The Proliferation Regulation Effect On Silencing PLCe Expression By RNA Interference In The Bladder Cancer Cells

PART ONEObjective:To investigate PLCεexpression in the transitional cell carcinoma of bladder (TCCB) and its clinical significance.Methods: The expression of PLCεin 27 cases of TCCB and 12 cases of normal bladder tissues were detected by RT-PCR, and its relation with tumor clinicopathotogical characteristics was analyzed.Results: The expression of PLCεin TCCB tissues were significantly higher than that in normal bladder tissues (p<0.01).The overexpression of PLCεwas correlated with tumor invasion-related clinicopathological features, such as clinical stage (p<0.01). But it was not closely associated with high pathological grade (p>0.05).Conclusion: PLCεwas over expression in TCCB tissues, which play an important role in bladder carcinogenesis development.It may act as a new diagnosis index of TCCB. PART TWOObjective: To construct shRNA expression plasmid against PLCεgene and establish a stable and efficient transfection method of bladder cancer cell line. To observe the inhibition of PLCεexpression in bladder cancer cell line BIU–87 cells.Methods: Potential RNAi oligonucleotides of PLCεwere selected on appropriate web site. Then these oligonucleotides were synthesized and two pGenesil-PLCεplasmids were constructed using pGenesil vector with GFP. The plasmids were digested by SalI and sequenced to confirm the inserted sequence. After the constructed plasmids had been transfected into bladder cancer cell line (T24,5637,BIU-87),transfection efficiency were calculated by fluorenscence microscope at different time and plasmid-liposome proportion. RT-PCR, Western blot analysis were taken to know about inhibition of PLCεexpression in BIU-87 cellsResults: It was confirmed by endonuclease digesting and DNA sequencing that the recombinant plasmids had been constructed successfully.And the stable and efficient transfection method was established,the best transfection time was 48h and the most suitable plasmid-liposome proportion was 1:2.The result of RT-PCR and Western blot showed that pGenesil-PLCεplasmids had inhibited PLCεexpression of BIU–87 cells obviously. The inhibition rate of PLCεmRNA was 78.08% and 75.96%, and that of protein expression was 79.93% and 72.16%. Conclusion: pGenesil-PLCεstably transfected into bladder cancer cells and effectively inhibited PLCεexpression in BIU-87 cells, which lay the foundation for the role of PLCεin bladder cancer for further studies.PART THREE Objective:To study the proliferation regulation effect on silencing PLCεgene expression by RNA interference in TCCB cell line.Mehods: After transfected recombinant plasmids into TCCB cells by eukaryotic cell transfection technique,the influence on proliferation was investigated by MTT , the changes of PCNA were analyzed by immunocytochemical method,the distribution of cell cycle were analyzed by flow cytometry (FCM) ,and the expression of cyclinD1 gene was detected by RT-PCR.Results: After transfected the specific recombinant plasmids, PCNA expression was significantly decreased,and the analysis of cell cycle indicated that cells of G0/G1 phase were increased,and G2/M phase cells were decreased strikingly, cells were blocked at G0/G1 phase, the cell proliferation was inhibited obviously, but the expression of cyclinD1 gene was not changed.Conclusion: PLCεplay an important role in proliferation of TCCB cells, which may be a potential target of biological treatment on TCCB in the future.