The Study On The Function And Mechanism Of MiR-214during The Angiogenesis Of Bladder Cancer

Objective:To explore the role and molecular mechanism of miR-214during angiogenesis of bladder cancer.Methods:1. The expression of miR-214in bladder cancer cells and10pairs of bladder carcinoma and their counterpart normal tissues was measured by qRT-PCR.2. The mimics and inhibitor of miR-214were constructed and then transduced into bladder carcinoma cell line-5637to elevate or reduce the expression of miR-214in this cell line, and the culture medium was collected respectively. HUVE cells were cultured in these culture medium, and the difference of the capacity in angiogenesis was observed.3. We identified Fibulin-5mRNA as one direct target of miR-214by Luciferase reporter assay.4. The expression of Fibulin-5protein was tested with Western Blot in bladder cancer cell line-5637after the expression of miR-214was reduced through the transfection of miR-214inhibitor into the cell line.5. bladder cancer cell line-5637was transduced with miR-214mimics,vectors expressing Fibulin-5and both of them respectively. Those cell culture medium was collected respectively to culture HUVE cells, and the difference of the angiogenesis capacity was observed.Results:1. The expression of miR-214in bladder carcinoma tissues and cell lines was higher than that in negative contrast respectively, and the difference was statistically significant (p<0.05).2. Cell culture of5637cells over express miR-214helps stimulate angiogenesis much greater than that of cells with low expression of miR-214, The difference was statistically significant(P<0.05).3. the expression ratio of the couple Luciferase in5637cells co-transfected with FBLN5-3’UTR-WT vectors and miR-214 mimics was obviously lower than the control cells(P<0.05). while there is no difference in that ratio in cells co-transfected with FBLN5-3’UTR-MUT vectors and miR-214mimics with respect to control cells.4. The expression of Fibulin-5was up regulated in5637cells with miR-214downregulation compared to the control one.5. Our study also found that the role of stimulating angiopoiesis of culture medium highly expressed miR-214was the most powerful, the second was the culture medium co-expression Fibulin-5and miR-214,and the last was the culture medium over express Fibulin-5.Conclusions:miR-214contributes to angiogenesis in bladder cancer by targeting Fibulin-5.