摘要
背景和目的
鼻咽癌(nasopharyngeal carcinoma, NPC)在我国南部,尤其在广东省高发,其发病率可达25/10万。2008年全世界新增鼻咽癌病例84400人,死于鼻咽癌患者51600人,其中大部分在我国南方。鼻咽癌的治疗方案是以放射治疗为主的综合治疗,早期病例治疗效果较好。远处转移鼻咽癌预后不良,中位生存时间仅12~20个月,化疗是转移性鼻咽癌的主要治疗方法,但治疗效果差,反应率仅有50~80%,仅能延长鼻咽癌患者中位生存期5-11个月。鼻咽癌总的远处转移率可达25~30%,5年内死亡的首诊未转移病例有2/3归因于远处转移。因此,明确鼻咽癌的转移机制并由此探索新的治疗方法对延长鼻咽癌患者的生存时间具有重要意义。
血管生成是指从已有的毛细血管或毛细血管后静脉发展而形成新的血管,是恶性肿瘤的主要特征之一。与正常人体组织一样,恶性肿瘤也需要氧气和营养成分进行新陈代谢,同时还需排出二氧化碳和有害代谢产物,所以血管生成对于肿瘤至关重要。肿瘤发展至1~2mm3,就需要新生血管的滋养才能维持生长,否则就处于静止状态。新生血管基底膜的通透性较高,肿瘤细胞可直接穿透血管进入血流发生转移。因此,血管生成是肿瘤生长和转移的基础,抑制血管生成即可有效控制肿瘤,增强化疗药物的疗效,延长患者的生存时间,是肿瘤分子靶向治疗的重要方法。目前,已运用于临床的抗血管生成分子靶向药物如贝伐单抗(bevacizumab)和索拉非尼(sorafenib)等在卵巢癌、乳腺癌、肾癌和肝癌等恶性肿瘤的晚期治疗中已取得较好的效果,有效延长了患者的生存时间。研究者使用贝伐单抗对1528例卵巢癌患者进行Ⅲ期临床试验,结果显示治疗进行至第42月时,贝伐单抗可延长无进展生存期近4个月。
Zeste基因增强子同源物2(enhancer of zeste ho mo log2, EZH2)是poly comb group (PcG)基因家族的重要成员,与EED (embryonic ectoderm development)、 SUZ12(suppressor of zeste12)和RbAp46/48共同组成PRC2,将靶基因组蛋白H3K27甲基化,导致靶基因沉默,是分子靶向治疗的潜在靶标。研究显示,与局限性前列腺癌和良性前列腺肿瘤相比,EZH2在转移性前列腺癌中表达升高并可促进前列腺癌的生长和转移;EZH2还与前列腺癌的预后有关,是前列腺癌的癌基因。EZH2在乳腺癌中同样表达上调并可增强乳腺癌细胞的侵袭能力;EZH2还可通过抑制乳腺癌细胞雌激素受体a (estrogen receptor a, ERa)表达降低乳腺癌的治疗敏感性。同时,EZH2在白血病、卵巢癌、肝癌等恶性肿瘤中也存在表达上调情况,并与这些肿瘤的发生、生长、转移和预后密切相关。最近研究还发现EZH2可能通过促进血管生成影响肿瘤的生长和转移。
EZH2对肿瘤血管生成的作用已见诸多报道。研究者发现EZH2在卵巢癌内皮细胞中表达升高,并可通过抑制血管抑制蛋白1(vasohibin1, VASH1)表达促进卵巢癌血管生成。而在EZH2的上游,血管内皮生长因子(vascular endothelial growth factor, VEGF)通过转录因子E2F上调了EZH2的表达。EZH2在恶性胶质瘤内皮细胞中的表达也明显上调,EZH2基因沉默可抑制人脑微血管内皮细胞(human brain micro vascular endothelial cells, HBMVECs)迁移和成管能力,恶性胶质瘤中VEGF通过miR-101靶向调控EZH2而形成VEGF/miR-101/EZH2轴,促进血管生成。另外,近期的一项研究显示成纤维细胞生长因子-2(fibroblast growth factor2, FGF-2)同样通过miR-101调控EZH2,促进肿瘤的血管生成。
本课题组前期通过RT-qPCR方法检测了18例鼻咽癌和16例鼻咽炎标本,发现鼻咽癌标本中EZH2mRNA表达升高,为鼻咽炎的2.12倍。而且,EZH2可调控鼻咽癌细胞的细胞周期并促进其增殖。随后,本课题组进一步发现EZH2表达与鼻咽癌的临床分期、转移和预后关系密切,是鼻咽癌的独立预后指标。我们课题组的研究结果也得到其它文献证实,Hwang等通过RT-PCR和Western blot方法发现EZH2在鼻咽癌中表达升高,并与肿瘤的临床分期和患者生存期缩短有关;Tong等发现EZH2在鼻咽癌细胞中通过靶向抑制E-cadherin表达促进鼻咽癌转移。上述研究表明EZH2可促进鼻咽癌细胞增殖和侵袭,并与肿瘤的分期、转移、复发、生存期缩短和预后不良密切相关,是鼻咽癌治疗的潜在靶标,但是EZH2对鼻咽癌生长和转移的促进作用是否通过影响肿瘤血管生成实现尚未见报道。
因此,本研究通过免疫组化(immunohistochemistry, IHC)方法检测鼻咽癌患者组织标本EZH2和CD34表达,分析EZH2与肿瘤内微血管密度(intratumor microvessel density, MVD)的相关性,初步探索EZH2与鼻咽癌血管生成的关系;随后通过改变EZH2的表达水平,在体外细胞增殖、侵袭和成管实验,以及体内鸡胚绒毛尿囊膜(chicken embryo chorioallantoic membrane, CAM)血管生成模型和裸鼠肝包膜下移植瘤模型实验中,阐明EZH2对鼻咽癌血管形成的影响;最后通过RT-qPCR和ELISA检测血管生成相关因子,初步探讨EZH2影响鼻咽癌血管生成的下游基因,为进一步揭示EZH2影响鼻咽癌生长和转移机制奠定基础,同时对鼻咽癌分子靶向治疗的靶点研究也有积极的参考意义。
方法
1、细胞株和实验动物
293T细胞、人脐静脉内皮细胞(human umbilical vein endothelial cells, HUVECs)细胞购自广州弗尔博公司,5-8F、6-10B为南方医院耳鼻喉科实验室冻存。293T细胞用含10%FBS的DMEM培养基,5-8F、6-10B、HUVECs细胞用10%FBS的1640培养基,常规培养传代。鸡胚,中国黄鸡,6日龄种蛋,购自广东粤禽育种有限公司。实验用裸鼠,BALB/c-nu/nu雌性裸鼠,购自中山大学实验动物中心,SPF条件下分笼饲养。
2、组织标本
69例初诊未治疗非角化未分化型鼻咽癌标本均取自2004年9月~2008年12月就诊于南方医院并行鼻咽部活检患者,标本的获取经过医院伦理委员会批准。
3、免疫组化
免疫组化按3步法常规进行染色,EZH2染色结果判定以胞核染成棕黄色为阳性细胞,染色结果以半定量的染色指数(staining index, SI)评估,染色指数(SI)为染色范围和染色强度乘积,染色强度(staining intensity)分为:0分:阴性;1分:弱阳性;2分:阳性;3分:强阳性。肿瘤细胞染色范围(positive tumor cell area)以染色癌细胞占癌细胞总数百分比计算:0分:癌细胞未见染色;0分:≤10%;1分:11~25%;2分:26~50%;3分:51~75%;4分:>76%。MVD计数参照文献报道进行,CD34染色后,先用低倍镜(×40~100)扫视玻片,寻找血管密度最高的区域,即“热点”("hotspots"),后在高倍镜下(×400)计数视野内被染色的血管数,即为MVD。
4、病毒包装、转染
空载质粒pLVTHM以及稳定过表达和沉默EZH2基因的两个慢病毒表达载体为南方医院耳鼻喉科实验室保存。采用293T细胞常规病毒包装,转染鼻咽癌细胞,流式细胞分选(fluorescence activated cell sorter, FACS)绿色荧光蛋白(green fluorescent protein, GFP)阳性细胞,构建稳定转染细胞株
5、RT-qPCR
分别采用Trizol、TaKaRa RT试剂盒和复能公司All-in-OneTM qPCR Mix试剂盒进行总RNA提取、逆转录和荧光定量PCR反应,反应体系和实验操作按照说明书进行。以2-△△α表示表示各基因的相对表达。
6、Western blot
RIPA buffer裂解细胞,Pierce公司BCA Protein Assay Reagent Kit进行蛋白定量,计算出待测样品的蛋白浓度。常规SDS-PAGE电泳,PVDF膜湿转法转膜,一抗4℃孵育过夜。二抗(1:3000)室温杂交。化学发光暗室X胶片显影、定影及扫描。
7、MTT assay
胰酶消化HUVECs细胞,每孔接种细胞2×103个,设对照孔及调零孔,24小时后吸净培基,稳定转染细胞上清与全培1:1混合液继续培养1-4天,于培养0-4天(0、24、48、72小时)后加入20μL MTT(5ug/mL)和DMSO,酶标仪检测各孔OD值。
8、Transwell invasion assay
在Boyden小室上室加入200μL稀释的Matrigel胶,过夜干燥,HUVECs细胞分别用不同细胞上清混合液连续培养4天,1×105/孔接种细胞,下室用分别加入细胞上清混合液,继续培养16小时。0.1%结晶紫溶液染色,拍照,计数细胞。
9、Tube formation assay
在96孔板中各加入501μL预解冻Matrigel胶,HUVECs细胞分别用不同细胞上清混合液连续培养4天,2×104/孔接种细胞,细胞上清混合液继续培养18小时,拍照,采用Image Pro Plus6.0软件,计数比较管状结构相对长度。
10、CAM血管生成模型
鸡胚用70%乙醇擦拭干净,置入超净台,小心去除鸡胚气室端外壳、外膜和内膜,开窗大小约2×2cm,暴露绒毛尿囊膜,放置硅胶圈1枚,2×106/鸡胚种植细胞,第2日小心取出硅胶圈,继续孵育5日。取出鸡胚,拍照,滴加10%福尔马林杀死鸡胚并固定尿囊膜,30mmin后剪下尿囊膜,倒置显微镜下(×40)计数移植瘤新生血管数(vessels number, VN)
11、裸鼠肝包膜下鼻咽癌移植瘤模型
以1%戊巴比妥钠按照40mg/kg腹腔注射麻醉裸鼠,肝包膜下1.0×106/只接种细胞,24天后取出荷瘤裸鼠肝脏及肺脏组织,PBS冲洗2次,分离肝脏移植瘤,拍照,称重;固定,常规石蜡包埋,CD34免疫组化染色。
12. ELISA
细胞上清VEGF蛋白浓度采用R&D公司Human VEGF Valukine ELISA Kit试剂盒检测,按照说明书操作。
13、统计学处理
采用SPSS13.0统计软件进行分析。计量资料均采用x±s表示。计量资料两组间比较采用两独立样本的t检验(independent-samples t test),等级资料两组间比较采用Mann-Whitney U检验,MTT增殖曲线采用析因方差分析(factorial design ANOVA)比较,单独效应分析采用One-way ANOVA比较;相关分析采用Spearman法。检验水准α=0.05。
结果
1、鼻咽癌EZH2表达与MVD相关性分析
采用免疫组化方法,检测69例非角化未分化型鼻咽癌组织中EZH2及CD34的表达,计算MVD。结果显示:EZH2高表达组(SI>3,32例)MVD为(36.7+13.8),低表达组(SI<3,37例)为(24.0+9.0),高表达组MVD显著高于低表达组(t检验,t=-4.46I,P<0.001);Spearman秩相关分析显示,EZH2染色指数(SI)与MVD存在中度正相关关系(r=0.480,P<0.001)。
2、EZH2影响鼻咽癌体外血管生成
采用慢病毒稳定转染方式,分别沉默5-8F细胞EZH2基因,过表达6-10B细胞EZH2基因,构建5-8F Lv-vector、5-8F Lv-shEZH2;6-1OB Lv-vector、6-10B Lv-EZH2两组共4个稳定转染细胞株。RT-qPCR方法检测EZH2表达改变,结果显示:5-8F Lv-shEZH2细胞的EZH2表达相对于5-8F Lv-vector细胞下调73.1%(t检验,t=14.922,P<0.001),6-10F Lv-EZH2细胞EZH2表达相对于6-10B Lv-vector细胞则上调了5.6倍(t检验,r=-23.927, P=0.002)。Western blot检测验证了EZH2的沉默和过表达效果,表明稳定转染细胞株构建成功,结果可靠,可进行后续实验。
分别取EZH2基因沉默/过表达细胞上清培养诱导HUVECs,通过MTT方法检测鼻咽癌细胞EZH2表达对HUVECs细胞增殖能力的影响。研究结果显示:相对于5-8F Lv-vector,5-8F Lv-shEZH2细胞上清诱导的HUVECs细胞生长速度下降(析因方差分析,F=495.314,P=0.000);相对于6-1OB Lv-vector,6-10B Lv-EZH2细胞上清诱导的HUVECs细胞OD值显著升高,生长速度加快(析因方差分析,F=532.764,P=0.000),表明鼻咽癌细胞EZH2基因过表达促进HUVECs增殖,沉默则抑制HUVECs增殖。
采用Tran swell invasion方法检测鼻咽癌EZH2表达对HUVECs侵袭能力影响,结果显示:EZH2基因沉默5-8F Lv-shEZH2细胞上清诱导的HUVECs穿膜细胞数为(93.8+23.0),5-8F Lv-vector细胞上清组则为(149.9+21.3),EZH2沉默使HUVECs穿膜细胞数减少37.4%(t检验,t=9.811,P<0.001),表明HUVECs细胞侵袭力下降;EZH2基因过表达6-10B Lv-EZH2细胞上清诱导的HUVECs穿膜细胞数为(174.9+27.5),空载对照6-10B Lv-vector细胞上清组为(118.8±17.7),EZH2基因过表达使HUVECs穿膜细胞数增加47.2%(t检验,t=9.811,P<0.001),表明HUVECs细胞侵袭力增强。
采用Tube formation方法检测鼻咽癌EZH2表达对HUVECs分化成管能力影响,结果显示:相对于5-8F Lv-vector,5-8F Lv-shEZH2细胞上清诱导的HUVECs相对小管长度由(110.1土21.4)缩短到(19.9+7.8),缩短81.9%(t检验,t=15.338,P<0.001),表明HUVECs分化成管能力减弱;相对于6-10BLv-vector,6-10B Lv-EZH2细胞上清诱导的HUVECs相对小管长度由(100.9±23.4)增加到(152.7±±31.2),增加51.3%(t检验,t=-5.143,P<0.001),表明HUVECs侵袭能力增强,该结果表明鼻咽癌细胞EZH2基因过表达促进HUVECs分化成管,沉默则抑制HUVECs分化成管。
3、EZH2影响鼻咽癌体内血管生成
将稳转细胞株接种在CAM血管生成模型上和裸鼠肝包膜下,分别建立鼻咽癌CAM血管生成模型及裸鼠肝包膜下移植瘤模型,检测EZH2表达对鼻咽癌细胞体内肿瘤血管生成能力的影响。CAM血管生成模型研究结果显示:种植肿瘤细胞6日后均可见移植瘤形成,EZH2基因沉默的5-8F Lv-shEZH2细胞移植瘤新生血管数(26.0±±7.1)较空载对照(38.1±±8.2)减少37.8%(t检验,t=3.524,P=0.002):EZH2基因过表达的6-10B Lv-EZH2细胞移植瘤新生血管数(41.3±6.4),较对照细胞(34.2±6.7)增加26.6%(t检验,t=-2.425,P=0.026)。裸鼠肝包膜下鼻咽癌移植瘤模型研究结果显示:接种后第24天实验结束时,EZH2基因沉默的5-8F细胞肝脏移植瘤体积明显缩小,重量下降86.4%(t检验,t=3.266,P=0.027)。免疫组化染色方法检测移植瘤MVD,研究结果显示,EZH2基因沉默的5-8F Lv-shEZH2细胞MVD(13.0+4.3)较空载对照(29.6±12.1)下降56.1%(t检验,t=2.896,P=0.020)。
4、EZH2影响鼻咽癌血管生成作用机制的初步研究
通过RT-qPCR方法检测稳定转染鼻咽癌细胞株,初步筛选血管生成相关基因,研究结果显示:EZH2基因沉默的5-8F Lv-shEZH2细胞VEGF表达下调65.4%(t检验,t=27.175,P=0.000),而EZH2基因过表达的6-10B Lv-EZH2细胞VEGF表达升高4.87倍(t检验,t=8.680,P=0.001)。ELISA方法检测稳定转染鼻咽癌细胞上清,研究结果显示:EZH2基因沉默的5-8F Lv-shEZH2细胞上清VEGF浓度显著下降(t检验,t=13.431,P=0.000);EZH2基因过表达的6-10B Lv-EZH2细胞上清VEGF浓度显著升高(t=-10.427,P=0.000),提示鼻咽癌中VEGF可能是EZH2的下游基因,EZH2促进鼻咽癌血管生成可能通过VEGF实现。
结论
1、EZH2与鼻咽癌MVD呈正相关。
2、体外过表达鼻咽癌细胞的EZH2可促进内皮细胞HUVECs的增殖、侵袭和分化成管;EZH2基因沉默可抑制HUVECs的增殖、侵袭和分化成管。
3、在体内,CAM血管生成模型与裸鼠肝包膜下移植瘤模型研究均表明EZH2促进鼻咽癌血管生成。
4、VEGF可能是EZH2的下游基因,EZH2可能通过上调VEGF表达促进鼻咽癌的血管生成。
Background and object
Nasopharyngeal carcinoma (NPC) is a primary nasopharyngeal mucosa malignant tumor. It is a common tumor in the south of China, the incidence rate of it is very high and reaches the top in Guangdong Province. According to the latest statistics of Jemal, there were an estimated84,400incident cases of NPC and51,600deaths in2008all through the world. However most of these patients were in southern China. The first choice of treatment for NPC is radiotherapy, which achieving a5-year overall survival of about80%in patients without metastasis. The predominant mode of failure of NPC is unquestionably distant metastases. It is well known that metastatic NPC is associated with very poor prognosis and the median survival is merely12~20months. Chemotherapy is the main treatment for metastatic NPC, but a first-line doublet chemotherapy (platinum based) achieves merely50~80%response rates with a median time to progression of only5to11months. On initial diagnosis, metastasis is only found in just5~7%of patients, However metachronous metastases are discovered in the course of evolution, usually within3years of treatment. The overall rate of metastasis is25~30%. The mechanism underlying the metastasis of nasopharyngeal carcinoma remains poorly understood, however, and requires additional elucidation. Further study in this field may lead to promising therapy for metastatic NPC.
Angiogenesis is the process by which new vessels grow toward and into a tissue. This creation of a blood supply is one of the key characteristics of malignant tumors, as the rapidly dividing cells need oxygen and lots of nutrients to continue growing. Angiogenesis is a prerequisite for the development and growth of solid tumors beyond1~2mm3Along with this, angiogenesis also enables tumor cells to spread through the bloodstream to distant sites. Therefore angiogenesis is essential for tumor growth and metastasis, and controlling tumor-associated angiogenesis is a promising tactic in limiting cancer progression. Interferon-a, angiostatin, and endostatin are examples of the first generation of angiogenesis inhibitors, while compounds such as bevacizumab and sorafenib are examples of current clinically used compounds. They are licenced to treat various cancers, including liver, kidney, breast and ovarian. Many results demonstrate that they prolong progression-free survival (PFS) and overall survival (OS) in patients with advanced cancer. For example, the use of bevacizumab prolongs the median progression-free survival by about4months in patients with advanced epithelial ovarian cancer in a phase Ⅲ trial. Angiogenesis is a common target for those seeking potential cancer therapies.
EZH2is the catalytic subunit of Polycomb repressive complex2(PRC2), which represses genes by trimethylating the core histone H3lysine27(H3K27me3) at and around the promoter regions of target genes. EZH2enhances neoplastic transformation and proliferation, is overexpressed in many cancers, and is strongly associated with tumor metastasis. It is found that the expression of EZH2is markedly increased in metastatic prostate cancer relative to localized prostate cancer or benign prostate. Additionally, other results confirmed the relationships between dysregulated expression of EZH2and metastatic prostate. Overexpress of EZH2have been found to lead to metastasis of many other malignant tumor, for example, breast cancer, ovarian cancer, liver cancer, and so on. However, Recent studies show that EZH2might influence tumor proliferation and metastasis through promoting angiogenesis of tumor.
Overexpression of EZH2has also been described in ovarian carcinoma endothelial cells. Silencing EZH2with small interfering RNA blocks endothelial cell migration and tube formation in vitro. The increase in endothelial EZH2is a direct result of VEGF stimulation by a paracrine circuit that promotes angiogenesis and silencing vasohibinl (VASH1). Another study has also found that EZH2overexpressed in endothelial cells of glioblastoma and plays an important role in the angiogenesis process of glioblastoma. Inhibition of EZH2may prove therapeutic in cancer through anti-angiogenic mechanisms.
Our previous studies have shown that the expression level of EZH2is upregulated in NPC. We have measured the mRNA levels of EZH2in NPC specimens. The results demonstrated that the average expression level of EZH2was upregulated by2.12-fold in NPC specimens than in normal nasopharyngeal tissues. We also successfully demonstrated that overexpression of EZH2promotes the proliferation of NPC cells through regulation of cell cycle regulatory proteins. In a subsequent study, we found that EZH2overexpression correlates with aggressiveness of metastatic NPC. This has been confirmed by numerous experimental studies reported recently. One of these reports suggested a critical role of EZH2in the control of NPC cell invasion and/or metastasis by repressing E-cadherin.
The effect of EZH2on angiogenesis in NPC, however, remains unkown. We hypothesized that EZH2may promote tumor growth and metastases via angiogenesis in NPC. However, related studies have not been reported as we know. We then designed this study to investigate the role of EZH2in the angiogenesis of NPC.
Materials and Methods
1. Cell culture, chicken eggs and BALB/c-nu/nu mice
293T and HUVECs cell line were purchased from Focusbio Company (Guangzhou, China).293T were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with10%FBS and1%PS, and HUVECs were cultured in RPMI1640. NPC cell lines5-8F and6-10B were maintained in our lab and cultured in RPMI1640supplemented with10%fetal bovine serum (FBS) and1%Penicillin-Streptomycin (PS) solution. All cell were cultured at37℃in a humidified atmosphere of5%CO2. Fertilized6-day-old embryo of yellow chicken eggs were purchased from Yueqin Company (Guangzhou, China) and incubated at37℃with60%humidity. Female nude mice (BALB/c-nu/nu,4to6weeks old,18to22g in weight) were purchased from Center of Experiment Animal of Sun Yat-sen University and were maintained under pathogen-free conditions (specific pathogen-free level).
2. Clinical specimens
Primary NPC biopsy specimens were obtained from Nanfeng Hospital (Southern Medical University, Guangzhou, China). All NPC specimens were classified as undifferentiated nonkeratinizing type. Informed consent was obtained from all patients, and the research protocols were approved by the Ethics Committee of Nanfang Hospital.
3、I mmuno histoc he mistry
Formalin-fixed, paraffin-embedded tissues of transplanted tumors were sectioned at4-mm thickness and analyzed for EZH2and CD34expression. For EZH2expression, a staining index (SI) was calculated as the product of intensity (0to3) and positive tumor cell area (0to4,≤10%,11%to25%,26%to50%,50%to75%,>75%). For MVD, the number of CD34-positive vessels was counted in four selected hotspots in a×400field. MVD was defined as the mean count of micro vessels per four field areas.
4. Production of lentivirus and cell transfection
The transfer vector and the packaging plasmids were co-transfected into293T cells using lipofectamine2000precipitation. The supernatant was harvested48-72h post-transfection. Then, the viral particles were concentrated by ultracentrifugation, and the viral pellets were resuspended. The packaged lentivirus was used to infect NPC cell lines. The cells were observed using a fluorescence microscope to detect green fluorescent protein (GFP) at48h post-transfection. Following fluorescence-activated cell sorting, the GFP-positive cells were isolated, and the cell lines were established.
5. Real-time PCR
Total RNA and small RNA were extracted from cells and tissues. The RNA was reversely transcribed into cDNA. RT-qPCR was performed using All-in-OneTM qPCR Mix. All samples were normalized to internal controls and fold changes were calculated through relative quantification (2-ΔΔCt).
6. Western blot
Total protein was isolated and quantitated using BCA assay. The protein lysates were separated by10%SDS-PAGE, and electrophoretically transferred to PVDF membrane. Then, the membrane was incubated with antibodies and detected by chemilumineseenee.
7. MTT assay
A total of2×103HUVECs were seeded in a96-well plate and the medium were removed after24hour. Media containing50%supernatantas of stable transfected NPC cell were then added to the HUVECs and allowed them to grow for4days. For MTT assay, cells were incubated in20μl of5mg/ml solution of MTT at37℃for4h and lysed in150μl dimethyl sulfoxide (DMSO) at room temperature for10min. The absorbance in each well was measured at490nm by a microplate reader.
8. Transwell cell invasion assay
HUVECs were cultured in media containing50%supernatants of stable transfected NPC cells for4days before assay. Invasion assays were then performed in Transwells with an8.0-μm pore size and coated with BD Matrigel Basement Membrane Matrix. A total of1×105HUVECs in serum-free medium were added to the upper wells. Media containing50%supernatants of stable transfected NPC cell were added to the lower wells. Cells that migrated through the filter after16h were stained and counted by phase microscopy.
9. Tube formation assay
HUVECs were cultured in media containing50%supernatants of stable transfected NPC cell for4days before assay. In vitro tube formation assay was then performed. Matrigel (BD) was added on96-well plates and polymerized by incubating for30min at37℃. A total of2×104HUVECs were seeded onto Matrigel coated plates and incubated in media containing NPC cell supernatants. After8h incubation, randomly chosen fields were photographed at×100magnification.
10. Chick CAM assay
HUVECs were also cultured in media containing50%supernatants of stable transfected NPC cells for4days before assay. Shells of tile chicken eggs were opened on day6and silicon rings were applied onto the chorioallantois membrane (CAM). Then a total of2×106NPC cells were transplanted onto CAM and incubated at37℃with60%humidity to generate tumors on the CAM. Silicon rings were removed24h later. Then on days12CAMs were photographed.
11. Animal experiment
Mice were maintained under pathogen-free conditions (specific pathogen-free level). Then mice were anaesthetized with1%pentobarbital sodium (40mg/kg) before surgery. Primary tumors were established by direct injection of1×106stable transfected NPC cells directly into the liver as indicated. After24days mice were sacrificed and tumors were dissected.
12. Statistical analysis
SPSS13.0software was used for statistical analysis. Data were presented as X±s. The results of MTT assay were analyzed by Factorial design analysis of variance. The relationship between EZH2and MVD was explored by Spearman's correlation. The result of Rank data was analyzed Mann-Whitney U test. Of measurement data, independent-samples t test and one-way ANOVA was used. P values of<0.05were considered statistically significant.
Result
1. Expression of EZH2is correlated with MVD.
Expression of EZH2and CD34were analyzed by IHC in sixty-nine non-keratinizing undifferentiated nasopharyngeal carcinoma tissues. MVD was calculated. The MVD of the EZH2high expression group (SI>3) was (36.7±13.8), which was higher than the EZH2low expression group (SI<3,24.0±9.0, t=-4.461, P=0.000). The SI of EZH2showed a moderate positive correlation with MVD (r=0.480, P=0.000). This result indicates that EZH2might promote angiogenesis in NPC.
2. EZH2promotes angiogenesis in NPC in vitro
We selected the NPC5-8F and6-10B cells, which excellently exhibited similar clinical phenotypes of NPC, as the model for further experiments. By using lentiviral vectors, duplex shRN A targeted EZH2was transfected into5-8F cells. Meanwhile, a lentivirus vector was used to upregulate the expression of EZH2in6-10B cells. The results of RT-qPCR showed that the expression of EZH2was downregulate by73.1%in5-8F Lv-shEZH2cells when compared with5-8F Lv-vctor (control lentivirus vector), and the expression of EZH2was upregulated by5.6-fold in6-10B Lv-EZH2cells when compared with6-10B Lv-vctor (control lentivirus vector). Result of Western blot assay confirmed these silencing and overexpression effect of EZH2. All these data demonstrated that the stable transfection could effectively upregulate or downregulate the expression of EZH2.
We then added media containing50%supernatants of stable transfected NPC cells to the HUVECs before MTT assay. The result of MTT assay displayed that HUVECs cultured by5-8F Lv-shEZH2supernatant grew slower significantly when compared with5-8F Lv-vetor (F=495.314, P=0.000), and HUVECs cultured by6-10B Lv-EZH2supernatant grew faster significantly when compared with6-10B Lv-vector (F=532.764, P=0.000). It indicates that EZH2overexpression in NPC promoted cell growth of HUVECs, and EZH2downregulation inhibited cell growth.
We next cultured HUVECs with media containing50%supernatants of stable transfected NPC cells for4days, and then detected the effect of EZH2on HUVECs by transwell cell invasion assay. We calculated numbers of HUVECs passed the membrane and found that the number of HUVECs was (149.9±21.3) in the5-8F Lv-vetor supernatant group and (93.8±23.0) in the5-8F Lv-shEZH2supernatant group. The number of HUVECs cultured by5-8F Lv-shEZH2supernatant decreased by37.4%(t=9.811, P=0.000). The cell number of HUVECs was (118.8±17.7) in the6-10B Lv-vetor supernatant group and (174.9±27.5) in the6-10B Lv-EZH2supernatant group. It increased by47.2%in the6-10B Lv-EZH2supernatant group (t=9.811,P=0.000).
Similar results were found in tube formation assay which was carried out after HUVECs were cultured with cell supernatants for4days. It was found that the relative tube length of HUVECs cultured by5-8F Lv-shEZH2supernatant decreased by81.9%when compared with5-8F Lv-vetor supernatant (t=15.338, P=0.000), and the relative tube length of HUVECs cultured by6-10B Lv-EZH2supernatant increased by51.3%when compared with6-10B Lv-vetor supernatant (t=-5.143, P=0.000).
3. EZH2promotes angiogenesis in NPC in vivo
Chick CAM assay was carried out with the stable transfected NPC cells. After6days of incubation, new blood vessels of transplanted tumors calculated. New blood vessels number of transplanted tumors markedly reduced by37.8%in5-8F Lv-shEZH2group compared with the5-8F Lv-vector control group (t=3.524, P=0.002). Meanwhile, compared with6-10B Lv-vector, new blood vessels number of transplanted tumors markedly increased by26.6%in EZH2over express ion6-10B Lv-EZH2group (t=-2.425, P=0.026).
In animal experiment, mice were sacrificed and xenograft were dissected at24days after cell transplantation. The results indicate that when compared with5-8F Lv-vector control, the average weight of xenograft of the5-8F Lv-shEZH2group was markedly reduced by86.4%(t=3.266, P=0.027). Then IHC assay was carried out and MVD of xenograft was calculated. The results displayed that MVD of the5-8F Lv-shEZH2group was markedly reduced by56.1%, as compared with control (t=2.896, P=0.020).
Both the experiments in vivo suggested that EZH2promotes angiogenesis in NPC.
4. EZH2promotes angiogenesis in NPC though upregulation of VEGF
The result of RT-qPCR showed that the expression of VEGF downregulated by65.4%in EZH2silencing5-8F Lv-shEZH2cells (t=27.175, P=0.000), and upregulated by4.87times in EZH2overexpressed6-10B Lv-EZH2(t=-8.680, P=0.001). To fatherly confirm this result, we examined the concentration of VEGF in supernatants of stable transfected NPC cells by ELISA. Significant decrease of VEGF concentration was detected in EZH2silencing5-8F Lv-shEZH2cell supernatants (t=13.431, P=0.000), and an increased of VEGF concentration was detected in supernatants of EZH2overexpression6-10B Lv-EZH2cells (t=-10.427, P=0.000). The results show that VEGF could be a downstream gene of EZH2in NPC, and EZH2might promotes angiogenesis inNPC though upregulation of VEGF expression.
Conclusion
1. Expression of EZH2showed a positive correlation with MVD in NPC.
2. The results of our in vitro studies suggested that upregulation of EZH2in NPC cell promoted cell growth, invasion and tube formation of HUVECs, and downregulation of EZH2inhibited cell growth, invasion and tube formation of HUVECs.
3. It was also proved that EZH2promoted angiogenesis in NPC in vivo.
4. Our results suggest that VEGF could be a downstream gene of EZH2in NPC, and EZH2might promote angiogenesis though upregulation of VEGF expression.
引文
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