基于cDNA微阵列技术Ⅱ、Ⅲ期肠癌转移相关基因的筛选、鉴定及应用研究

【作者】 姜争；

【导师】 蔡三军； 杜祥； 徐烨；

【作者基本信息】 复旦大学 ， 肿瘤学， 2010， 博士

【摘要】 第一部分：基因CXCL10、TCF7L1、TMEMl6J及SLC17A9在Ⅱ、Ⅲ期肠癌中的表达、异常甲基化及预后相关研究目的：利用全基因组表达谱芯片技术筛选出与Ⅱ、Ⅲ期大肠癌复发、转移相关基因,在此基础上采用单盲筛选的方法进一步确定四个基因-CXCL10、TCF7L1、TMEM16J和SLC17A9进行深入研究。本实验的目的是在Ⅱ、Ⅲ期结直肠癌组织及肠癌细胞株中检测这些基因的表达、甲基化状态及探讨其潜在的临床应用价值。方法：利用Real time PCR(qPCR)及RT-PCR技术检测64例(36例复发、28例未复发)Ⅱ、Ⅲ期肠癌冰冻组织及6株肠癌细胞四种基因的mRNA表达水平,同时,采用免疫组织化学法及Western blot等分子生物学技术检测118例(78例复发、40例未复发)Ⅱ、Ⅲ期结直肠癌石蜡包埋组织样本及6株肠癌细胞四种基因的蛋白质表达水平,并与患者的临床病例特征及预后随访资料相结合,检测基因表达与肿瘤临床病理指标的相关性及进行预后生存分析。此外,我们对47例(23例复发、24例未复发)冰冻Ⅱ、Ⅲ期肠癌组织运用BSP及MSP技术进行TCF7L1启动子区域CpG岛的甲基化检测,用以探讨其表观遗传学调控机制。结果：在64例(36例复发、28例未复发)Ⅱ、Ⅲ期肠癌冰冻组织标本中CXCL10、TMEM16J和SLC17A9等三个基因在未复发人群的mRNA表达水平显著高于复发人群(P<0.05),而基因TCF7L1的结果却与之相反,呈现复发组高水平表达(P<0.05),在蛋白质水平的检测中,我们也得到了相同的结果。此外,6株肠癌细胞的mRNA及蛋白质检测也获得了同样的结果。在随后进行的基因表达与各临床病理学指标的相关性研究中我们发现,CXCL10的蛋白表达与患者的性别、年龄及肿瘤的组织分级关系密切(P<0.05);TCF7L1的蛋白表达与组织学分级和肿瘤侵袭深度相关(P<0.05)；而基因TMEM16J和SLC17A9的mRNA表达水平与肿瘤的TNM分期联系紧密(P<0.05)。结合患者预后随访资料进行的生存分析结果显示,高表达TCF7L1、低表达CXCL10、TMEM16J或SLC17A9的患者其无复发生存期明显较对应的人群短(P<0.05)。而在对TCF7L1基因启动子区域CpG岛的甲基化检测中我们发现,其启动子区的甲基化是TCF7L1表达调控机制中的频发事件,而异常的低甲基化可能是导致TCF7L1表达升高的主要机制之一。结论：我们的研究结果表明,CXCL10、TCF7L1、TMEM16 J和SLC17A9四种基因均可能是具有评估Ⅱ、Ⅲ期肠癌患者预后转归价值的潜在性标志物,其表达水平往往能够预示肿瘤的复发及转移,而对于癌基因TCF7L1,启动子区的甲基化可能是其正常的表观遗传学调控机制之一,异常的低甲基化状态可能与肿瘤的复发、转移相关。第二部分：Ⅱ、Ⅲ期肠癌转移相关基因TMEM16J和SLC17A9的功能研究目的：通过前述研究我们已经验证基因TMEM16J和SLC17A9的表达与Ⅱ、Ⅲ期肠癌患者的复发显著相关,当前关于这两类基因的功能,尤其研究其在肿瘤组织中的作用的报道相对较少,本研究的目的在于深入探讨TMEM16J和SLC17A9在结直肠癌发生、发展等病理生理过程中所起的生物学作用。方法：我们提取肠癌组织标本的总RNA为模板,通过RT-PCR获取全长目的基因,经限制性内切酶酶切后与质粒pcDNA3.1(+)连接构建重组体,进行测序以确保目的基因无突变,然后将重组质粒导入6株肠癌细胞株(SW480、Caco-2、HCT116、SW620、LoVo和Colo205),对转染细胞进行RNA提取后,qPCR进行mRNA水平验证,证明TMEM16J及SLC17A9表达水平对比未转染细胞显著升高。对转染重组质粒的细胞株进行软琼脂集落形成试验、生长曲线、细胞周期、凋亡检测以及细胞侵袭、粘附等多种肿瘤体外生物学行为观测。此外,我们通过将转染重组质粒、空载体的两种肠癌细胞株分别注射于裸鼠皮下,建立动物模型进行在体内肿瘤的生长和侵袭情况的观察。结果：通过qPCR及测序检验证实我们成功将无任何点突变的基因TMEM16J及SLC17A9重组质粒导入到肠癌细胞株,并进行稳定传代,软琼脂集落形成试验结果显示,转染TMEM16J及SLC17A9重组体的细胞株其平均集落形成率明显低于转染空载体的细胞株,而且细胞形成的大集落较空载体组少、克隆形成时间较后者晚、克隆存活时间较后者明显缩短。在对转染细胞生长曲线的观测中我们发现,转染TMEM16J及SLC17A9的细胞生长明显减慢。通过流式细胞仪对转染细胞进行的细胞周期及凋亡检测分析显示,转染TMEM16J及SLC17A9的细胞处于S+G2+M增殖期的细胞数明显低于未转染TMEM16J及SLC17A9的细胞,且S期细胞减少,而凋亡细胞比例明显高于后者。在细胞侵袭及粘附实验中我们发现转染TMEM16J及SLC17A9的细胞粘附能力明显升高而侵袭能力显著下降。此外,动物模型观察证实了转染TMEM16J及SLC17A9的细胞所形成的肿瘤原发灶大小均明显少于未转染TMEM16J及SLC17A9的细胞。结论：研究结果表明,TMEM16J及SLC17A9基因能够抑制肠癌细胞的生长及增殖,降低癌细胞侵袭能力,从而在生物学功能上支持了其作为潜在性肿瘤复发标志物运用于临床检测的可能。更多还原

【Abstract】 Part I:The expression, hypomethylation and prognostic significance of CXCL10, TCF7L1, TMEM16J and SLC17A9 in stageⅡandⅢcolorectal cancerObjectives:In previous study, we have identified 4 genes that differently expressed in colorectal cancer (CRC) tissues with recurrence and those without recurrence using gene expression profiling assays. This study was aimed to investigate the relationship between the expression of 4 genes and their methylation status, and their potential roles in predicting prognosis of patients with stageⅡandⅢcolorectal cancer.Methods:To examine mRNA expression of 4 genes, real-time quantitative polymerase chain reaction (qPCR) and reverse transcriptase PCR was performed in 36 colorectal cancer tissues with recurrence and 28 without recurrence, and in 3 CRC-metastasis-derived cell lines (SW620, LoVo, Colo205) and 3 primary-CRC-derived ones (SW480, Caco-2, HCT116). Furthermore, protein was evaluated using immunostaining in 118 paraffin-embedded specimens and Western blot in 6 colorectal cancer cell lines, and the correlations between clinicopathologic factors and disease-free survival time and gene expression were analyzed. In addition, the methylation status of the CpG islands in TCF7L1 promoter was also detected by Bisulfite genomic sequencing (BGS) and methylation-specific polymerase chain reaction (MSP).Results:CXCL10, TMEM16J and SLC17A9 mRNA was down-regulated in both CRC tissues with recurrence and metastasis-derived cell lines but TCF7L1 mRNA was up-regulated. The expression level of TCF7L1 was unrelated with gender, age, tumor grade, lymphvascular or perineural invasion and other parameters. However, it was positively related to disease-free survival time, histological type and depth of invasion (P=0.002,0.038,0.020). The expression level of TMEM16J and SLC17A9 were positively related to TNM stage. CXCL10 expression was significantly associated with gender, age and tumor grade. Moreover, lower CXCL10, TMEM16J and SLC17A9 and higher TCF7L1 expression indicated poorer survival rate, respectively (P＜0.05, log-rank test). Multivariate analysis also showed they were independent prognosticators in CRC. Moreover, it was found that up-expression of TCF7L1 is significantly associated with its promoter CpG island hypomethylation.Conclusion:These findings indicate that up-regulation of TCF7L1 causing by hypomethylation or down-expression of any one of CXCL10, TMEM16J or SLC17A9 might play an important role in metastasis and predict poor prognosis of patients with stageⅡandⅢCRC patients.Chinese Library Classification:R73Part 2:Study on biological functions of human TMEM16J and SLC17A9 genesObjectives:In foregoing study, we have validated the expression TMEM16J and SLC17A9 genes were significantly associated with recurrence of stageⅡandⅢcolorectal cancer. Currently, the data concerning the biological functions of both genes were relatively insufficient, especially in cancer. This study was aimed to further investigate the role of TMEM16J and SLC17A9 genes in tumorigenesis of colorectal cancer.Methods:we designed to determine the impact of over-expression of TMEM16J and SLC17A9 by stable transfection of pcDNA3-gene expression vector on cell growth, cell cycle, apoptosis, cell adhesion and migration as well as invasion in human clorectal cancer lines (SW620, LoVo, HCT116, Caco-2). In addition, to further examine the effects of TMEM16J and SLC17A9 over-expression on tumor growth and metastasis, we performed the in vivo assay using an orthotopic xenograft tumor model in the athymic mice. We examined the primary tumor growth and the metastasis.Results:Over-expression of TMEM16J and SLC17A9 prolong cell doubling time or inhibit growth of colorectal cancer cells lines, including SW620, HCT116, LoVo and Caco-2. This inhibition of cell growth was associated with the regulation of cell cycle, increasing of apoptosis. Over-expression of TMEM16J and SLC17A9 elevated cellular capability of adhesion and reduced capability of invasion and migration in LoVo and HCT116. Additionally, the volume of tumor in nude mice was dramatically decreased by TMEM16J and SLC17A9 over-expression especially in LoVo cells (P<0.05).Conclusion:These findings indicate that the expression of TMEM16J and SLC17A9 decreased in human colorectal cancer cells as invasive and metastatic potential increased, indicating possible involvement of TMEM16J and SLC17A9 in invasive and metastatic phenotypes of colorectal cancer cells. TMEM16J and SLC17A9 have extensive effects on colorectal cancer cells in vitro and in vivo by inhibiting cell growth, redistributing cell cycle, promoting apoptosis, reducing invasion in vitro and metastasis in vivo. Both of them are new anti-oncogenes.更多还原