JNK2 Regulates Apoptosis And Autophagy Through P53 In Bladder Cancer

Background: Bladder cancer is one of the most common malignancies of the urinary system,with an increasing morbidity in China.The recurrence rate of non-muscle invasive bladder cancer is about 50%,and 5-year survival rate of muscle invasive bladder cancer remains less than a half even if treated with radical cystectomy combined chemotherapy.Besides,the prognosis of bladder cancer has not improved for decades.A comprehensive understanding of the carcinogenesis and progression of bladder cancer is urgently needed to advance treatment.Previously,we compared m RNA expression in tumors and normal bladder tissues from orthotopic rat model of bladder cancer through gene expression microarray.Among these differentially expressed genes,JNK2 exhibits both tumor promoter and tumor suppressor actions depending on tumor type,but its function in bladder cancer is still unknown.Objective:(1)To verify the expression of JNK2 protein in bladder cancer tissue and adjacent normal tuissue by IHC and Western Blot.To analyze JNK2’s correlation with pathological stage,grade and find its effect on prognosis by Kaplan-Meier analysis.(2)To verify JNK2’s correlation with P53 in T24 cells and clinical specimen by co-IP and Western Blot.With plasmid and si RNA to up-regulate and knock down JNK2,we analyzed JNK2’s influence on P53-mdm2 and ubiquitination of P53 by co-IP.We also constructed P53 mutant plasmid and confirmed the phosphorylation site of P53 with JNK2.Besides,we used Western Blot and FCM to detect JNK2’s influence on apoptosis through P53 and conducted MTS assay to analyze JNK2’s influence on MMC.(3)To explore JNK2’s correlation with LC3 II in T24 cells and clinical specimen by Western Blot.We detected JNK2’s role on autophagy by Western Blot and m RFP-GFP-LC3 adenovirus in different JNK2 levels.We also analyzed P53 and autophagy with plasmid and si RNA to up-regulate and knock down target protein.Finally,with 3-MA to inhibit autophagy,we explored apoptosis and cell death of T24 cells induced by MMC and the potential mechanism.Results:(1)JNK2 protein levels were lower in rat and human bladder cancer tissues than in normal tissues.In TURBT patients,JNK2 had a negative correlation with pathological grade,and positive with pedicle.Furthermore,lower JNK2 expression was associated with poorer overall survival among patients who underwent radical cystectomy.(2)JNK2 had a positive correlation with P53.When JNK2 was upregulated,P53-mdm2 decreased and the ubiquitination of P53 also reduced.When JNK2 down-regulated or p-JNK inhibited,P53-mdm2 increased and so did ubiquitination of P53.JNK2 phosphorylates P53 at Thr81,which was crucial to P53 stability.JNK2 increased apoptosis through P53.Decreased expression of JNK2 in T24 cells conferred resistance to cell death induced by mitomycin C.(3)JNK2 had a positive correlation with LC3 II.Over-expression of JNK2 lead to increasing of autophagy,while down-regulation of JNK2 resulted in decreasing of autophagy.When autophagy inhibited by 3-MA,MMC induced severe apoptosis in T24 cells.Conclusion:(1)JNK2 protein was down-regulated in bladder cancer and was associated with poorer overall survival among patients who underwent radical cystectomy.(2)JNK2 phosphorylated P53 at Thr-81,thus protecting P53 from MDM2-induced proteasome degradation.Decreased expression of JNK2 in T24 cells conferred resistance to cell death induced by mitomycin C.(3)JNK2 induced autophagy though P53.MMC combined with 3-MA will cause severe apoptosis in T24 cells.