Effects Of PLCΕ Specific SHRNA On Proliferation Of Human Bladder Cancer BIU-87 Cell S

PART ONE RESEARCH OF PLCΕSPECIFIC SHRNA ON HUMAN BLADDER CANCER BIU-87 CELLS IN VITROObjective:To study the proliferation regulation effect on silencing PLCεgene expression by short hairpin RNA in BIU-87 cells.Mehods: After transfected pGenesil-PLCεrecombinant plasmids into BIU-87 cells by eukaryotic cell transfection technique,G418 were used to screen positive clone cells,select monoclone and culture expandly. The expression of PLCεmRNA and protein in transfected cells were detected by RT-PCR and Western blot,the influence on proliferation was investigated by MTT,the distribution of cell cycle were analyzed by FCM. BIU-87 cells that were transfected with pGenesil-NP recombinant plasmids and normal saline were set as negative control group and blank control group.Results: The pGenesil-PLCεrecombinant plasmids was successfully transfected into BIU-87 cells and stable transfection cells were obtained, it can effectively reduce the expression of PLCεmRNA and protein, the reduce rate of mRNA and protein were 46.65% and 35.68% respectively,the G0/Gl cycle was increased and G2/M cycle was decreased in transfected BIU-87 cells, the cellular proliferation was also lower in transfected BIU-87 cells. Conclusion : The pGenesil-PLCεrecombinant plasmid can successfully transfected into BIU-87 cells and can effectively inhibit the expression of PLCεmRNA and protein, it also can control the cell cycle and inhibit the cellular proliferation of BIU-87 cells.PART TWO RESEARCH OF PLCΕSPECIFIC SHRNA ON HUMAN BLADDER CANCER BIU-87 CELLS IN XENOGRAFT NUDE MICEObjective:To explore the inhibitory effect of tumor growth by silencing PLCεgene with short hairpin RNA on BIU-87 cells in xenograft nude mice.Methods:The stable transfection BIU-87 cells were transplanted into nude mice to establish xenograft tumors,the model mice were divided into experimental group,negative control group and blank control group. The tumor volume was monitored,the tumor quality was measured,the protein expression of cyclinD1 was detected by immunohistochemical.Results: The growth of Xenograft tumor in experimental group was slower than control groups,the size and weight of Xenograft tumors in experimental group were significantly lower (p <0.01), the inhibition rate was 71.95%, the protein expression of cyclinD1 in experimental group was down-regulated in experimental group.Conclusion:Silencing PLCεgene with the shRNA technology can decrease cyclinD1 protein expressions,inhibit the tumor growth in the human bladder cancer model. PART THREE EFFECT ON BCL-2/BAX GENE EXPRESSION OF IMPLANTED HUMAN BLADDER CANCER CELLS IN NUDE MICE BY SILENCING PLCΕGENE WITH SHRNA TECHNOLOGYObjective:To explore the effect on cell Bcl-2/Bax gene expression of nude mice model with implanted bladder cancer BIU-87 cells by applying shRNA technique to silence PLCεgeneMethods:Mice were subcutaneously implanted with 2×106 BIU-87 cells. Once tumors reached a definite size,the model mice were divided into experimental group,negative control group,and blank control group,the mice were allocated to receive either PLCε-shRNA or non-silencing shRNA vector or physiological saline(0.9 % NaCl solution) respectively.The tumor volume was monitored;the tumor weight was measured;the pathological changes of carcinoma were observed;the expression of PLCεmRNA and protein were detected by RT-PCR and Western Blot;the protein expression of Bcl-2,Bax and p-Akt1/ Akt1,p-Bad/Bad were detected by Western Blot.Results: The size and weight of tumors in experimental group were significantly lower than control groups(P <0.05). In experimental group the signs of cell apoptosis were fond under light microscope. The expression of PLCεmRNA and protein were obviously lower than the control groups(P <0.05).The protein expression of Bcl-2 and Bax in experimental group were down-regulated in experimental group(P <0.05). We found that knockdown of PLCεdid not affect the protein expression of Akt1 and Bad,and p-Akt1,p-Bad protein could not expressed in each group.Conclusion:Silencing PLCεgene with the shRNA technology can decrease PLCεgene and Bcl-2 protein expressions ,increase Bax gene expressions,induce cell apoptosis and inhibit the tumor growth in the human bladder cancer model.