MiR-188-5p/miR-141-3p Activate Akt Pathway Modulate Bladder Cancer Progression By Target Inhibiting PTEN

Objective: Radical operation is the main therapeutic method for bladder cancer to get better survival and cured.Post-operation bladder perfusion chemotherapy is the main way to decrease the recurrence rate and improve survival time.The recurrence rate of invasive bladder is high and the pathological grade may be increased with recurrence.The chemoresistance of invasive bladder cancer is a key factor in high recurrence rate and low long-time survival rate.Located between two genes or within genes,miRNAs control proliferation,EMT,invasion,migration and chemoresistance in bladder cancer.In this study,we observed the expression of mir-188-5p and mir-141-3p in bladder cancer tissues and cell lines to detect the correlation with clinical pathological characteristics,thus to observe the proliferation,EMT,invasion,migration and cisplatin effect under mi R-188-5p and miR-141-3p involvement,to detect the mechanisms that miR-188-5p and miR-141-3p activated Akt pathway via suppressing PTEN and to observe the synergistic effect of mi R-188-5p and miR-141-3p.We aim to evaluate the correlation of mi R-188-5p and mi R-141-3p in bladder cancer and the effect on malignant functions,to observe the synergistic effect of them.In vitro and in vivo experiments were used to identify mi R-188-5p and miR-141-3p inhibited PTEN to activate Akt modulating bladder cancer proliferation.We provided the research basis for early screening,diagnosis and prognosis of bladder cancer and to show experimental foundation for target therapy and combined therapy.Methods: 1.QRT-PCR was used to detect the expression of miR-188-5p and miR-141-3p in bladder cancer tissues and cells.The correlation of miR-188-5p,miR-141-3p with clinicopathological characteristics and survival was analyzed.The correlation of miR-188-5p and miR-141-3p in cancer tissue was analyzed by Pearson Rank correlation.2.MiR-188-5p and mi R-141-3p inhibitors was transfected into 5637 cells to downregulate miR-188-5p or miR-141-3p.Half-dose mi R-188-5p and miR-141-3p inhibitors were co-transfected.Proliferation,migration and invasion were detected by CCK8 assay,would healing assay and Transwell assay.3.MiRNA inhibitor and mimics were transfected into 5637 cells to downregulate or upregulate miR-188-5p and miR-141-3p,thus to observe TGF-β induced EMT and detect the EMT related genes expression.4.Luciferase reporter assay was used to identify miR-188-5p and miR-141-3p targeting PTEN respectively.Inhibitor or mimics were used to identify the regulating effect of mi R-188-5p and mi R-141-3p to PTEN.5.5637 cells were transfected to upregulate miR-188-5p and miR-141-3p,half-dose miR-188-5p/141-3p mimics were transfected into 5637 cells to observe whether upregulated PTEN could reverse the miR-188-5p and mi R-141-3p induced migration and invasion.6.MiR-188-5p and miR-141-3p targeted PTEN to activate Akt pathway was observed.pAkt/Akt was used to define the activity of Akt pathway.7.The effect of down or up-regulating PTEN on Akt phosphorylation was observed to verify that Akt was the down pathway of PTEN.8.Akt Inhibitor Ⅷ was given as the Akt inhibitor and CCK was introduced to observe miR-188-5p and miR-141-3p induced proliferation.Ki67 expression was detected by Western blot.9.MiR-188-5p mimics,miR-141-3p mimics and miR-188-5p/141-3p mimics stable half-dose transfection 5637 cells were dealt with CDDP and cell survival rate was observed after 24 hours to evaluate the upregulation’s effect on cisplatin chemotherapy.10.In vivo experiments were conducted to verify the effects of miR-188-5p,miR-141-3p and miR-188-5p/141-3p on bladder tumors: nude mice were transplanted with miR-188-5p inhibitor,miR-141-3p inhibitor and miR-188-5p/141-3p inhibitor stable half-dose transfected 5637 cells.Growth curve was drawn and HE staining was used to testify tumor formation.Volume and weight were compared.11.PTEN,Akt and pAkt were detected in tumor tissue of nude mice.PAkt/Akt was used to define the activity of Akt pathway and detect Ki67.Results: 1.MiR-188-5p and mi R-141-3p were higher in bladder cancer tissues than in cancer adjacent tissues(P<0.0001),miR-188-5p and miR-141-3p were higher in bladder cancer cells(5637、T24、BIU87)than in bladder epithelial cell(SV-HUC-1)(P<0.05);2.MiR-188-5p and miR-141-3p in patients with tumor diameter≥1cm,lymph node metastasis and metastasis M1 were higher than in patients with tumor diameter<1cm,lymph node negative and metastasis M0 and positively correlated with TNM stage;3.Pearson Rank correlation analysis showed that miR-188-5p and miR-141-3p were positively correlated(r=0.7651,95%CI: 0.606～0.865,P<0.0001);4.MiR-188-5p and miR-141-3p high expression correlated with poor survival(P < 0.01);5.Down-regulating miR-188-5p or miR-141-3p inhibited 5637 cells proliferation,invasion and migration.Half-dose combined down-regulation showed synergistic effect;6.Down-regulating miR-188-5p and miR-141-3p inhibited TGF-β induced EMT and up-regulating miR-188-5p and miR-141-3p promoted EMT.Half-dose co-transfected miR-188-5p/141-3p showed synergistic effect;7.MiR-188-5p and mi R-141-3p targeted PTEN 3’-UTR end directly.They inhibited PTEN expression;8.MiR-188-5p and miR-141-3p induced 5637 cell migration and invasion by suppressing PTEN expression;9.Up-regulating miR-188-5p and miR-141-3p activated Akt phosphorylation in 5637 cells.Once combined with pcDNA3.1-PTEN,Akt phosphorylation was down-regulated and PTEN reversed miR-188-5p and miR-141-3p induced Akt pathway activating;10.Akt is a downstream gene of PTEN;11.Inactivating Akt pathway inhibited miR-188-5p and miR-141-3p induced 5637 cell proliferation;12.Up-regulating miR-188-5p and miR-141-3p reduced CDDP’s inhibiting effect on 5637 cells and half-dose co-transfected miR-188-5p/ miR-141-3p show synergistic effect;13.Down-regulating mi R-188-5p,miR-141-3p and miR-188-5p/141-3p inhibited tumorigenesis in nude mice and PTEN protein was increased in tumors(P<0.05),pAkt/Akt ratio was decreased(P<0.001)and Ki67 was decreased(P<0.05).These data were more obvious in half-dose co-transfected group.Conclusion: 1.MiR-188-5p and mi R-141-3p are up-regulated in bladder cancer tissue and cells,which indicates mi R-188-5p and miR-141-3p are negatively correlated with survival rate and positively correlated with poor prognosis,tumor diameter,lymph node metastasis and adverse clinicopathological characteristics.2.Down-regulating miR-188-5p and miR-141-3p inhibited bladder cancer 5637 cell proliferation,migration,invasion and EMT.They show synergistic effect.3.MiR-188-5p and miR-141-3p activate Akt pathway by depressing PTEN to promote bladder cancer cell proliferation,invasion and migration.4.Up-regulated miR-188-5p and miR-141-3p reduce CDDP’s inhibiting effect on bladder cancer cells.The two mirs show synergistic effect.5.Down-regulating miR-188-5p and mi R-141-3p inhibits tumorigenesis in nude mice.Half-dose combined down regulation shows better effect.