【作者】 甘丽虹；

【导师】 王良静； 姒健敏；

【作者基本信息】 浙江大学 ， 内科学， 2011， 硕士

【摘要】 结肠癌是一种常见的消化系统恶性肿瘤,其发生发展涉及癌基因活化和抑癌基因失活。越来越多的证据表明表观遗传学改变与抑癌基因失活密切相关。表观遗传学改变主要包括DNA甲基化、组蛋白乙酰化和染色质重塑等,其中DNA高甲基化是抑癌基因失活的最重要机制之一。对表观遗传学的研究不但可以揭示结肠癌新的发病机制,并且还有助于疾病的早期临床诊断和预后判断。ZIC1是我们前期通过cDNA芯片技术筛选到的,具有胃癌抑制作用的重要基因,并且发现一系列结肠癌细胞中ZIC1沉默表达或表达明显下调,这种低表达与启动子区DNA高甲基化密切相关。ZIC1编码锌指转录因子,与脊椎动物发育密切相关,尤与脑组织和神经系统发育相关。近年来发现ZIC1还参与肿瘤的发生,如ZIC1与髓母细胞瘤、纤维瘤、脂肪肉瘤和子宫内膜癌的发生密切相关。本课题中,我们通过检测结肠癌组织和癌旁组织中ZIC1 mRNA的差异表达,并检测ZIC1启动子甲基化情况,以期探讨ZIC1在结肠癌中表达调控的可能机制及基因启动子甲基化检测对结肠癌临床诊断的潜在价值；同时通过外源性过表达ZIC1,明确其对结肠癌细胞的增殖、凋亡和细胞周期的影响及信号通路调控机制；最后采用表达谱芯片技术筛选ZIC1基因调控的下游关键基因并进行相关验证。方法和材料：1.使用实时定量RT-PCR(qRT-PCR)检测24例结肠癌及相应癌旁相对正常组织中ZIC1基因mRNA表达水平；用甲基化特异性PCR(MSP)检测40例结肠癌及癌旁相对正常组织的ZIC1基因启动子区DNA甲基化情况。2.细胞转染ZIC1和筛选后,采用克隆形成实验和细胞存活实验(MTS)观察过表达ZIC1对结肠癌细胞HCT116和HT29增殖的影响,采用流式细胞仪分析ZIC1对细胞凋亡和细胞周期的影响。3.利用Western blot检测ZIC1对增殖PI3K,MEK/Erk通路蛋白(Akt,Erk1/2)和凋亡调控通路关键蛋白(Bad,Bcl-xl,Caspase-3)表达的影响。4.HCT116细胞稳定转染pCDNA3.1-ZIC1和pCDNA3.1空白载体后,利用基因表达谱芯片技术检测人类肿瘤发生相关的多功能基因的变化情况,对其中10个候选靶基因分别在稳定转染ZIC1的HCT116和HT29细胞中进行qRT-PCR验证。结果：1.qRT-PCR结果显示：结肠癌组织中ZIC1 mRNA表达水平较癌旁相对正常组织明显下调(Wilcoxon配对检验,p=0.0001)。MSP检测发现结肠癌组织发生ZIC1启动子高频率甲基化(85%,34/40),而癌旁相对正常组织没有发生甲基化。2.过表达ZIC1后结肠癌细胞株(HCT116和HT29)克隆形成数较空载体对照组明显减少(p<0.01)。MTS增殖检测亦发现转染ZIC1后结肠癌细胞株(HCT116和HT29)的存活细胞数较空载体对照组明显减少(p<0.01)3.流式细胞仪检测发现转染ZIC1后,HCTl 16和HT29细胞的凋亡水平较空载体组显著增加(p<0.05),而细胞周期无明显改变(p>0.05)。转染ZIC1后结肠癌细胞(HCT116和HT29)增殖通路相关活性蛋白p-Akt,p-Erk1/2表达水平明显下调；凋亡级联反应蛋白Bad和活化片段Caspase3(cleaved-Caspase3)表达水平明显上调,而抗凋亡蛋白p-Bad和Bcl-xl表达水平下调。4.通过mircoarray技术筛选ZICl可能的下游调控基因,分析得到337个下调基因,95个上调基因。基因功能归类分析结果显示,差异表达的基因主要参与细胞增殖、细胞凋亡、细胞迁移、转录调控和信号转导等重要细胞活动。并对其中10个重要的靶基因(CCNA2, IGFBP3, ANGPT2, GADD45B, LAMB2, LAMB3, MALAT1, PNMA2,RPA4和TACSTD2),分别在稳定转染的HCT116和HT29细胞中进行了qRT-PCR验证,其结果与基因表达谱芯片分析吻合。结论：1.ZIC1在结肠癌组织中表达下调,并与启动子DNA高甲基化密切相关。2.体外试验证明ZIC1可抑制结肠癌细胞增殖,这一效应与抑制PI3K/Akt和MEK/Erk信号通路有关。过表达ZIC1可激活Bcl-xl/Bad/Caspase3信号级联反应,促进结肠癌细胞凋亡。3.ZIC1可能通过调控多个细胞生命活动相关功能基因,从而影响结肠癌的发生发展。更多还原

【Abstract】 Colon cancer (CRCs) is one of the most common malignant tumors in digestive diseases system. The development of CRCs is associated with the activation of oncogenes, as well as the inactivation of tumor suppressor genes. There were growing evidences suggested that the inactivation of tumor suppressor genes was closely related to the epigenetic changes, which including DNA methylation,, histone acetylation, and chromatin remodeling and so on. Among these, DNA methylation is considered as one of most important mechanisms. Study on these epigenetic events not only may reveal new pathogenesis of colon cancer, but also contribute to explore novel biomarkers of early detection and prognosis prediction of CRCs.We have previously found that ZIC1 is one of novel tumor suppressor candidates in gastric cancer through cDNA microarray. ZIC1 expression was also found to be silenced or significant downregulation in a panel of colon cancer cell lines, and DNA hypermethylation of ZIC1 promoter was related with its downregulation expression. ZIC1 gene encodes zinc finger transcription factor and plays a crucial role in development of nervous system. Recently, studies have demonstrated that ZIC1 participated in tumor progression, including medulloblastomas, desmoid tumors, liposarcomas and endometrial cancers.In this study, we sought to determine the possible regulation of ZIC1 exprssion and its potential utility of promoter methylation as a biomarker for differenential dignosis between colon cancer and adjacent non-tumor tissues. We evaluate the effect on proliferation, apoptosis and cell cycle of colon cancer cells by ectopic expression of ZIC1, and investigate the signaling pathways of these regulations. Finally, we testify the downstream targets of ZIC1 by cDNA microarray screening and qPCR validation.Materials and Methods:1. ZIC1 mRNA expression of 24 paired colon cancer and adjacent non tumor tissues were analyzed by quantitative RT-PCR (qRT-PCR), and ZIC1 promoter methylation status were detected by methylation specific PCR (MSP) in 40 paired colon cancer and adjacent non-tumor tissues.2. To determine the effect on cell proliferation, we performed colony formation and cell viability assays in HCT116 and HT29 which tranfected with ZICl or empty vector. Meanwhile, we analyzed the effect on cell apoptosis and cell cycle after ectopic exprssion of ZIC1 by flow cytometry analysis.3. We determined the expression of the PI3K/Akt, MEK/Erk pathway kinases (Akt, Erkl/2) and the apoptosis regulatory proteins (Bad, Bcl-xl, Caspase-3) by western blot.4. Microarray studies were employed to identify differential expression of those genes involving in tumorigenesis by ZIC1. We validated 10 candidate target genes in stable transfection cell lines (HCT116 and HT29) by qRT-PCRResult:1. QRT-PCR results showed that the level of ZIC1 mRNA was significantly decreased in colon cancer tissues relative to adjacent non-tumor tissues (Wilcoxon.matched paired tests, p=0.0001,n=24). Furthermore, methylation of ZIC1 gene promoter was frequently detected in primary tumor tissues (85%,34/40), but not in adjacent non-tumor tissues. 2. The number of surviving colonies formed was significantly reduced by overexpression ZIC1 in colon cancer cells (HCT116 and HT29) when compared with the control vector transfectants (p<0.01). Moreover, MTS assay showed that the survival cells were markedly decreased in cells (HCT116 and HT29) transfected with ZIC1 when compared with vector (p<0.01). Ectopic expression of ZIC1 could induce cell apoptosis, while not affect on cell cycle.3. Ectopic expression of ZIC1 could suppress the expression of phosphorylation of Erk1/2 and Akt, as well as phosphorylation of Bad and Bcl-xl, while activate the Bad and cleaved-Caspase-3.4. Results revealed that 337 genes were downregulated and 95 genes were upregulated by ZIC1 in colon cancer cells. Many of these genes have been reported to play important roles in cell proliferation, cell apoptosis, cell migration, transcriptional regulation and signal transduction. We validated the expression pattern in 10 selected genes (CCNA2, IGFBP3, ANGPT2, GADD45B, LAMB2, LAMB3, MALAT1, PNMA2, RPA4, TACSTD2) in colon cancer cells HCT116 and HT29 transfected with ZIC1 by qRT-PCR. These datas were concordant with that obtained from the microarray analysis.Conclusion:1. Downregulation of ZIC1 is associated with promoter DNA hypermethylation in colon cancer tissues.2. ZIC1 could inhibit cell proliferation by inactiviation of PI3K/AKT and MEK/Erk pathways, and induced cell apoptosis by regulating Bcl-xl/Bad/Caspase-3 cascades.3. ZIC1 may participate in the progression of colon cancer by modulating those functional genes involved in cellular activities.更多还原