Effects Of DJ-1 On Migration And Invasion Of Bladder Cancer Cells T24 And UC-3

Objective and background:With the rapid increase of incidence rate and mortality of Bladder Cancer,the impact of bladder cancer on human health has attracted the attention of urologists,and It has become the focus of medical staff research.With the development of minimally invasive techniques,surgery has become the first choice for bladder cancer treatment,The development and potential of gene therapy for bladder cancer need further study.DJ-1 is a new mitogen dependent oncogene,Which is found to be closely related to initiation,progression and prognosis in a variety of malignant tumors[1,2].Phosphatase and tensin homolog deleted on chromosome ten(PTEN)controls a variety of processes related to cell survival,proliferation and invasion[3].In our previous experiments,we found that the expression of DJ-1 gene in bladder cancer tissues was significantly higher than that in normal tissues.The correlation between DJ-1 protein and PTEN protein in bladder cancer is negative.In this study,the authors will explore the relationship between the expression of cell model related proteins,migration and invasion of bladder cancer cells and cycle changes by constructing DJ-1 gene overexpression and short hairpin RNAsh DJ-1 bladder cancer cell model,and explore the relationship between DJ-1 and PTEN gene,as well as the mechanism of Affecting Migration and invasion of T24 and UC-3 human bladder cancer cells.Methods: 1.The T24 bladder cancer cells and UC-3 bladder cancer cells in the experiment were purchased from the Shanghai cell bank of China Academy of Sciences and cultured in 10% fetal bovine serum 1640 medium(1% penicillin and streptomycin).2.T24 and UC-3 bladder cancer cell lines with DJ-1 gene overexpression and short hairpin RNAsh DJ-1 were constructed by lentivirus.3.The transfection efficiency of DJ-1 was detected by Western blot.4.The changes of invasion and migration ability of T24 and UC-3 cells were measured by Transwell.5.Flow cytometry was used to measure cell cycle changes(cell cycle analysis was provided by the school of life sciences,Chongqing Medical University).6.The changes of PTEN protein was measured by Western blot,Western blot also used to measured the downstream protein β-catenin of wnt/β-catenin pathway and the phosphorylation level of FAK.Results:For the sake of exploring the mechanism of DJ-1 produces an influence on bladder cancer cell migration and invasion,we first measured the expression of PTEN protein in T24 and UC-3 two bladder cancer cell lines.As the results reflect that the expression level of PTEN protein was remarkably higher in T24 cells than that in UC-3 bladder cancer cells(Fig 5).Therefore,T24 bladder cancer cells and UC-3 bladder cancer cells was selected as the experimental subjects.Then,we established stable DJ-1 gene interference sequences and over expression sequences.After successful sequencing(as shown in Figure 2,4),we introduced them into viruses and transfected T24 and UC-3 bladder cancer cell lines with virus.According to different treatments,they were divided into blank control group(CTRL),overexpression group(p EGFP),overexpression control group(EV),interference group(si DJ-1),interference control group(sh-NC).The transfection efficiency was detected by Westerblot.The results showed that in the interference group,the DJ-1 gene in T24 and UC-3 bladder cancer cell lines was successfully silenced compare with sh NC group(P < 0.05,Fig 6,7).In the overexpression group,DJ-1 gene of the two cell lines was successfully overexpressed compared with the EV group(P <0.05,Fig 6,7).Flow cytometry analysis showed that compared with the control group,silencing or overexpression of DJ-1 gene had no significant effect on the G0 / G1 and S cell cycle progression of the two cell lines(P > 0.05,Fig 9).After transfection,we also measured the effect of DJ-1 on cell migration and invasion by Transwell experiment.The results showed that compared with the interference control group,the migration and invasion of T24 bladder cancer cells with DJ-1 gene silence were significantly inhibited(P < 0.05,Fig 9),while the migration and invasion ability of sh DJ-1 cells in UC-3 bladder cancer cells did not show significant changes compared with that in the irradiated group(P > 0.05,Fig 8).In T24 bladder cancer cells,the p EGFP group increased cell invasion and migration ability compared with the EV group(P < 0.05,Fig 9),In UC-3 bladder cancer cells,overexpression of DJ-1 showed no obvious change in migration and invasion.(P>0.05,Fig 9).For the sake of studying the relevance between DJ-1 and PTEN,Westernblot were used to measure the expression level of DJ-1 and PTEN in the two bladder cancer cell lines.The results reveal that there were obviously negatively correlated between the expression levels of DJ-1 and PTEN in T24 bladder cancer cell lines(Fig 7).For the sake of investigating whether the migration and invasion of bladder cancer cells are affect by DJ-1 through downregulating PTEN.We detected the alterations of protein β-catenin downstream of wnt/β-catenin pathway and the phosphorylation level of FAK in two cell lines,The results showed that in stable interfering cells,the phosphorylation level of FAK gene in T24 bladder cancer cells decreased compared with the control group,and the FAK protein showed a consisitent and significant upregulation(P < 0.05,Fig 10),while the DJ-1silence did not significantly affect the FAK expression of UC-3 bladder cancer cells(P > 0.05,Fig 11).In stable overexpression of two bladder cancer cells,The phosphorylation of FAK gene in stable overexpressing T24 bladder cancer cells was obviously higher compared with EV group,and the expression of FAK protein was down regulated(P < 0.05,Fig 10),while the over expression of DJ-1 did not show any obvious influence on the expression of FAK in UC-3cells(P > 0.05,Fig 11).Compared with the EV group the expression ofβ-catenin gene in T24 bladder cancer cells was down regulated in p EGFP group(P < 0.05,Fig 10),The expression of β-catenin in sh DJ-1 group was obviously higher than that sh-NC group(P < 0.05,Fig 10).The over expression or interference of DJ-1 expression in UC-3 bladder cancer cells had no significant effect on the expression of β-catenin protein(P > 0.05,figure 11).Conclusion: 1.Our group guess that DJ-1 could change the expression of PTEN that influence proliferation and invasion of T24 bladder cancer cells,thereby effecting wnt/β-catenin pathway and the phosphorylation of FAK.2.There is no significant influence on the cell cycle of bladder cancer cells whether Up regulation or silencing of the expression of DJ-1.