The Search Of Mechanism For ARPC4 Regulate The Cytoskeleton On Bladder Cancer Invasion And Metastasis

PART 1 Expression of ARPC4 in bladder urothelial carcinoma and its relationship with lymph nodeObjective: To investigate the expression of ARPC4 in bladder urothelial carcinoma(BUC)and to explore the relationship between the expression of ARPC4 and the clinicopathological parameters and lymph node metastasis of bladder urothelial carcinoma.Methods: A total of 228 tissue specimens from BUC patients who underwent radical cystectomy from January 2012 to January 2015,40 cases of lymph node and 40 cases of normal bladder tissue specimens were collected as controls,and all patients clinical data were collected.All of specimens for construction of tissue microarray and subsequently subjected to immunohistochemical staining of ARPC4 expression.All of specimens for construction of tissue microarray and subsequently subjected to immunohistochemical staining of ARPC4 expression.ARPC4 expression in BUC tissues was analysis which associated with age,sex,tumor size,tumor stage,tumor grade,the number of tumor and lymph node metastases,BUC lymph node metastasis factor was analysis with multivariate logistic regression.Results: Immunohistochemical data showed that ARPC4 protein was mainly expressed in the cytoplasm and nucleus of positive cells and ARPC4 expression was significantly higher in BUC than in normal bladder tissues(84.3% and 27.5%,respectively,P <0.001).ARPC4 expression in BUC tissues was associated with tumor size,multiplicity,tumor stage,tumor grade,and lymph node metastases(P<0.05).Logistic regression analysis demonstrated that only the expression of ARPC4 was independent risk factor for lymph node metastasis of BUC(P<0.05).Conclusion: These findings suggested that ARPC4 protein was significantly upregulated in BUC tissues compared with that of normal bladder tissue and ARPC4 expression was an independent predictor for lymph node metastasis.PART 2 ARPC4 gene silencing inhibits proliferation and migration of bladder cancer cell line T24Objective: To study the regulation of ARPC4 on the biological behavior of bladder cancer T24 cells,To investigate the impact of silencing ARPC4 gene on pseudopodia,skeleton,proliferation,invasion and migration of bladder cancer T24 cells.Methods: si RNA-ARPC4 interference fragment was transfected with liposome,construction of bladder cancer T24 cell line with stable low expression of ARPC4,the transfection efficiency of si RNA-ARPC4 was detected by semi-quantitative RT-PCR,Western blot was used to detect the expression of ARPC4 and downstream cofilin-1 protein after transfection.The experiment was set up 3 groups: si RNA-ARPC4 laboratory group,si RNA-NC negative control group and si RNA-N blank control group,bladder cancer T24 cell proliferation,invasion,migration and cell pseudopod and cytoskeleton were detected by cell counting kit(CCK-8)method,Transwell Chambers,scratch assay and Cell immunofluorescence assay respectively.Results: The expression of ARPC4 m RNA in bladder cancer T24 cells transfected with si RNA-ARPC4 was significantly down-regulated,compared with si RNA-NC negative control group and si RNA-N blank control group,the difference was statistically significant(P <0.05),the inhibition rate of ARPC4 m RNA expression was 73.68%.The expression of ARPC4 and downstream cofilin-1 protein were significantly lower than that of si RNA-NC negative control group and si RNA-N blank control group,and the difference was statistically significant(P <0.05),the proliferation and migration ability of bladder cancer T24 cells in si RNA-ARPC4 group were lower than those in si RNA-NC negative control group and si RNA-N blank control group,the difference was statistically significant(P <0.05),and cell pseudopodia formation disorder,resulting in reduced biological behavior of T24 cells may be related to the regulation of ARPC4 on the formation of pseudopods.Conclusion: Silencing ARPC4 gene can significantly inhibit the proliferation,invasion,migration and pseudopodia formation of bladder cancer T24 cells.