Research Of RNAi PLCepsilon Induced Apoptosis In Human Bladder Cancer Cell Lines

PartⅠConstruction and indentity of PLCεshRNA enkaryotic expression vectorObjective:An eukaryotic expression vector containing PLCepsilon shRNA(short hairpin RNA) was constructed and indentified.Methods:1.After designing and checking shRNA rank of aim directly at PLCepsilon of objective gene and negative contrast, to design and synthesis corresponding shRNA. The shRNA and plasmid pGenesil-1 of fluorescence protein expression connect to construct pGenesil-PLCε1,pGenesil-PLCε2 and negative plasmid pGenesil-NP.2.According to behavior that pGenesil-1 plasmid producted green Fluorescence after expression, to observe and count green fluorescent intensity to judge the best transfection time and the suitable ratio of plasmid to liposome.3.RT-PCR and Westernblot were used to detect PLCεexpression in experiment and control group.Results:1.The recombinant plasmid was constructed and transfected successfully.2.To observe the green Fluorescent cells and conclude that when the transfection time is 48h and the ratio of plasmid to liposome is 1:2 the intencity of fluorescence is best.3.The PLCεmRNA expression is lower in positive plasmid group than in negative group and blank group by RT-PCR(p<0.01), we got the same result in the PLCεprotein expression by Westernblot(p<0.01).Conclusion:Transfection of PLCepsilon shRNA recombinant plasmid could inhibit expression of PLCεeffectively in human bladder cancer cell line T24 and BIU-87.PartⅡResearch of Transfected PLCεshRNA recombinant plasmid induced apoptosis in bladder cancer cell linesObjective:Apoptosis was measured in transfected bladder cancer cell lines by recombinant plasmid.Methods:1.Flow cytometry was used to detect the cell cycle and cell apoptosis.2.RT-PCR was used to measure Bcl-2,Bax mRNA expressions.3.The expression of NF-κB P50 mRNA was detected by in situ hybridization.4.Electron microscope was used to observe the morphological changes.Results:1.Flow cytometric profiles revealed that positive plasmid transfection led to the increase of the cell percentage of G0/ G1phase(p<0.05)and the cell percentage of apoptosis(p<0.05). 2.Bcl-2 mRNA expression determined by RT-PCR was down -regulated(p<0.05) after positive transfection,with Bax mRNA expression up-regulated(p<0.05).3.The NF-κB P50 mRNA ex- -pression was measured down-regulated after positive trans-fe -ction by in-situ hybridization(p<0.05).4.Distinct morphology changes of cell apoptosis were observed by electron micro- scope.Conclusion:Transfection of PLCepsilon shRNA could induce apoptosis of bladder cancer cell line.The mechanisms may be changing tumor cell cycle,down-regulating the expression of Bcl-2,NF-κB and up-regulating the expression of Bax. PLCepsilon may be one of the up stream regulatory factors of NF-κB signal pathway.