Expression And Correlation Of AR,PAK6 And P53 In Bladder Cancer

Objective: androgen receptor(AR),p21-activated kinase 6(PAK6)and P53 genes are closely related to human cancer,but their biological roles in bladder cancer have not been fully demonstrated.The purpose of this study was to investigate the expression and difference of AR,PAK6 and P53 in bladder cancer and normal bladder mucosa.The relationship among the three was analyzed to further reveal the interaction of the three in the occurrence and progression of bladder cancer.To explore the relationship between the expression of the three factors and the number of bladder cancer tumors,clinical stage,pathological grade,recurrence and metastasis and other clinicopathologic features.To verify the potential value of AR,PAK6 and P53 as tumor molecular markers in bladder cancer.Methods:(1)Immunohistochemical staining method(Envision)was used to detect the positive expression rates of AR,PAK6 and P53 proteins in the bladder cancer group and the normal bladder mucosa group.The clinicopathological features of AR,PAK6 and P53 protein expression in bladder cancer were analyzed,as well as the correlation between AR and PAK6 and P53 protein expression in bladder cancer.(2)The expression level of PAK6 m RNA in normal bladder cell line(SV-HUC-1)and two bladder cancer cell lines(T24,BIU-87)was detected by real-time quantitative PCR(qPCR),and PAK6 overexpression plasmid was constructed.(3)The constructed PAK6 overexpression plasmid was transfected into cells by transient method,PAK6 overexpression group(OE),and empty plasmid was transfected as negative control group(NC),qPCR was used to detect the transfection efficiency of cells in each group.(4)Effects of up-regulated CCK-8 and cell scratch detection of PAK6 on the proliferation and migration of BIU-87 bladder cancer cell lines.(5)qPCR was used to detect the effect of up-regulated PAK6 on the expression of AR m RNA and P53 m RNA in BIU-87 cell line.(6)The effect of up-regulated PAK6 on the expression of AR and P53 proteins in BIU-87 cell line was detected by Western blotting.Results:(1)The positive expression rates of AR,PAK6 and P53 in the bladder cancer group were 78.1%(50/64),45.5%(20/64)and 70.3%(45/64),respectively.The positive expression rates of AR,PAK6 and P53 in the normal bladder mucosa group were as follows: 25%(16/64),84.4(54/64),43.8%(28/64),the differences were statistically significant(P< 0.05),that is,the expression levels of AR and P53 proteins in the bladder cancer group were significantly higher than those in the normal mucosa group,and the expression levels of PAK6 proteins in the bladder cancer group were lower than those in the normal mucosa group.(2)The expression of AR protein was correlated with the pathological grade,tumor stage,and the presence of metastasis of bladder cancer(P<0.05).The expression of PAK6 protein was associated with the recurrence of bladder cancer(P<0.05).The expression of P53 protein was correlated with pathological grade,tumor stage,recurrence and metastasis(P<0.05).(3)Correlation analysis was conducted on the expression levels of AR,PAK6 and P53 proteins in bladder cancer tissues.There was a negative correlation between the expression levels of AR and PAK6 proteins in bladder cancer(rb=-0.525,P<0.01),AR protein was negatively correlated with P53 protein expression in bladder cancer(rb=-0.711,P<0.01).(4)The expression level of PAK6 m RNA in SV-HUC-1,BIU-87 and T24 cell lines was detected by qPCR,and the expression level in BIU-87 cell line was the lowest(0.03±0.025),the difference was statistically significant(F=14.74,P<0.01).(5)Compared with NC group,m RNA level of PAK6 in OE group was significantly increased,and the difference was statistically significant(P<0.01),proved that PAK6 transfection was successful.(6)The results of CCK-8 showed that,compared with the NC group,the cell proliferation activity was significantly increased within 1-5 days after transfection with PAK6 overexpression reagent,and the difference was statistically significant(P<0.001).(7)Scratch test showed that PAK6 overexpression could enhance the migration ability of bladder cancer cells,and the difference was statistically significant(P<0.001).(8)After up-regulation of target gene PAK6,AR m RNA in BIU-87 cell line was significantly decreased compared with NC group,and the difference was statistically significant(P<0.05);PAK6 m RNA in BIU-87 cell line was significantly higher than that in NC group,and the difference was statistically significant(P<0.01);P53 m RNA in BIU-87 cell line was significantly decreased compared with NC group,and the difference was statistically significant(P<0.01).(9)After up-regulation of the target gene PAK6,the expression of AR protein in BIU-87 cell line was significantly decreased compared with NC group,and the difference was statistically significant(P<0.0001);The expression of PAK6 protein in BIU-87 cell line was significantly higher than that in NC group,and the difference was statistically significant(P<0.0001);The expression of P53 protein in BIU-87 cell line was significantly lower than that in NC group,and the difference was statistically significant(P<0.01).Conclusion:(1)The expression of AR,PAK6,P53 and the clinicopathologic features of bladder cancer showed that: The expression of AR protein is correlated with the pathological grade,tumor stage and metastasis of bladder cancer,the expression of PAK6 protein is correlated with the recurrence of bladder cancer,and the expression of P53 protein is correlated with the pathological grade,tumor stage,recurrence and metastasis of bladder cancer,suggesting that the three proteins AR,PAK6 and P53 play an important role in the development,invasion and metastasis of bladder cancer.(2)Inhibition of PAK6 expression can reduce the proliferation and migration ability of bladder cancer cells.(3)There is a negative correlation between the expression of AR protein and PAK6 protein in bladder cancer tissues,and the expression of AR protein and P53 protein in bladder cancer tissues.In cell experiments,the up-regulation of PAK6 can inhibit the expression of AR and P53 in bladder cancer,and AR,PAK6 and P53 may promote the progress of bladder cancer in the aspects of cell proliferation and migration.The combined detection of AR,PAK6 and P53 will provide a theoretical basis for the diagnosis,treatment and improvement of the prognosis of patients with bladder cancer.