【作者】 王雪刚；

【导师】 章小平；

【作者基本信息】 华中科技大学 ， 泌尿外科， 2013， 博士

【摘要】 第一部分肾透明细胞癌miRNA表达谱的检测及miR-200c与肾癌临床病理参数之间的相关性分析目的：研究肾透明细胞癌与癌旁正常组织miRNA表达谱的差异,分析(?)miR-200c与肾透明细胞癌临床病理参数的相关性,为肾透明细胞癌的诊断和鉴定诊断提供有用的参考指标,为治疗效果评估和预后预测提供依据。方法：我们利用包含了851个人类成熟(?)niRNAs探针的生物芯片来检测5对肾透明细胞癌组织与癌旁正常组织中miRNAs的表达,并利用统计学分析方法分析癌组织与癌旁组织miRNAs的表达差异,按筛选条件fold change>2, P<0.05列为表达差异显著。然后利用实时定量PCR法进一步验证25对肾透明细胞癌组织与癌旁正常组织中miR-200c的表达变化,并利用统计学分析方法分析其与病理参数之间的关系。结果：microarray芯片共发现118个miRNAs在肾透明细胞癌与癌旁正常组织中存在表达差异,其中74个miRNAs表达具有显著差异。74个miRNAs中有30个miRNAs在肾透明细胞癌组织中表达明显上调,另外44个miRNAs表达明显下调。在另外的25对组织中,实时定量PCR显示相对于癌旁正常组织,miR-200c在肾透明细胞癌组织中的表达下降明显,符合(?)nicroarray芯片的结果,但miR-200c的表达变化与肾透明细胞癌病理参数间未见明显相关性。结论：肾透明细胞癌与癌旁正常组织的miRNAs表达谱具有明显差异,可以为肾透明细胞癌的诊断和与其他肿瘤的鉴别诊断提供有用的参考指标。miR-200c在癌组织中表达明显下调,有可能扮演抑癌基因的角色。第二部分miR-200c抑制肾癌细胞增殖的机制研究目的：我们的microarray芯片结果显示肾透明细胞癌组织中(?)miR-200c的表达量明显低于癌旁正常组织,有作为抑癌基因的潜能。深入研究miR-200c在肾癌细胞增殖中的作用及相关机制探讨,为未来肾癌靶向治疗的开发提供理论基础。方法：首先利用实时定量PCR检测三株肾癌细胞中miR-200c的表达量,筛选合适的细胞株,利用包装了hsa-miR-200c的慢病毒感染细胞建立稳定过表达miR-200c的肾癌细胞株模型；然后利用这个细胞模型探索miR-200c对肾癌细胞增殖的影响,MTT法测定细胞增殖曲线,PI染色和流式细胞仪分析细胞周期；再通过生物信息学方法预测与细胞增殖相关的目标靶基因,利用实时定量PCR、Westernblot和荧光素酶报告基因法进行靶基因的验证；最后动物体内实验进一步证实(?)miR-200c对肾癌细胞增殖的影响。结果：在SN12-PM6、786-0和A-498细胞中,miR-200c的表达量明显低于25个癌旁正常组织的平均表达量。我们利用慢病毒感染SN12-PM6和786-0建立的肾癌细胞模型是成功的,绿色荧光达90%以上,能稳定过表达miR-200c。MTT法测定细胞增殖曲线发现(?)miR-200c可以抑制肾癌细胞的生长,以72小时最明显。PI法染色后流式细胞仪检测发现miR-200c可以使肾癌细胞停滞在G0/G1期,而拮抗miR-200c的表达后,细胞增殖抑制作用能被逆转。Westernblot法证明(?)miR-200c组CDK2的表达量明显低于miR-Ctr组,荧光素酶报告基因法证明CDK23’UTR区存在(?)miR-200c的结合位点。最后动物实验证明miR-200c组肾癌原位移植瘤的肿瘤重量要明显小于miR-Ctr组。结论：miR-200c能在转录后水平调控CDK2的表达,使肾癌细胞停滞在G0/G1期,从而抑制肾癌细胞的增殖。这为未来肾透明细胞癌靶向治疗的开发研究提供理论基础第三部分miR-200c调节上皮间质转化参与肾癌细胞侵袭迁移的初步研究目的：本部分拟研究miR-200c能否调节上皮间质转化过程,起到抑制肾癌细胞侵袭转移的作用,这将有助于深入了解肾癌的转移过程,并可以鉴定到有意义的治疗靶标。方法：利用第二部分中建立的稳定过表达miR-200c的肾癌细胞株模型,应用(?)answell法检测过表达miR-200c的肾癌细胞侵袭转移能力的改变。实时定量PCR法检测SN12-PM6、786-0细胞和25对肾透明细胞癌组织与癌旁正常组织样本中ZEB1和E-cadherin的表达,分析肾癌细胞和肾癌组织中miR-200c、ZEB1和E-cadherin三者的关系。最后运用实时定量PCR和Westernblot法检测过表达miR-200c后肾癌细胞ZEB1、E-cadherin在mRNA和蛋白水平上表达的变化。结果：过表达miR-200c后,相比miR-Ctr组,miR-200c组肾癌细胞的侵袭和迁移能力明显下降。在肾癌细胞和肾癌组织中,miR-200c的表达量与ZEB1成负相关,与E-cadherin成正相关。实时定量PCR法结果显示miR-200c组ZEB1mRNA的表达量要明显低于miR-Ctr组,E-cadherin mRNA的表达量则明显上升。在蛋白水平上,westernblot结果显示miR-200c组ZEB1的表达明显下降,E-cadherin的表达则明显上升。结论：miR-200c可直接靶向作用于ZEB1,降低ZEB1的表达间接使E-cadherin的表达升高,调节上皮间质转化从而抑制肾癌细胞的侵袭和转移。更多还原

【Abstract】 Part one:Analysis of the miRNAs expression profiles between renal cell carcinoma and adjacent normal tissueObjective:To investigate different expression profiles of miRNA between clear cell renal cell carcinoma (ccRCC) and adjacent normal renal tissue. Aim to provide a useful reference for the diagnosis and identification indicators for assessing treatment effect of renal cell carcinoma.Methods:By using a miRNA microarray platform which covers a total of851human miRNAs, the expression profiles of miRNA in five coupled ccRCCs and normal renal tissues were screened. Then miR-200c with significant downregulation was confirmed by quantitative RT-PCR in twenty pairs of ccRCC and matched normal renal tissue, and we analysised the correlation between miR-200c and renal cancer clinicopathological parameters by t-test.Results:We found that118miRNAs were differentially expressed between five coupled ccRCCs and normal renal tissues, of which74miRNAs were significant difference (fold change>2, P<0.05).30miRNAs were significantly up-regulated in ccRCCs and the other44were down-regulated, In twenty pairs of ccRCC and its matched normal renal tissue, miR-200c was significantly down-regulated in ccRCC, but no correlation with renal cancer clinicopathological parameters.Conclusion:Our results demonstrated that the different miRNA expression profiles could be a potential reference for the diagnosis and differential diagnosis of renal cell carcinoma. MiR-200c which is commonly down-regulated in ccRCC specimens may affect the development of renal cell carcinama as a potential tumor suppressor gene. Part two:The mechanism investigation of miR-200c on involving in cell proliferation of renal cancer cellsAbjective:In part one, we observed the expression of miR-200c in renal clear cell carcinoma was significantly lower than the adjacent normal tissue. Here, we continue to study the role of miR-200c in renal cell carcinoma proliferation.Methods:First, the miR-200c expression of three renal cancer cell lines was detected by qRT-PCR. To investigate the effect of miR-200c in tumor cells, SN12-PM6and786-0cells were transduced with pGCSIL-GFP-hsa-miR-200c to establish cell lines model stably expressing miR-200c. The transduction efficiency was determined by counting fluorescent cells and total cells from6random fields for each condition. Growth Curves and FACS Assays were used to study the role of miR-200c in cell proliferation in this model. Then we used bioinformatic predictions to determine possible targets of miR-200c and confirmed this prediction using qRT-PCR, westerblot, fluorescent reporter assay.Results:We found that miR-200c was significantly down-regulated in SN12-PM6,786-0and A498cell lines compared with normal tissues. The transduction efficiency was over90%in SN12-PM6cells and786-0cells. qRT-PCR indicated that compared to the control, SN12-PM6miR-200c cells and786-0miR-200c cells had26.4-fold and18.1-fold higher miR-200c expression, respectively. Growth curves and FACS assays indicated that ectopic expression of miR-200c suppressed cell growth and induced arrest in the G0/G1phases of cell cycle in renal cancer cells. When miR-200c was overexpressed, protein level of CDK2was markedly reduced. Conversely, inhibiting miR-200c expression resulted in up-regulation of CDK2protein level. We then confirmed that CDK2, whose3’UTR contained the potential binding site of miR-200c, was the candidate target gene using a fluorescent reporter assay. Furthermore, miR-200c suppressed tumor growth of SN12-PM6cells in nude mice.Conclusion:Our results showed that miR-200c could induce renal cancer cell arrest in G0/G1phases and inhibited cell proliferation through directly regulating the expression of CDK2at the post-transcriptional level. Part three:MiR-200c modulates the epithelial-to-mesenchymal transition in human renal cell carcinoma metastasisObjective:The aim of the present study was to investigate the possible roles of miR-200c in regulating metastasis and to identify its target genes in the renal carcinoma cells.Methods:To validate the involvement of miR-200c dysregulation in migration and invasion, migration test and invasion test were performed to test the effects of miR-200c. Then we measured the expression of miR-200c, ZEBl, and E-cadherin in SN12-PM6cells,786-0cells, and20pairs of human RCC tissues by qRT-PCR. We used bioinformatic predictions to determine possible targets of miR-200c and confirmed this prediction using qRT-PCR, westerblot.Results:Functional assays demonstrated that restoration of miR-200c significantly inhibited migration and invasion of SN12-PM6and786-0cells in vitro. Genome-wide gene expression analysis and TargetScan database studies showed that ZEB1which has been shown to promote tumor invasion and migration through E-cadherin gene silencing, is a promising candidate target gene of miRD200c. Overexpression of miR-200c in SN12-PM6and786-0cells was concurrent with downregulation of ZEB1and upregulation of E-cadherin mRNA and protein. The expression of miR-200c was inversely correlated with that of ZEB1but positively correlated with that of E-cadherin in SN12-PM6cells,786-0 cells, and20pairs of human RCC tissues.Conclution:These observations indicate that miR-200c could regulate ZEB1at the post-transcriptional level to modulate the EMT, inhibiting migration and invasion in SN12-PM6and786-0cancer cells.更多还原