PLCε Regulates Aerobic Glycolysis Through STAT3/LDHA Pathway And Promotes Proliferation In Bladder Cancer Cells

Objective:Currently,urinary bladder cancer(UBC)is one of the common malignant tumors in the urinary system.Aerobic glycolysis is one of the prominent characteristics of malignant tumors,which provides energy for the growth of tumor cells.Lactate dehydrogenase A(LDHA)is one of the key enzymes regulating glycolysis.Signal transduction and activator of transcription 3(STAT3)has been proved to participate in glycolysis and promote tumorigenesis and development.Phosphatidylinositol-specific phospholipase C epsilon(PLCε)is highly expressed in a variety of tumors,which can promote the occurrence and progression of tumors.Our previous studies found that PLCεknockdown could significantly down-regulate the expression of LDHA and inhibit the activation of STAT3 in bladder cancer T24 cells.However,it is not clear whether PLCεcan participate in aerobic glycolysis and proliferation of bladder cancer T24 cells through regulating STAT3 and LDHA.Based on the previous studies,we use lentivirus vector carrying PLCεshRNA to study whether PLCεparticipates in aerobic glycolysis and proliferation of bladder cancer cells through STAT3 and LDHA pathways,and its regulation mechanisms.Methods:1)Sixty-four patients in Department of Urology,the First Affiliated Hospital of Chongqing Medical University from January 2017 to December2017 who were pathologically diagnosed as bladder cancer were enrolled in this study.64 specimens of bladder cancer and 42 specimens of tissue adjacent to bladder cancer were used to study the expressions of PLCεand LDHA by immunohistochemistry.The expression of PLCεand LDHA in bladder cancer tissues and adjacent tissues were statistically analyzed.The correlation between expression of PLCεand LDHA in bladder cancer was analyzed,and the correlations between the expression of PLCε,LDHA and clinical pathological parameters were conducted.Western blot(WB)and Real-time quantitative PCR(RT-qPCR)were used to detect the protein and mRNA expression of PLCεand LDHA in bladder cancer tisssue and adjacent tissues from 21 patients.The correlation between protein and mRNA expression of PLCεand LDHA in bladder cancer tissues was analyzed.2)Using targeted PLCεand non-targeted short hairpin RNA(shRNA)to establish bladder cancer T24 cells with stable knockdown of PLCεand its control cell lines.T24 cells were divided into Blank group,sh-NC group(non-targeted shRNA group)and sh-PLCεgroup(targeted PLCεshRNA group).RT-qPCR was used to detect the expression of PLCεmRNA in each group.Western blot was used to detect the protein expression of PLCεand LDHA.Glucolysis kit was used to detect the glucose consumption and lactate production of cells in each group.CCK-8 was used to evaluate cell proliferation.Simultaneously,siRNA was constructed and delivered to knock down expression of LDHA in T24 cells.Glucose consumption,lactate production and proliferation of bladder cancer T24 cells were observed after knocking down LDHA and PLCε.Eukaryotic vector for LDHA overexpression was constructed.The glucose consumption,lactate production and proliferation of T24 cells were examined after overexpression of LDHA,and its effect on PLCεknockdown mediated changes of T24 cells were also observed.3)Phosphorylation of STAT3(p-STAT3)and total STAT3 expression in each group were detected by western blot.The expression of phosphorylated STAT3 and LDHA were detected by western blot in each group after treatment with STAT3 inhibitor Stattic(5μM and 10μM)and STAT3 agonist IL-6(20 ng/ml).Glucose consumption,lactate production and proliferation of bladder cancer T24 cells were observed at the same time.The effects of IL-6 on PLCεknockdown mediated changes of T24cells were observed.Bioinformatics database JASPAR was used to predict whether LDHA was a target gene for transcription factor STAT3,and to predict the possible STAT3 binding sites of LDHA promoter.Chromatin immunoprecipitation assay(ChIP)was used to validate the predicted results.Double luciferase reporter assay further confirmed the effect of STAT3 on LDHA expression.4)Fifteen BALB/c nude mice were randomly divided into 3 groups(n=5 each group).BALB/c nude mice were subcutaneously injected with T24 cells of Blank group,sh-NC group and sh-PLCεgroup on the left hip.The size of xenograft tumor in nude mice was measured regularly.One month later,all nude mice were sacrificed and the xenograft tumors were harvested and weighed.Immunohistochemistrical staining was performed to detect the expression of PLCε,p-STAT3 and LDHA in each group.Results:1)Immunohistochemistrical staining showed that the positive rates of expression of PLCεand LDHA in bladder cancer tissues were significantly higher than those in adjacent tissues,respectively(76.6%vs 31.0% and 79.7%vs 28.6%;χ~2 test;P<0.001).Immunohistochemistrical staining scores of PLCε and LDHA(3.672±0.211;3.859±0.193,n=64)in bladder cancer tissues were significantly higher than the scores of PLCε and LDHA(1.738±0.205;2.095±0.215;n=42)in adjacent tissues.The expression of PLCεwas positively correlated with the expression to LDHA in bladder cancer tissues.Western blot and RT-qPCR also showed that the expression of protein and mRNA of PLCεand LDHA in bladder cancer tissues increased significantly in comparison with adjacent tissues of bladder cancer.And both protein and mRNA levels of PLCεshowed positive correlation with those of LDHA in bladder cancer tissues.There was no significant correlation between PLCεand LDHA and clinicopathological parameters.2)RT-qPCR showed that the expression of PLCεmRNA in sh-PLCεgroup was significantly decreased,compared with Blank group and sh-NC control group(P<0.01).Knockdown of PLCεin bladder cancer T24 cells decreased the protein expressions of PLCεand LDHA.Knockdown of PLCεsignificantly inhibited glucose consumption,in comparison to sh-NC group(3.559±0.179 vs 2.422±0.184 nmol/min/1x10~6 cells).In addition,knockdown of PLCεin bladder cancer T24 cells also reduced lactate production compared to the sh-NC control group(3.919±0.291 vs 6.210±0.323 nmol/min/1x10~6 cells).CCK-8 assay showed that PLCεknockdown significantly inhibited the proliferation of T24 cells.Furthermore,LDHA knockdown also inhibited proliferation and glycolysis of in T24 cells as PLCεknockdown,and combined knockdown of PLCεand LDHA showed a synergistic inhibition effect on T24 cells.Overexpression of LDHA up-regulated glucose consumption,lactic acid production and proliferation in T24 cells,and reversed the decrement of glucose consumption,lactic acid production and proliferation mediated by knockdown of PLCε.3)Knockdown of PLCεinhibited phosphorylation of STAT3 in bladder cancer T24 cells without change of total STAT3 expression.The inhibitor against STAT3(Stattic)inhibited the STAT3 phosphorylation and LDHA expression in T24 cells.Furthermore,Stattic also inhibited proliferation and glycolysis of T24 cells.Moreover,the STAT3 agonist IL-6mediated phosphorylation of STAT3 and also up-regulated the expression of LDHA,reversed the downregulation of LDHA expression induced by knockdown of PLCε.IL-6 up-regulated glucose consumption,lactic acid production and proliferation in T24 cells,and reversed the decrement of glucose consumption,lactic acid production and proliferation caused by knockdown of PLCε.Bioinformatics database JASPAR predicted that LDHA might be one of the target genes for STAT3,and predicted three sites that might bind to transcription factor STAT3.ChIP results showed significant enrichment of LDHA promoter with exogenous STAT3immunoprecipitated by HA antibody.STAT3 could bind to the promoter of LDHA.Dual luciferase reporter assays validated that the binding of LDHA promoter with STAT3 enhanced LDHA expression.4)Bladder cancer T24 cells of each group were subcutaneously injected in BALB/c nude mice,and all mice showed tumor growth.The xenograft tumors in nude mice showed slower tumor growth and smaller volume of tumor size in sh-PLCεgroup compared with both blank and sh-NC groups.On the 30~thh day after inoculation of T24 cells,the weight of the xenograft tumor in sh-PLCεgroup was significantly lighter than that of the control group.Compared with the control group,the expressions of PLCε,p-STAT3 and LDHA in sh-PLCεgroup was significantly decreased,which were consistent with the results in vitro.Conclusions:1)The expression of protein and mRNA of PLCεand LDHA in bladder cancer tissues was significantly higher,and there was a positive correlation between them.2)PLCεparticipated in aerobic glycolysis of T24 cells through LDHA and promotes glucose consumption,lactic acid production and proliferation.3)STAT3 participated in the regulation of LDHA by PLCε.STAT3directly regulated LDHA expression at transcriptional level.4)PLCεknockdown inhibited the growth of xenograft tumor of T24cells in nude mice.Knockdown of PLCεdown-regulated the expression of PLCε,p-STAT3 and LDHA in xenograft tumors.