The Role Of Small Ubiquitin-related Modifier-specific Protease 2 And MicroRNA-495 In The Occurrence And Development Of Bladder Urothelial Carcinoma

Objectives:1.To evaluate the effect of SENP2 on the expression of SENP2 in normal bladder epithelial cells and different grade malignant cancer cell lines and the effect of SENP2 on EMT of bladder cancer cell line,and to explore its clinical value as a therapeutic target.2.To explore the specific mechanism of SENP2 inhibiting EMT of bladder cancer cell line through inhibiting TGF-?/Smad signaling pathway,so as to provide a more adequate basis for SENP2 as a therapeutic target treating bladder cancer.3.To study the role of miR-495 in the development and progression of bladder cancer and to study the specific target gene of miR-495 inhibition,and to provide theoretical basis for further clinical treatment.Materials and Methods:1.(1)qRT-PCR was used to detect the expressions of SENP2 mRNA in non-invasive bladder urothelial carcinoma cell lines RT4 and DSH1,and invasive bladder urothelial carcinoma cell lines TCCSUP and T24.(2)The mRNA expressions of E-cadherin in non-invasive bladder cancer cell lines RT4 and DSH1 as well as the invasive bladder cancer cell lines TCCSUP and T24 were detected by qRT-PCR,the relationship between the expression of E-cadherin and SENP2 was analyzed.(3)Over expression of SENP2 and siRNA silencing techniques were used to detect the differential expression of E-cadherin,N-cadherin and fibronectin mRNA and protein level in bladder cancer cells.2.(1)Western blots were used to detect the phosphorylation of Smad2/3 which is downstream signal molecules of TGF-? in different SENP2 expression levels.(2)Double luciferase reporter gene system was utilized for the detection of bladder cancer cells TGF-?1 uptake ability.(3)The expressions of E-cadherin mRNA in bladder cancer cells with different SENP2 expression levels and different time after co-cultured with TGF-?1 were detected by qRT-PCR.(4)Transwell was used to detect invasive abilities of bladder cancer cells with different SENP2 expression levels and the different TGF-?1 environments.3.(1)qRT-PCR was used to evaluate the miR-495 expression difference between in bladder cancer cell lines J82,T24,UMUC3 and TCCSUP and normal bladder epithelium SV-HUC-1.(2)BrdU assay was used to detect the effects of miR-495 overexpression and miR-495 inhibition on proliferation of bladder cancer cell lines.(3)Transwell was used to detect the effect of different miR-495 expression on the invasive ability of bladder cancer cells.(4)The double luciferase reporter gene assay was used to detect the combining ability of miR-495 on PTEN mRNA 3'-UTR.(5)Western blot was used to detect the effect of different miR-495 expression on PTEN protein expression.Results:1.(1)The mRNA expression of SENP2 in bladder cancer cell line was significantly lower than that in normal bladder urothelial cells,and the expression of SENP2 mRNA in bladder invasive urothelial carcinoma cell line was significantly lower than that in bladder non-invasive urothelial carcinoma cell line.(2)The mRNA expression level of E-cadherin in bladder cancer cell line was significantly lower than that in normal bladder urothelial cells,and the expression level of E-cadherin was positively correlated with SENP2.(3)The expression of E-cadherin mRNA and protein in T24 cells after overexpression of SENP2 was significantly higher than control group,while the mRNA and protein expression of N-cadherin and fibronectin were significantly decreased compared with those in the control group.The expression of E-cadherin mRNA and protein in RT4 cells after suppression of SENP2 was significantly lower than control group,while the mRNA and protein expression of N-cadherin and fibronectin were significantly increased compared with those in the control group.2.(1)TGF-?1 can significantly increase the phosphorylation level of Smad2/3 protein in T24 and RT4 cells,while overexpression of SENP2 can inhibit the phosphorylation of Smad2/3 and thus inhibit TGF-?/Stmad signaling pathway.(2)After co-cultured with TGF-?1 The intracellular expression of TGF-?1 in SENP2 overexpression T24 and RT4 cells was significantly lower than that of the low SENP2 expression T24 and RT4 cells and the SENP2-mut overexpression T24 and RT4 cells.(3)After co-cultured with TGF-?1,the expression of E-cadherin in SENP2 overexpression T24 cells and RT4 cells was higher than the control group.(4)High expression of T24 cells can significantly reduce the invasive ability of T24 cells,overexpression of SENP2 in T24 cells can reverse the enhanced invasive ability induced by co-culture of TGF-?1.3.(1)The expression of miR-495 in bladder cancer cell lines J82,T24,UMUC3 and TCCSUP was higher than that in normal bladder epithelial cells SV-HUC-1.(2)Overexpression of miR-495 can significantly increase the proliferation of J82 and T24 bladder cancer cells,and inhibition of miR-495 expression can inhibit the two kinds of bladder cancer cells in the value-added ability.(3)Overexpression of miR-495 can improve the invasive ability of J82 and T24 bladder cancer cells,while knockdown of miR-495 can inhibit the invasive ability of J82 and T24 bladder cancer cells.(4)Compared with the control group,miR-495 could significantly reduce the fluorescence activity of wt-PTEN-3'-UTR-pGL3 but not reduce the fluorescence activity of mut-PTEN-3'-UTR-pGL3.(5)MiR-495 can inhibit the protein expression of PTEN in J82 and T24 bladder cancer cells.Conclusions:1.The expression of SENP2 in bladder epithelial cancer cells is significantly lower than that in normal bladder epithelial cells,and it is negatively correlated with the degree of malignancy.SENP2 inhibits the development of bladder cancer by inhibiting the level of EMT in bladder cancer.2.SENP2 inhibits TGF-?1-induced EMT by suppressing the SUMOylation of TGF-?/Smad signaling pathway in bladder cancer cells,vand so that suppresses the invasion of bladder cancer cells.3.The expression of miR-495 in bladder urothelial carcinoma cell line is significantly higher than that in normal bladder epithelial cells.miR-495 can promote the development of bladder cancer by inhibiting the expression of tumor suppressor gene PTEN,and provides a new target for further improving the early diagnosis of bladder cancer and clinical treatment.