Effects Of RhoGDI2 Gene On Clinicopathological And Prognosis And Its Downstream Signaling In Bladder Cancer

Objective: To investigate the effect of RhoGDI2 gene on the metastasis and prognosis of bladder cancer,and to explore its downstream pathway.Method: 1.The expression of RhoGDI2 protein in tissue microarray was detected by immunohistochemistry,and the correlation between the expression of RhoGDI2 protein and the pathology/prognosis of bladder cancer was analyzed.2.The data of the whole genome expression profile and the clinical data of the bladder cancer tissue were downloaded from the database of the Cancer Genome Atlas(TCGA).To analyze the correlation between the expression level of RhoGDI2 gene and the clinicopathological and prognosis of bladder cancer patients.Using TCGA database of bladder cancer gene expression profiles and DAVID online tools to enrich RhoGDI related downstream signalingpathway.3.Construction of RhoGDI2 positive and negative expression cell lines by lentivirus transfection,and Western blotting was used to verify the possibility of RhoGDI2 downstream pathway.4.Western blotting was used to confirm the correlation between the expression of RhoGDI2 and the enriched signal pathway in 18 cases of bladder cancer.A total of 16 related indicators need to be examined: RhoGDI2,Cdc42,Rac123,Phosphor-Rac1/cdc42(ser71),RhoA,RhoB,RhoC,Raf1,Ras,Rock2,MEK1/2,,Erk1,Vav2,Vav3,MLC2,and P-MLC2(S19).Result:1.Bladder cancer tissue microarray analysis results: There was a significant negative correlation between T2 ~ T3 stage,T1-2 ~T3-4 stage,metastasis and RhoGDI2(P<0.05).There was a significant positive correlation between the prognosis and RhoGDI2(P<0.05).2.TCGA data analysis results:RhoGDI2 was negatively correlated with the T2~T3 stage and II~III clinical stage,and there was a significant negative correlation with pathological grade(P<0.05),and there was no significant correlation with the invasion of the myometrium(P>0.05).There was a significant positive correlation between the prognosis and RhoGDI2(P<0.05).The median survival time,median tumor-free survival time,5year survival rate and 5year disease-free survival rate of RhoGDI2 high expression group were significantly longer than those of low expression group(P<0.05).A total of 5960 differentially expressed genes related to RhoGDI2 were screened from the whole gene expression profile of bladder cancer in TCGA database.After inclusion of David's online tools,there were 105 signals associated with RhoGDI2,among which there were 9 genes related to Cdc42,RhoA and Rac,and the genes contained in RhoGDI2 were positively correlated with RhoGDI2 expression.Inaddition,there are 18 pathways involved in the Ras gene,and there is a negative relationship between the gene and the Rho GDI2 gene.3.Validation of RhoGDI2 regulatory pathway by in vitro cell Western blotting,RhoGDI2,Cdc42,Rac123,RhoA,Ras and MLC2 in T24-RhoGDI2 cells were significantly higher than those in control group,and significantly lower in T24-RhoGDI2-RNAi than control group(P <0.05).The expression of Rock2,Erk1,Vav2 and Vav3 was significantly decreased in T24-RhoGDI2-RNAi cell line(P<0.05),MEK1/2 and P-MLC2 were significantly decreased in T24-Rho GDI2 group(P<0.05).The expression of RhoC in T24-RhoGDI2-RNAi and T24-RhoGDI2 was significantly higher than that in controlgroup(P<0.05),Phosphor-Rac1/cdc42(ser71),Rho B and Raf1 were not expressed in the5 cell lines.4.Bladder cancer tissue specimens Western blotting validation RhoGDI2 regulatory pathway:(1)In ancer group,the expression of CDC42,Rac123,RhoA,Ras(P21 Waf / Cip1(12D1),Erk1,Vav2,MLC2 and RhoGDI2 were significantly positively correlated in Spearmon correlation analysis(P<0.05),while MEK1/2,P-Mlc2 expression is contrary to the above indicators,Rock2 and RhoGDI2 there is no significant correlation(P<0.05).(2)There was no significant correlation between Rho GDI2 and related protein in normal group(P>0.05).(3)The expression of RhoGDI2 and RhoA in cancer group was significantly lower than that in normal group(P<0.05),Rock2,Ras,MEK1/2,Erk1,Vav2,MLC2,P-Mlc2 were significantly up-regulated in cancer group(P<0.05).There was no significant difference in the expression of Rac123 and CDC42 in the cancer group and the normal group(P>0.05).(4)In the RhoGDI2 low expression group,the expressionof RhoGDI2,Cdc42,Rac123 and RhoA in the cancer group was significantly lower than that in the normal group(P<0.05),while MEK1/2 was significantly higher than that in the control group(P<0.05).And in the high expression of RhoGDI2 group,the expression of Cdc42 and Ras in the cancer group was significantly higher than that in the normal group(P<0.05).In the cancer group,Rock2,Erk1,Vav2,MLC2,P-MLC2,whether in high Rh OGDI2 expression or low expression group,were up-regulated relative to the normal group(P<0.05).Conclusion:1.RhoGDI2 is a key factor in pathological stage,clinical stage,pathological grade,metastasis and prognosis of bladder cancer.2.RhoGDI2 may play a major role in regulating the downstream signaling pathways through regulation of Rho GTP2 and that pathway are Vav-RhoA-ROCK-MLC and Ras-MEK-Erk,and ultimately inhibit the development of bladder cancer and improve the prognosis of biology Effect,so the gene and its signal pathway may become a new target for bladder cancer targeting therapy.