The Study Of Expression And Function Of MiR-141in Bladder Urothelial Carcinoma

Part one Detection of expression of miR-141in bladder urothelial carcinoma tissues and cell linesObjective Detection of expression of miR-141and EMT associatied proteins in bladder urothelial carcinoma tissues and different cell lines.Methods Detected the expression of miR-141of20bladder urothelial carcinoma samples and cell lines with real-time quantitative PCR, and analysis the correlation between expression level of miR-141and clinical diameters. Evaluated the EMT situation of primary tumor, metastatic lymphnode and cell lines by detecting expression of proteins associated with EMT,with western-blot. Analysis the expression level of miR-141in tissues and cell lines in different situation of EMT.Results Compared with normal urothelial tissue and tissue adjacent to tumor, expression of miR-141in urothelial carcinoma increased significantly. Compared with superficial tumors,expression of miR-141in muscle invasive tumors decreased. Compared with primary tumors, expression of miR-141in metastatic lymphnode decreased also. Compared with HTB9and T24cell lines, expression of miR-141in UMUC3cell line that with heavily EMT decreased significantly.Conclusions Expression of miR-141decreased along with the rise of clinical stage and grade of bladder urothelial carcinoma. Along with the rise of extent of EMT, expression of miR-141decreased in both bladder tumor tissue and cell lines.miR-141and EMT may both play important roles in progression of bladder tumor. Part two Construction of lentiviral vector with pri-mir-141and stable transfection into human bladder cancer cell line UMUC3Objective Construction of miR-141over expression bladder cancer cell line,provided the cellular basis for further study.Methods Synthesised pri-mir-141sequence according to miRBase and cloned it into pLVX-IRES-ZsGreenl vector to build overexpression plasmid, confirmed by restriction enzyme cut and sequencing. Lentiviral packaging were performed in miR-141over expression plasmid and blank control plasmid.UMUC3cell line were infected with different kinds of lentiviral particles, and real time quantitative PCR was used to examine expression of miR-141of UMUC3-141,UMUC3and UMUC3-neg.Results Result of restriction enzyme cut and sequencing showed pYr-LVX-pri-mir141expression plasmid were constructed successfully. Fluorescence detection and titer determination of293T cells indicated that lentiviral packaging were done. Real time quantitative PCR results demonstrated that we had built UMUC3-141cell line that overexpressed miR-141and UMUC3-neg cell line for negative control.Conclusions Restriction enzyme cut and nucleotide sequencing results showed that the expression vector pYr-LVX-pri-mir141successfully constructed; miR-141overexpression cell line UMUC3-141and control cell line UMUC3-neg were established. Part three Effects of over expression of miR-141on biological behaviors of bladder cancer cell line UMUC3Objective Evaluate effects of over expression of miR-141on biological behaviors of bladder cancer cell line UMUC3.Methods Cell scratch repair experiments, transwell invasion chamber experiments, MTT detection, colony formation assay and flow cytometry apoptosis detection were used to detect the invasion, proliferation, colony formation and anti-apoptotic ability of bladder cancer cell line UMUC3、 UMUC3-neg and UMUC3-141.Results48hours later, scratch repair rate of UMUC3-141were decreased compared with UMUC3,MUC3-neg;36hours later, the number of cells penetrated through the matrigel of the UMUC3-141cells were decreased compared with UMUC3,MUC3-neg; MTT detection displayed proliferation of UMUC3-141cell line was significantly lower than the other two cell lines; three weeks later,the number of colony-forming of UMUC3and UMUC3-neg were higher than UMUC3-141; apoptosis detection showed no significant difference in the three cell lines.Conclusions Overexpression of miR-141in bladder cancer cell line UMUC3could reduce the ability of invasion, metastasis and proliferation;but no significant change in its anti-apoptotic ability. Part four Preliminary study of the relation of miR-141,EMT and Wnt signaling pathway during progression of bladder cancer.Objective Explore intrinsic mechanisms that overexpression of miR-141influences the biological behavior of bladder cancer cell lines.Methods Bioinformatics were used to predict possible target genes of miR-141;Western-blot was used to examine protein changes of the predicted target genes, and the proteins that changed were recognized as the final target genes of miR-141; Real-time quantitative PCR was used to detect mRNA level of the final target; EMT-associated protein and Wnt signaling pathway protein changes were detected with western blot. Results ZEB2and E3F2protein coding genes were selected as target genes of miR-141; Western-blot assay showed expression level of protein ZEB2and E3F2were down-regulated along with the over expression of miR-141; expression level of E-cadherin was up-regulated and down-stream proteins of Wnt signaling pathway:CyclinDl and MMP-7were down-regulated along with the over expression of miR-141.Conclusions miR-141could regulate the behavior of bladder cancer cell line UMUC3through target gene ZEB2and E2F3; miR-141regulated proliferation,invasion and metastasis of UMUC3not only through reversing EMT, but also inhibiting Wnt signaling pathway.