The Investigation On Inhibition Of RNAi-Mediated PLC-E To Invasive Power Of Human Bladder Cancer Cells

Objective: Silence the gene expression of PLCepsilon in Transitional Cell Carcinoma of the Bladder(TCCB) through the method of RNA interference(RNAi), in order to observe the change of PLC-εmRNA Transcription and protein level and to realize the effects of PLCepsilon shRNA transfection on invasive power of human bladder cancer cell lines. Finding the function of PLC-εsignal way in metastasis and invasive power maybe can bring a new method of gene therapy for bladder cancer.Methods:①Construction of recombinant plasmid carrier: after designing and checking siRNA rank of aim directly at PLCepsilon of objective gene and negative contrast, to design and synthesis corresponding shRNA(short hairpin RNA, shRNA). The shRNA and plasmid pGenesil-1 of fluorescence protein expression connect to construct PLCε1,PLCε2 and hollow plasmid NP. 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 .②Tests of the inhibition of PLCepsilon RNAi to metastasis and invasive power of invitro cultured TCCB cell lines named T24 and BIU-87 after the transfection of experiment group and control group . Tanscriptive level of PLCepsilon mRNA was detected by RT-PCR and protein level of PLCepsilon and PKCalfa was detected by western-blot, protein level of PKCalfa was detected by western-blot too. To detect the protein expression of MMP-2 and MMP-9 in cells ,after RNA interference use the immunohistochemical method and gelatin enzymography(including 72 kD typeⅣcollagenase and 92 kD typeⅣcolagenase analyzed). Invasive power of T24 and BIU-87 were measured before and after transfection by the membrane invasion culture system(Transwell chamber). Results:①Rank analyzing and gelose electrophoresis confirm succession of constructed PLCepsilon1 and PLCepsilon2 and NP.②observe 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.③The result of RT-PCR and western-blot displayed that the cells transfected with PLCepsilon1 and PLCepsilon2 vectors had lower expression of PLCepsilon mRNA,PLCepsilon protein than the control groups cells transfected with NP and cells without transfection.④PKCalfa protein in the cell lines transfected with PLCepsilon1 and PLCepsilon2 vectors had lower expression than the control.⑤In the membrane invasion culture system the invasion number of cells transfected with PLCepsilon1 and PLCepsilon2 are both lower than the the control groups cells transfected with NP and cells without transfection .The cells transfected with PLCepsilon1 and PLCepsilon2 vectors had lower gelatin enzymography expressing than the control groups cells transfected with NP and cells without transfection, and the result of immunohisto- chemical method were same as the gelatin enzymography.Conclusion:The PLCepsilon1 and PLCepsilon2 constructed can efficiently effect at PLCepsilon gene of human TCCB cell lines. Transfection of shRNA PLCepsilon1 and PLCepsilon2 might decrease invasive power of bladder cancer cell lines T24 and BIU-87 through the way of PLCepsilon to PKCalfa so as to inhibit the development of bladder cancer.And this study may inaugurate a new way for the geen treatment of TCCB.