Mechanism Studies Of MiR-200c On Inhibiting Cell Proliferation, Invasion And Metastasis Of Renal Cancer Cells

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.