Phospholipase CE Regulate Cell Invasion Through JAK/Stat3Pathway In Bladder Cancer Cell Lines

Objective To study the expression of phospholipase C epsilon in transitional cell carcmoma of the bladder and the relationship with clinicopathologyMethod RT-PCR was employed to detect the expression of PLCε in30pairs of fresh TCCBs and corresponding nontumor bladder tissues, relationship between PLCε expression and tumor clinicopathotogical characteristics was also analyzed.Result Overexpression of PLCε is frequently observed in TCCB, the expression of PLCε mRNA was related with clinical stage(P<0.01),but not associated with high pathological grade(P>0.05).Conclusion PLCε expression is significantly elevated in TCCB, Clinical association analysis reveals that PLCε overexpression is positively correlated with the tumor histological stage, implying its involvement in TCCB development. Indicating that PLCε may act as a new diagnosis index for TCCB. Objective To construct the recombinant adenovirus vector with a short hairpin RNA(shRNA) against Phospholipase C epsilon (PLCε) gene by PAdEasy system,and for gene therapy of bladder cancer.Method The U6expression promoter and shRNA of pGenesil-PLCε and pGenesil-NP, which were constructed and identified in our previous experiment,were subcloned to pAdTrack shuttle plasmid. The recombinant shuttle plasmid pAdTrack-U6-PLCε was linearized by Pme I for homologous recombination with pAdEasy-1in AdEasier. The positive clone was identified by enzyme digestion and DNA sequence analysis. After linearization by pac I, the recombinant adenovirus DNA plasmid pAdEasy-U6-PLCε was transfected into HEK-293cells for packaging. The recombinant adenoviruses were amplified in HEK-293and the titers were determined. RT-PCR、Western blot and immunofluorescence was used to detect the expression of PLCε in bladder cancer cells infected with the adenovirus.Results The pAdTrack-U6-PLCε and pAdeasy-U6-PLCε plasmids had been successfully constructed as verified by enzyme digestion and DNA sequence analysis.the titer of the Ad-U6-PLCε was1.5×1012after package and amplification in HEK-293cells. PLCε decreased significantly in BIU-87and T24cells after infection by the recombinant virus(p<0.05).Conclusion We have successfully constructed the recombinant adenovirus Ad-U6-PLCε targeting PLCε gene, which provides a basis for investigating the role of PLCε gene in the ongoing of bladder cancer after using RNA interference. Objective To detection the effect of PLCε shRNA on bladder cancer cell metastasis and to analyze the possible molecular mechanism.Methods PCR and western blot were used to detect MMP-2, MMP-9, VEGF expression after adenovirus infection, cell invasiveness was assessed using transwell system, activation of STAT3after adenovirus was also detected by western blot.Results Both BIU-87and T24cells exhibited lower mRNA an protein expression of MMP-2, MMP-9and VEGF in Ad-U6-PLCε groups, a dramatic decrease in STAT3phosphorylation was detected in cells infected with Ad-U6-PLCε. Using an in vitro transwell assay, we observed a significant suppression of cell invasion in Ad-U6-PLCε groups compared to respective Ad-U6-NP or untreated cells in both BIU-87and T24cells, and the differencewas significant (P<0.05).Conclusion PLCε shRNA may suppress bladder cancer cells invasion by downregulating MMP-2,MMP-9and VEGF, and JAK/STAT3signal pathway maybe involved. Objective Assessed the effects of JAK/STAT3pathway inhibitor AG490on the invasiveness of BIU-87and T24cells and the expression of MMP-2,MMP-9,VEGF and p-STAT3protein, in order to confirming that PLCε shRNA inhibited invasion of BIU-87、T24cells through JAK/STAT3pathway.Methods BIU-87and T24cells were treated with AG490in three concentrations (low, medium, high), the protein level of p-stat3、 MMP-2,MMP-9,VEGF were assessed by western blot. In vitro invasion assay was performed to detect invasion pattern of cells on AG490treatment.Results AG490treatment significantly inhibited protein levels of VEGF,MMP-2,MMP-9and p-STAT3in both cell lines in a dose-dependent manner, cell invasiveness changes occurred in concert with changes in protein levels.Conclusion AG490inhibited the invasiveness of BIU-87、T24cells by downregulating MMP-2、MMP-9and VEGF via the JAK/STAT3pathway in bladder cancer cell lines.