者,miR-10b表达高于非转移组。这一研究还发现。乳腺癌细胞中miR-10b的表达,正是由转录因子Twist启动的。
Twist属于碱性的螺旋-环-螺旋(Helix-loop-helix)蛋白家族中高度保守的转录因子,可以调节胚胎发育过程中的组织重建,并赋予细胞迁移的能力,也是促进多种肿瘤的转移与侵袭的关键基因,在鼻咽癌的转移中也发挥重要作用。Horikawa T.等发现LMP-1能够诱导转录因子Twist,促进鼻咽癌转移和侵袭。据此我们提出研究假说:miR-10b受LMP-1/Twist信号通路调控,在鼻咽癌进展过程中发挥促进转移的功能。
为了证实这一假说,我们首先检测miR-10b在鼻咽癌组织和不同的鼻咽癌细胞中的表达水平,分析miR-10b表达与细胞转移能力的关系,并观察LMP-1和Twist的表达变化对miR-10b的影响。利用pre-miR-10b慢病毒表达载体,在鼻咽癌细胞中过表达miR-10b,或转染miR-10b抑制剂阻断miR-10b,分析其表达变化对细胞周期、增殖、凋亡,移动、侵袭和体内转移能力的影响,以初步探讨鼻咽癌转移中miR-10b的功能和作用机制。
方法
1.鼻咽癌组织和细胞中miR-10b的检测
收集鼻咽癌组织标本45例(其中转移26例,未转移19例),慢性鼻咽炎组织16例作对照。所有新鲜鼻咽癌组织标本分为2份,一份经10%甲醛固定,包埋、切片免疫组化染色检测LMP-1表达情况。另一份标本液氮冻存,制作冰冻切片,用原位杂交检测miR-10b表达。
2.鼻咽癌细胞中miR-10b的检测
长期携带EB病毒基因的人类低分化鼻咽癌细胞株C666-1,高分化鼻咽癌细胞CNE1,低分化鼻咽癌细胞CNE2、HONE1、HNE-1细胞,以及SUNE-1细胞的高转移亚株5-8F和不转移亚株6-10B,永生化鼻咽上皮细胞株NP69细胞,8株细胞分别采用荧光定量PCR和原位杂交检测miR-10b表达。
3.沉默及过表达LMP-1表达
合成能够转录出LMP-1干扰模板的双链DNA片段,插入shRNA的表达载体pAVU6+27,获得pAVU6+27/shLMP-1质粒。以空pAVU6+27载体为对照,分别转染C666-1细胞后,通过G418筛选和巢式PCR检测获得LMP-1阴性的亚株C666/shLMP-1。含有N-LMP-1 cDNA的质粒pCR~(?)Ⅱ-TOPO/LMP-1,用Lipofectamine-2000转染细胞,48h后RT-PCR检测LMP-1mRNA水平。
4.miRNA提取和Real time PCR检测
按照mirVana miRNA isolation kit说明书进行操作,富集分子量小于200nt的小分子RNA。按照TaqMan MicRNA RT Kit说明书操作,采用TaqMan MicRNAAssays中的逆转录引物,以cDNA为模板,进行逆转录反应,U6 RNA作为内对照。
5.制备慢病毒载体稳定过表达miR-10b
根据miR-10b前体pre-mir-10b(MI000681)的序列,在5'端和3'端引入Mlu I和Cla I的酶切位点,合成全长102bp的pre-mir-10b转录模板,连接转化,挑取重组阳性克隆,经PCR、Mlu I和Xba I双酶切鉴定,鉴定正确的质粒送DNA测序。采用脂质体法将慢病毒包装系统中3种质粒按比例共转染293FT细胞,收集上清液,离心过滤后,得到慢病毒悬液。病毒悬液连续稀释法计算病毒滴度。慢病毒pLVTHM/miR-10b和空病毒载体pLVTHM感染鼻咽癌细胞。采用流式细胞仪分选出GFP(+)细胞扩大培养。Real PCR检测miR-10b表达水平。
6.siRNA及anti-miR转染
细胞融合度达到40%左右,更换无血清培养基。将anti-miR充分溶解于不含有血清的转染专用培养基Opti-MEM中。阳离子脂质体Lipofectamine2000用Opti-MEM稀释后,与siRNA或anti-miR溶液混合,轻轻振荡后,置于室温20min,加入细胞培养板。37℃孵育4h后,加入30%胎牛血清FBS,继续培养24h,48h,72h后,荧光定量PCR方法检测miR-10b表达水平的变化。
7.miR-10b报告基因质粒的构建和双荧光报告实验
以基因组DNA为模板扩增出将含有miR-10b种子区互补序列的片段作为miR-10b结合位点,将片段插入psiCHECK-2质粒海肾荧光素Renilla下游XhoI和NotI位点之间。用Lipofectamine-2000将psiCHECK-2/miR-10b-binding质粒anti-miR,同时转染细胞,24h后按照Dual-Luciferase Reporter Assay System的说明进行样品的Luciferase活性检测,根据荧光素比值:Luciferaseactivity(Renilla/Firefly)变化分析miRNA inhibitor抑制miR-10b的效果。
8.细胞增殖实验
细胞以每孔1×10~3个细胞接种于96孔培养板中,每孔体积200μl,每组5孔,同时设空白对照(仅加培养基),分别培养1-5天。每孔加入5mg/ml的MTT20μl,37℃继续培养4h后终止培养,加入DMSO,酶标仪上570nm测定各孔吸光度值(OD值),以相对应OD比值表示细胞增殖能力大小。各组取5孔平均值,绘制增殖曲线。
9.流式细胞实验
C666/shLMP-1和C666/shLMP-1/miR-10b细胞,0.25%胰酶消化,PBS洗涤3次后调整细胞浓度为5×10~5/ml的细胞悬液,离心后,按照BD公司荧光染色试剂盒说明书,进行荧光标记,流式细胞仪进行细胞周期检测。
10.划痕实验
在6孔细胞培养板中,每组细胞作3个复孔。待细胞密度达到90%左右,吸干培养基,用同样大小的加样枪头,垂直在培养孔中部轻轻划过,制造多条相互平行的划痕,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。每个孔随即选取9个有划痕穿过的视野,在不同时间点,对越过划痕边缘向空白出移动生长的细胞进行计数,独立实验重复3次。
11.侵袭实验
采用美国BD公司的肿瘤细胞基底膜穿透实验系统Biocoat MatrigelInvasion Chambers,按照说明书操作,C666-1细胞胰酶消化后,以无血清培养基种植与小室内5.0×10~3/孔,24孔细胞培养板,每孔中预先加入含有10%胎牛血清的培养基0.5ml,然后将培养杯置入孔中培养。不同时间后取出小室,棉签拭去基底膜正面的细胞,将部分小室基底膜沿杯底剪下翻转,用甲苯胺蓝对能够穿透基底膜的细胞进行染色,显微镜下计数。
12.体内转移实验
建立肝被膜下种植肺转移动物模型。4～6周龄BALB/c nu/nu裸鼠48只,鼻咽癌细胞调制成2×10~6/ml细胞悬液,用1ml注射器于裸鼠肝包膜下缓慢注入0.1ml细胞悬液,全层缝合腹壁。其中36只裸鼠随机分成2组,分别接种C666/shLMP-1和C666/shLMP-1/miR-10b细胞,每组18只,其中每组随机取9只用于观察抗肿瘤效果,其余9只用于生存期观察。另外12只裸鼠分成2组,每组6只,分别接种过表达miR-10b的HNE1和HNE1/miR-10b细胞。
用于生存期观察的18只裸鼠,观察活体动物体内荧光分布,能够了解肿瘤细胞在体内扩散的情况。并继续饲养至自然死亡,记录生存期。用于观察抗肿瘤效果的18只裸鼠第三周末断髓处死,立即取荷瘤裸鼠肝脏及肺脏组织,PBC冲洗2次,用动物整体成像系统下观察带荧光的肿瘤组织分布情况,并拍照。荧光照相后的肝脏及肺脏组织,用10%甲醛固定,常规石蜡包埋HE染色,显微镜下观察肿瘤细胞在肝脏内成瘤情况,以及肺、腹膜等器官肿瘤转移情况。
13.统计学处理
采用SPSS 13.0软件对数据进行分析,均以P＜0.05表示差异有显著性意义。免疫组化和原位杂交结果采用X~2检验以及两独立样本非参数秩和检验Mann-Whitney U法进行统计学分析。荧光定量PCR和细胞周期实验,样本均数比较采用,两独立样本的t检验(independent-samples T test)或单因素方差分析(One-way ANOVA),多重比较采用SNK法(Student-Neuman-Keuls)。方差不齐者采用基于方差不齐的多重比较方法Dunnett's T3法进行。MTT实验、体外侵袭实验、划痕实验采用析因设计的方差分析。动物生存时间分析采用Kaplan-Meier法,肺转移、肝内转移和腹水情况的比较采用X~2检验。
结果
1.miR-10b的表达与鼻咽癌转移以及LMP-1的关系
①miR-10b表达与鼻咽癌发病
在全部45例鼻咽癌组织中,miR-10b表达率为80%(36/45),主要定位于细胞核,鼻咽慢性炎症组织中表达率为56.25%(9/16),二者无显著差异(X~2=3.441,P=0.097)。
②miR-10b表达与鼻咽癌转移
将45例鼻咽癌组织按照确诊时有无淋巴结或远处转移分为转移组(26例)和未转移组(19例),鼻咽癌转移组miR-10b的表达较强(Z=-3.243,P=0.001)。
③miR-10b表达与LMP-1
45例鼻咽癌组织中,LMP-1阳性率为37.78%(17/45),LMP-1(+)组miR-10b的表达较强(Z=-2.013,P=0.044)。在LMP-1阳性组的中转移者miR-10b表达高于未转移者(Z=-2.504,P=0.012),而LMP-1阴性组中转移和未转移者的miR-10b表达没有统计学差异(Z=-1.857,P=0.063)。
2.miR-10b表达水平与细胞转移能力和LMP-1表达的相关性
①.miR-10b在转移的鼻咽癌细胞中高表达
7种鼻咽癌细胞株与永生化人鼻咽上皮细胞NP69相比较,miR-10b在8株细胞中的表达存在显著差异(F=96.395,P=0.000),具备转移能力的C666-1和5-8F细胞表达水平高于其他5种不转移的鼻咽癌细胞。原位杂交显示C666-1和5-8F细胞miR-10b表达为阳性,6-10B和HONE-1细胞为阴性,与荧光定量PCR检测结果一致。
②miR-10b表达水平与LMP-1一致
RT-PCR结果显示,转染pCR~(?)Ⅱ-TOPO/LMP-1 C666/LMP-1细胞LMP-1mRNA较C666-1明显提高。转染C666/shLMP-1细胞中LMP-1稳定沉默。LMP-1沉默的C666/shLMP-1细胞miR-10b表达明显下降,而过表达LMP-1的C666/LMP-1的miR-10b表达提高明显提高,LMP-1含量与miR-10b表达呈一致变化。
3.miR-10b过表达鼻咽癌细胞株的建立
由于LMP-1是鼻咽癌主要的外源性癌基因,为了排除LMP-1基因表达变化对细胞的影响,我们利用LMP-1沉默的C666/shLMP-1细胞进行miR-10b的功能研究。首先利用慢病毒载体系统在中过表达miR-10b,制备了miR-10b稳定过表达的慢病毒载体pLVTHM/pre-miR-10b,感染C666/shLMP-1细胞48-72h后,阳性C666-1细胞发出绿色荧光,经流式细胞仪分选出带荧光的细胞经反复传代后,miR-10b水平比感染pLVTHM空载体C666/shLMP-1最多提高45倍,成为稳定过表达的亚克隆细胞C666/shLMP/miR-10b。
4.miR-10b过表达对C666-1细胞增殖和细胞周期的影响
MTT结果和流式细胞实验表明,C666/shLMP-1和C666/shLMP-1/miR-10b细胞的体外增殖能力及细胞周期均无显著性差异。
5.miR-10b过表达对鼻咽癌细胞体外侵袭和移动能力的影响
基底膜穿透实验和划痕实验表明过表达miR-10b的C666/shLMP-1/miR-10b细胞侵袭和移动能力较对照组增强。miR-10b过表达提高鼻咽癌细胞体外侵袭和移动能力
6.miR-10b过表达对鼻咽癌细胞体内侵袭、移动和生存时间的影响
接种C666/shLMP-1和C666/shLMP-1/miR-10b鼻咽癌细胞的裸鼠,3周后肝脏成瘤率达100%,其中C666/shLMP-1/miR-10b组动物全部出现种植瘤肝内广泛转移腹水出现时间较早(X~2=5.556,P=0.018),肺转移发生率均高于C666/shLMP-1组(X~2=5.844,P=0.016),生存时间较C666/shLMP-1组缩短(X~2=5.526,P=0.019)。而接种HNE1/miR-10b和HNE1组也可在局部成瘤,但均未发现肝内播散和肺转移。说明miR-10b过表达提高鼻咽癌细胞体内侵袭和移动能力。
7.miR-10b抑制物阻断miR-10b表达对鼻咽癌细胞转移能力的影响
荧光报告检测结果表明,miR-10b抑制物(anti-miR-10b)与转染后,只有C666/shLMP/miR-10b侵袭能力增强(t=-5.509,P=0.000),而C666/shLMP-1和C666/control侵袭能力无明显变化(图-6)。说明anti-miR-10b能够逆转由于miR-10b过表达造成的侵袭能力提高,而对于miR-10b表达很低的C666/shLMP-1没有影响,同样对于LMP-1阳性C666/control,单独抑制miR-10b也不足以改变其转移能力。
8.LMP-1通过Twist基因诱导miR-10b表达
利用RNA干扰抑制Twist的表达,分析LMP-1是否通过Twist启动miR-10b转录。Twist siRNA转染C666-1细胞72小时后,Twist mRNA含量下降超过87.4%。LMP-1(+)的C666/control(t=7.060,P=0.002)和C666/LMP细胞(t=7.564,P=0.002)中miR-10b均下降,而LMP-1(-)的C666/shLMP(t=-0.579,P=0.594)和C666/shLMP/miR-10b细胞(t=-0.090,P=0.932)miR-10b的含量没有明显变化。说明LMP-1诱导miR-10b的表达,很可能是通过转录因子Twist实现的。
结论:
1.miR-10b在转移的鼻咽癌组织和细胞中高表达,表达水平与EB病毒LMP-1基因相关。
2.miR-10b的过表达能够促进鼻咽癌细胞体外和体内转移能力,但对细胞增殖、凋亡及细胞周期没有影响。
3.LMP-1基因通过转录因子Twist调控miR-10b表达,鼻咽癌中可能存在LMP-1/Twist/miR-10b促转移信号传导通路。
4.miR-10b在鼻咽癌转移分子机制研究中有重要意义,可以作为判断预后指导治疗的生物学标记,但能否作为基因治疗靶点不能定论。
BACKGROUND AND OBJECTIVE
The incidence of nasopharyngeal carcinoma(NPC)is most prevalent in Southeast Asia,particularly in Southern China.NPC is distinctive among the head and neck carcinomas for its marked tendency to metastasis and invasion.It has been shown that at the time of diagnosis,60-85%of NPC patients already have clinically detectable aggressive metastasis in the regional lymph nodes,in distant organs such as the lungs and in bone.So far there is still no effective treatment for NPC at the stage of metastasis.As a result,prognosis of NPC is poor and the 5-year survival rate is less than 50%.Meanwhile,the mechanisms that control NPC metastasis remain poorly understood.
Epstein-Barr virus(EBV)encoding latent membrane protein-1(LMP-1)is a primary oncoprotein in NPC.It has been suggested to contribute to the highly metastatic nature of NPC.LMP-1 functions as a constitutively active tumor necrosis factor receptor(TNFR)and contributes to multiple aspects of NPC,mainly through activating a number of signaling pathways,including NF-kB,AP-1,ets-1,MAPKs, and JAK/STAT,and controlling the expression of metastasis related gene,such as E-cadherin,matrix metalloproteinase(MMPs),c-Met,VEGF,EGFR and COX-2. Knock down of LMP-1 reduces the metastasis abilities of NPC cells.
MicroRNAs(miRNAs)are 19-25 nt regulatory RNAs that participate in the regulation of various biological functions in numerous eukaryotic lineages,including plants,insects,vertebrate,and mammals.miRNAs can have pleiotropic effects on cell proliferation,apoptosis,and cell differentiation.They are generally believed to act by binding to imperfectly complementary sequences in the 3' untranslated region of the target genes,resulting in decreased translation or degradation of the target transcript. Bioinformatic and experimental evidence indicate that each miRNA may target several dozen to several hundred gene transcripts,thus allowing for coordinated and combinatorial regulation of multiple genes by miRNAs.More than 1000 miRNAs have been identified in humans so far,and many of the genes in the human genome are predicted to be subject to miRNA regulation.The expression of many miRNAs is usually specific to a tissue or developmental stage,and the miRNA expression pattern is altered during the development of many diseases miRNAs are integral to gene regulation,apoptosis,hematopoietic development,and the maintenance of cell differentiation.There are a growing number of examples of particular miRNAs whose expression is increased in cancer(i.e.,mir-21,the mir-17-92b cluster,mir-155,and mir-372/373)whereas the expression of other miRNAs is decreased in tumor versus normal cells.miRNAs that have been experimentally shown to directly induce tumor have been termed“oncomirs”,with the first example being the mir-17-92b cluster. Metastasis is a central problem in cancer,yet the underlying mechanisms remain poorly understood.The involvement of miRNAs in the development of metastases has come under intense scrutiny in recent years.Members of miR-17-92 family, miR-200 family,miR-205,miR-29C,miR-21 and Let-7 were proved to be involved in cancer metastasis.
Ma and coworkers from Robert Weinberg's group found that miR-10b initiated breast cancer invasion and metastasis.They also found that Twist,a metastasis promoting transcription factor,could induce miR-10b expression and demonstrated that miR-10b was an essential element in the Twist-induced metastasis program. Others' work showed that induction of Twist by an Epstein-Barr virus (EBV)-encoded human viral oncoprotein latent membrane protein-1(LMP-1)directly contributes to the metastatic nature of NPC.
Therefore,here we explored whether miR-10b could be induced by EBV oncogene LMP-1,and its association with Twist as well.We took advantage of an established pre-miR-10b lentiviral vector and RNA interference technology to determine the relationship between EBV LMP-1 and miR-10b,and how induction of miR-10b by LMP-1 might contribute to the highly metastatic feature of NPC.We first showed that LMP-1 could activate miR-10b,and possibly via Twist,and miR-10b stimulated cell migration and invasion in vitro.We showed next that in cell culture,blocking of miR-10b could directly inhibit the invasion behavior of miR-10b over-expressed NPC,but we did not find such effect in control cells without miR-10b over-expression.We established a NPC metastasis model in mice by transplanting miR-10b over-expressed EBV-positive NPC cells,C666/shLMP-l/miR-10b,in the livers of nude mice and observed lung metastasis.Metastasis was detected in lung and liver,while no metastasis was found when miR-10b over-expressed EBV-negative non-metastatic NPC cells were transplanted into mice.
This report suggested that miR-10b facilitates the metastasis of EBV related NPC cells,but miR-10b gene might temporarily not be considered as a therapeutic target for NPC metastasis.
MATERIALS AND METHODS
1.Expression characteristics of miR-10b in NPC tissues
This study was conducted on a total of 45 NPC samples(26 metastatic NPC and 19 non-metastatic)and 16 chronic nasopharyngitis samples.All the samples was divided into two.One part of the specimen was made into frozen-section for miR-10b in situ hybridization,the other for paraffin section and immunohistochemistry analysis of LMP-1.
2.Expression characteristics of miR-10b in NPC cells
Quantitive real-time PCR was used to detect the expression of miR-10b in 7 NPC-derived cell lines(HNE1,HONE1,CNE1,CNE2,5-8F,6-10B,C666-1,)and immortalized nasopharyngeal epithelial cells NP69.
3.miRNA isolation and Real-time PCR
Cells were collected and miRNAs were prepared using MirVana miRNA isolation kit and RT reactions were performed using TaqMan MicRNA RT Kit(ABI, USA).Real-time PCR was performed with TaqMan Universal Master Mix(ABI) using Mx3000P real-time PCR instrument(Stratagene,USA).U6 RNA was used as an endogenous control for miRNA detection.The data were analyzed using the standard curve analysis.
4.Knockdown and overexpression of LMP-1
To stably knockdown of LMP-1,C666-1 cells were transfected with shRNA vector pAVU6+27/shLMP-l targeting LMP-1(CATAGGCCTTGCTCTCCTTCTCC) using the lipofectamine 2000 reagent(Invitrogen)following the protocol provided by the manufacturer.Forty-eight hours later,cells were harvested and plated on 10 cm tissue culture plate,and clones stably expressing shRNA(C666/shLMP-l)were selected using 400μg/ml G418.To overexpression of LMP-1,C666-1 cells were transfected with N-LMP-1 cDNA containing vector pCR~?Ⅱ-TOPO/LMP-l(A kindly gift of professor Marie C.Lin from the Department of Chemistry,The University of Hong Kong,China)using the lipofectamine 2000 reagent following the protocol provided by the manufacturer.Forty-eight hours later,cells were harvested and LMP-1 mRNA was detected by RT-PCR.
5.Lentiviral vector mediated overexpression of miR-10b
To generate a miR-10b expression vector,miR-10b precursor pre-miR-10b was amplified by PCR and cloned into pLVTHM vector(GFP-encoding)and transfected with psPAX2 and pMD2.G into 293FT using lipofectamine 2000 to package lentivirus pLVTHM/miR-10b.Cells were infected.After 3 days,cells were sorted by BD FACSAriaⅡcell sorter(Becton-Dickinson Labware)to selectively amplify GFP-positive cells.miR-10b mRNA expression after infection of lentiviral vector was revealed by real-time PCR.
6.Oligonucleotide transfection
Twist siRNA(AAGCTGAGCAAGATTCAGACC),its control siRNA and anti-miR-10b miRNA inhibitor were transfected using Lipofectamine 2000 (Invitrogen)following the protocol provided by the manufacturer.
7.MTT assay
MTT assay was performed to assess the effect of miR-10b on cell proliferation. Cells(1×10~3cells/well)were plated in a 96-well plate and maintained in RPMI-1640 supplemented with 10%FBS.At 24,48,72,96 and 120h after seeding,culture medium was removed,cells were treated with 20μl sterile MTT dye(5mg/ml,Sigma, USA)for 4h at 37℃,and then 200μl of DMSO was added and thoroughly mixed for 30min.Spectrometric absorbance at wavelength of 570nm was measured on a microplate reader.
8.Flow cytometry
Cell cycle profiles are analyzed by flow cytometry.Cells(5×l0~5cells/well) were plated in a 6-well plate and were transfected.After 48 h,cells were collected and washed three times in PBS.After resuspension,solution A,B and C from fluorescence labeling kit were added to label DNA.Sample analysis was performed by flow cytometry(BD FACSAriaⅡcell sorter,Becton-Dickinson Labware).The cell cycle phase distribution was calculated from the resultant DNA histogram using Multicycle AV software(Phoenix Flow System,San Diego,CA,USA).
9.Wound healing assays
In vitro wound healing assay was carried out to determine the ability of cells to form membrane protrusion and cell migration.Equal numbers of cells(1×10~5)were seeded into six-well cell culture plates.When the confluence reached 90%,a single wound was created in the center of the 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 at different time points.A total of nine areas were selected randomly in each well under a 40×objective and cells in three wells of each group were quantified in each experiment.
10.In vitro Matrigel invasion assay
Cell invasiveness was determined in vitro by the ability of the cell to transmigrate a layer of extracellular matrix(ECM)in Matrigel in Biocoat Matrigel Invasion Chambers(Becton-Dickinson Labware,Bedford,MA).Cells were plated at a density of 5.0×10~3 cells/insert,respectively.Medium with 10%FBS was added to the lower chamber as a chemoattractant.After 24 h incubation,cells on the upper surface of the membrane were removed.Invasive cells,which were able to breach the 8μm pores and grow on the lower surface,were fixed in 100%methanol,stained with 1%Toluidine(Sigma),and counted under an inverted microscope(Leica,German). The cells were counted in three random optical fields(200×magnification)from duplicate experiments.
11.Construction of miR-10b reporter plasmids and luciferase assays
Complement fragment of miR-10b(ACAAATTCGGTTCTACAGGGTA)was cloned into psiCHECKTM-2 Vector(Promega,Madison,WI)downstream of the Renilla luciferase cDNA.Luciferase activity was assayed using Dual-Luciferase Reporter Assay System(Promega,USA)following the protocol provided by the manufacturer.
12.In vivo metastasis experiment
Nude mice(BALB/c nu/nu,4-6 weeks old,18-24 g in weight)were purchased from Charles River Labs(Wilmington,WA)and were maintained under pathogen-free conditions(specific pathogen-free level).Mice were divided into two treatment groups(n=18/group),primary tumors were established by direct injection of 2×10~6 C666/shLMP or C666/shLMP-l/miR-10b into the liver as previously described.Nine mice from each group were randomly chosen for long-term survival study on day 10.The remaining nine mice were sacrificed on day 21,and tissues were collected for pathologic analysis.GFP fluorescence images were observed under an in vivo fluorescence instrument.
13.Statistical analysis
All analyses were carried out using the SPSS 13.0 software package.Expression of miR-10b in relation to clinical data was analyzed with the Mann-Whitney U test. Comparison of mean was analyzed by one-way ANOVA and Student-Neuman-Keuls method for multiple comparison.Dunnett's T3 method was employed for heterogeneity of variance.Survival curves were plotted by the Kaplan-Meier method and compared by the log-rank test.The X~2 test for proportion was used to analyze the in vivo metastasis characteristics of different groups.Factorial analysis was utilized to analyze the results MTT,invasion and wound healing assays.The differences in wound migration and invasion indices between C666-1 cell clones were analyzed by the independented t test.A P value less than 0.05 was considered statistically significant.
RESULTS
1.miR-10b was over-expressed in metastatic NPC samples and was correlate with LMP-1 expression
In situ hybridization results showed that,although there is no difference of miR-10b expression between NPC(36/45)and chronic nasopharyngitis(9/16) samples(X~2=3.441,P=0.097),miR-10b was positively associated with NPC metastasis(Z=-3.243,P=0.001).Furthermore,expression of miR-10b was correlated with LMP-1 expressing(Z=-2.013,P=0.044).In LMP-1(+)samples,metastatic NPC present higher miR-10b rate than non-metastatic ones(Z=-2.504,P=0.012). However,the same thing didn't happened in LMP-l(-)cases(Z=-1.857,P=0.063).
2.miR-10b was over-expressed in metastatic NPC cells and its expression was correlate with LMP-1 expression
Using real-time PCR,miR-10b expression in 7 NPC-derived cell lines(HNE1, HONE1,CNE1,CNE2,5-8F,6-10B,C666-1,)and immortalized nasopharyngeal epithelial cells NP69 were examined.The results showed that the expression of miR-10b in 8 cell lines was significantly different from each other(F=96.395, P=0.000).And expression of miR-10b was significantly increased in the metastatic C666-1 and 5-8F cell comparing with the non-metastatic NPC cells.
In situ hybridization showed negative miR-10b expression in 6-10B,HONE and strongly positive in C666-1 and 5-8F which supported the real-time PCR results.
To test whether miR-10b was regulated by LMP-1,LMP-1 expression in C666-1 cells was down-regulated by RNAi and isolation of clones C666/shLMP stably expressing shRNA targeting LMP-1 or up-regulated by transfecting the expression plasmid pCR?Ⅱ-TOPO/LMP-l.Real-time PCR showed that when LMP-1 expression was down-regulated,the expression of miR-10b was correspondingly decreased(C666-1 vs C666/shLMP-l);when LMP-1 expression was up-regulated,the expression of miR-10b was also increased(C666-1 vs C666/LMP-1).These results correlated miR-10b expression with LMP-1 expression and aggressive tumor phenotypes.
3.Establishment of miR-10b over-expression clone
C666/shLMP was infected by pLVTHM/pre-miR-10b lentiviouas vector and were sorted by BD FACSAriaⅡcell sorter.the selected GFP~+ clone was named C666/shLMP/miR-10b,and miR-10b level of which is about 45 folds that of C666/shLMP.
4.Over-expression of miR-10b had no effect on cell proliferation and cell cycle
To determine whether over-expression of miR-10b could increased cell proliferation,we compared the growth rates of miR-10b and vector-transduced cells. We found no statistically significant differences between them by MTT proliferation assays.Similarly,no change in cell cycle was observed by flow cytometry analysis in miR-10b over-expressed cell.
5.Over-expression of miR-10b increased cell mobility and invasion
Active cell motility is a rate-limiting step of rumor cell invasion.The mobility of NPC cells was measured using a well-established in vitro wound-healing assay.The miR-10b expressing C666/shLMP-l cells displayed a significantly higher cell invasion activity as compared to the control C666/shLMP-l cells.At 8 h after wound formation,C666/shLMP-l/miR-10b cells had fully migrated towards the open wound.This result suggested that over-expression of miR-10b by lentiviral vector led to a significant increase in the migration of the cells.These results indicated that over-expression of miR-10b increased cell invasion,supporting a role for miR-10b in the metastasis of EBV positive NPCs.
Invasion of basement membranes by tumor cell is best approximated in vitro by evaluating the transmigration of a biologically active matrix such as Matrigel. Therefore,we measured the ability of these cells to transmigrate through the Matrigel membrane.We found that the miR-10b transfected C666/shLMP-l cells displayed a significantly higher transmembrane migration activity as compared to the mock transfected c666/shLMP-l cells.
6.miR-10b over-expression promoted NPC metastasis in vivo
To determine whether miR-10b over-expression could shorten the lifespan of the nude mice,the nude mice were divided into two treatment groups(n=18/group) with the primary tumors being established by either direct injection of 2×10~6 C666/shLMP-l or C666/shLMP-l/miR-10b under the liver capsule.Nine mice from each group were randomly chosen for long-term survival study.The average survival period of the miR-10b over-expressed group was significantly shorter than the C666/shLMP-l inoculation control group(X~2=5.526,P=0.019).
Liver morphologies from these two groups of nude mice in NPC metastasis study were then evaluated 21 days after tumor inoculation.In the nude mice study(n =9),livers from 77.8%of C666/shLMP-l/miR-10b and 33.3%of C666/shLMP-l treatment mice displayed multiple metastasized tumors of different sizes on the surface of the liver.Most of the control C666/shLMP-treated mice showed normal lung morphology with no signs of tumors,whereas the C666/shLMP/miR-10b treated mice clearly showed multiple lung tumors(X~2=5.844,P=0.016).In addition, C666/shLMP-l/miR-10b treatment also significantly enhanced the development of ascites fluid accumulations(X~2=5.556,P=0.018).Moreover,in miR-10b over-expressed non-metastatic HNE-1-treated mice,no indication of lung and liver metastasis could be found in these mice.
7.Inhibition of miR-10b expression
To determine the effect of lowering the expression of miR-10b,we transfected antisense oligonucleotides anti-miR-10b into NPC cell lines.Effective miR-10b silencing,which was demonstrated by luciferase assay,strongly suppressed the invasive behavior of miR-10b-highly-expressed C666/shLMP-l/miR-10b cells in ECM invasion assays.These results suggested that over-expression of miR-10b increased cell invasion,supporting a role of miR-10b in the metastasis of EBV positive NPCs.
8.Twist induced miR-10b expression in metastatic NPC cells
Transcription factor Twist has been newly implicated in miR-10b related dissemination of breast carcinoma cells.We explored whether Twist could affect miR-10b expression in NPC cells.Twist siRNA was used to knockdown the expression of Twist.After 72 h of transfection,miR-10b was decreased by 87.4%in C666-1 cells.Similar result was also observed in C666/LMP-1 cells.While in miR-10b over-expressed C666/shLMP/miR-10b cells,the expression of miR-10b did not change with Twist RNAi.These results demonstrated that Twist was involved in miR-10b expression in metastatic NPC cells.Because Twist has been demonstrated to be induced by LMP-1,LMP-1 might modulate miR-10b expression via Twist.
CONCLUSIONS
1.Expression of miR-10b is up-regulated in the metastasis NPC tissues and cells
2.Elevated miR-10b expression promotes NPC aggressiveness,likely by increasing the metastatic cells activities.
3.The LMP-1/Twist signaling pathways would promote metastasis partly by up-regulating miR-10b.
4.miR-10b could prove to be useful prognostic markers,but further investigation is needed to confirm whether it would be a therapeutic target for NPC metastasis needs.
引文
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16.Gregory PA,Bert AQ Paterson EL,et al.The miR-200 family and miR-205 regulate epithelial to mesenchymal transition by targeting ZEB1 and SIP1.Nat Cell Biol,2008, 10(5):593-601.
17.Connolly E,Melegari M,Landgraf P,et al.Elevated expression of the miR-17-92 polycistron and miR-21 in hepadnavirus-associated hepatocellular carcinoma contributes to the malignant phenotype.Am J Pathol.2008 Sep;173(3):856-64.
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22.Lee YB,Bantounas I,Lee DY,et al.Twist-1 regulates the miR-199a/214 cluster during development.Nucleic Acids Res.2009 Jan;37(1):123-8.
23.Yang J,Mani SA,Donaher JL,et al.Twist,a master regulator of morphogenesis,plays an essential role in tumor metastasis.Cell.2004 Jun 25;117(7):927-39.
24.Zhang Z,Xie D,Li X,et al.Significance of TWIST expression and its association with E-cadherin in bladder cancer.Hum Pathol.2007 Apr;38(4):598-606.Epub 2007 Jan 29.
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22.Lee YB,Bantounas I,Lee DY,et al.Twist-1 regulates the miR-199a/214 cluster during development.Nucleic Acids Res.2009 Jan;37(1):123-8.
23.Yang J,Mani SA,Donaher JL,et al.Twist,a master regulator of morphogenesis,plays an essential role in tumor metastasis.Cell.2004 Jun 25;117(7):927-39.
24.Zhang Z,Xie D,Li X,et al.Significance of TWIST expression and its association with E-cadherin in bladder cancer.Hum Pathol.2007 Apr;38(4):598-606.Epub 2007 Jan 29.
25.Song LB,Liao WT,Mai HQ,et al.The clinical significance of twist expression in nasopharyngeal carcinoma.Cancer Lett.2006 Oct 28;242(2):258-65.
26.Tsao SW,Tramoutanis G,Dawson CW,et al.The significance of LMPl expression in nasopharyngeal carcinoma,Semin.Cancer Biol.12(2002) 473-487.
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2.Woltering JM,Durston AJ.MiR-10 represses HoxB1a and HoxB3a in zebrafish[J].PLoS ONE.2008 Jan 2;3(1):e1396
3.Pearson,J.C.,D.Lemons,and W.McGinnis,Modulating Hox gene functions during animal body patterning[J].Nat Rev Genet,2005.6(12):893-904.
4.Veerla S,Lindgren D,Kvist A,et al.MiRNA expression in urothelial carcinomas:important roles of miR-10a,miR-222,miR-125b,miR-7 and miR-452 for tumor stage and metastasis,and frequent homozygous losses of miR-31[J].Int J Cancer.2009 May 1;124(9):2236-42
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