The Function And Mechanism Of Connexin43 Involving In The Development And Progression Of Bladder Cancer

Part? Expression of Cx43 in bladder cancer tissue is upregulatedObjective:To investigate whether Cx43 protein level in bladder cancer tissues and para-carcinoma tissues has changedMethods:Tissue samples were obtained from 52 bladder cancer patients from April2014 to October 2015 who underwent radical cystectomy in Urinary Surgery of affiliated hospital of Chengde Medical University.Immunohistochemical method was used to detect the changes of Cx43 protein in bladder cancer tissues and para-carcinoma tissues.Twelve pairs of bladder tumor tissues and their para-carcinoma tissues were selected from the above samples.The changes of mRNA and protein of Cx43 were detected by RT-PCR and immunoblotting.The association between Cx43 and bladder cancer was analyzed by immunohistochemistry and clinicopathological data.Results:1.The expression of Cx43 in the bladder cancer tissues was significantly higher than that in the para-carcinoma tissues(P <0.01).2.Immunohistochemistry showed that Cx43 was mainly located in the cytoplasm,and the staining was significant in the bladder cancer tissues(P<0.05).3.Clinicopathological analysis suggests that high expression of Cx43 is often associated with high-grade bladder cancer(P <0.05).Conclusion:Expression of Cx43 in bladder cancer tissue is upregulated and associated with high grade of bladder cancer.Part ? Influence of Cx43 protein on cisplatin antineoplastic effect in bladder cancer cellsObjective:To explore the influence of cisplatin chemotherapy sensitivity of silencing Cx43 gene expression by RNA interference in bladder cancer 5637 cell line.Methods:Bladder cancer 5637 cell and normal bladder epithelial SV-HUC-1 cell were cultured in vitro,expression of Cx43 was detected by Western blot in Bladder cancer 5637 cell and normal bladder epithelial SV-HUC-1 cell lines and then using immunofluorescence to detect localization of Cx43 in Bladder cancer 5637 cell line.Knockdown of Cx43 by SiRNA was transfected into bladder cancer 5637 cell then treated with cisplatin(3ug/ml),CCK-8(cell counting kit),Western blot and flow cytometry were detected the proliferation,apoptosis and the protein expression of connexin 43,Cleaved caspase-3 and Bcl-2.Results:Expression of Cx43 was significantly increased in Bladder cancer 5637 cell to compare with normal bladder epithelial cells and mainly localized in the cytoplasm(P=0.019).2.Proliferation of bladder cancer 5637 cells was reduced with increase of the concentration of cisplatin(0.75ug/ml,1.5ug/ml,3ug/ml,6ug/ml)and time(0 days,1 days,2 days,3 days)(P<0.001).The proliferation of Si RNA-Cx43 group was significantly decreased to Si RNA-Control group(P=0.02).Apoptotic ratio of SiRNA-Cx43 group and Si RNA-Control group were(63±4.58)% and(34.33±6.03)%,there are difference among these two groups(P<0.01).Western blotting results showed that the expression of Cx43 protein decreased(P<0.001)and cleaved caspase-3gradually increased(P<0.001)at the same concentration(3ug / ml)in different time points to treat with cisplatin in bladder cancer 5637 cells,and the expression of cleaved caspase-3 was significantly increased in the Si RNA-Cx43 group(P=0.001),but Bcl-2 was decreased in the SiRNA-Cx43group(P=0.005).Conclusion:Knockdown of cx43 protein expression in bladder cancer 5637 cell can enhance the sensitivity of cisplatin,which that under mechanism may be abnormal cytoplasmic localization of Cx43 protein to combine with mitochondria.Part ? Gap junction protein connexin43 deregulation contributes to bladder carcinogenesis via targeting MAPK pathwayObjective:To explore the effect of overexpression of Cx43 and knockdown of Cx43 on bladder cancer 5637 cells its possible mechanisms.Methods:We constructed stable lines in Bladder cancer 5637 cell line that overexpressed of Cx43 and analyzed for proliferation,apoptosis and cell cycle progression.Cx43 expression was also knocked down with siRNAs to confirm the functions of Cx43 in bladder cancer.Influence of Cx43 on MAPK pathway was examined by western blot.Results:Overexpression of Cx43 in bladder cancer 5637 cells increased cell proliferation,promoted cell cycle progression and inhibited apoptosis.Western blot showed that JNK and ERK pathways were dramatically activated in Cx43-overexpressed cells.Conversely,knockdown of Cx43 inhibited cell proliferation by increasing apoptosis and causing cell cycle arrest,concomitant with inhibition of JNK and ERK signaling.Conclusion:The expression change of Cx43 protein may affect the proliferation and apoptosis of cancer cells,and its mechanism may be related to the regulation of MAPK signal.