Plce Regulates Inflammatory Cytokine Release Via STAT3 Phosphorylation In Human Bladder Cancer Cells

PART ONE:CORRELATION OF THE PROTEIN EXPRESSION OF PLCε AND P-STAT3 IN TRANSITIONAL CELL CARCINIMA OF BLADDERObjective To study the expression of phospholipase Cε(PLCε) and signal transducers and activators of transcription 3(STAT3) in transitional cell carcmoma of the bladder (TCCB) and the relationship between with PLCε or p-STAT3 and various clinicopathological parameters.Method Expression of PLCε, p-STAT3 were determined by immunohistochemistry in 48 cases of bladder cancer tissues and 21 cases of adjacent tissues. Microscope was used to observe and capture images and mean density was analyzed by IPP6.0 software. Statistical analysis the relationship between with PLCε or p-STAT3 and various clinicopathological parameters.Result The expression of PLCs and p-STAT3 were higher in patient with bladder cancer than adjacent tissues(P<0.01).PLCε immunostaining in tumor cells was significantly associated with histological stage(P<0.05) but not with histological grade (P>0.05). P-STAT3 immunostaining in tumor cells was not associated with histological stage or histological grade(P>0.05). The analysis of spearman correlation showed that the increase of PLCε level was associated with the increase of p-STAT3 level (r=0.817,P<0.01).Conclusion The high expression of PLCε is closely correlated with high expression of p-STAT3 in TCCB tissues,which laid a foundation for further explore the role of PLCε and p-STAT3 in progress of bladder cancer.PART TWO:EFFECT OF PLCε ON EXPRESSION OF INFLAMMATORY-ASSOCIATED GENES IN BLADDER CANCER CELLSObjective To explore the effect of Ad-shPLCs on expression of inflammatory-associated genes-inT24 and-BIU-87 cells.Method PLCs positive adenovirus(Ad-shPLCs) and control adenovirus (Ad-HK) were infected to knockdown PLCs expression in human bladder cancer cell lines (BIU-87 and T24). qRT-PCR and western blot were used to detect PLCs expression.The expression of interleukin-6 (IL-6),tumor necrosis factor-a (TNF-a) and interleukin-1β (IL-1β)were measured by qRT-PCR and ELISA.The protein expression of TLR4, MyD88 were detected by western blot.Results The expression of PLCε were decreased using PLCε positive adenovirus (Ad-shPLCε) in T24 and BIU-87 cells.The result of qRT-PCR and ELISA showed that the mRNA and protein levels of IL-6,TNF-a and IL-1β in Ad-shPLCε group were lower than Ad-HK group. The differences have statistic significant(P<0.05).The expression of TLR4, MyD88 were decreased after infecting with Ad-shPLCs. The differences have statistic significant(P<0.05).Conclusion Knockdown of PLCe using RNA interference inhibit IL-6,TNFa, IL-1β expression and TLR4,Myd88 protein expression.PART THERE:KNOCKDOWN OF PLCε GENE INHIBITED INFLAMMATORY CYTOKINE RELEASE VIA INHIBITION STAT3 PHOSPHORYLATIONObjective To further explore the molecular mechanism of PLCε in regulating inflammation cytokine release.Methods T24 and BIU-87 cells were treated with LPS, Ad-shPLCε, LPS collaborate with Ad-shPLCε, ELISA were used to detect IL-6,TNFa, IL-1β secretion. The protein expression of p-STAT3, STAT3,p-ERK and ERK were detected by western blot after Ad-shPLCε treatment. T24 and BIU-87 cells were treated with different concentration of p-STAT3 inhibitor S3I-201,western blot was used to detect p-STAT3 expression. Then T24 and BIU-87 cells were treated with S3I-201 collaborate with Ad-shPLCε,western blot was used to detect p-STAT3 expression. T24 and BIU-87 cells were treated with LPS,Ad-shPLCs,LPS collaborate with Ad-shPLCε,western blot was used to test p-STAT3 expression.Results The result of ELISA showed that the protein levels of IL-6, TNFa and IL-1β in positive adenovirus collaborate with LPS treatment group were lower than LPS treatment group. The expression of p-STAT3 were decreased but STAT3,p-ERK,ERK. were not changed after infecting with Ad-shPLCε. STAT3 inhibitor S3I-201 inhibit p-STAT3 expression in dose-dependent manner.Combined treatment of STAT3 inhibitor S31-201 with Ad-shPLCε exhibited synergistic inhibitory effects on expression of p-STAT3.The result of western blot showed that the protein levels of p-STAT3 in Ad-shPLCε collaborate with LPS treatment group were lower than LPS treatment group.Conclusion Knockdown of PLCε inhibit proinflammatory cytokine release via LPS-induced STAT3 phosphorylation.