Effect of miR-527 expression on proliferation, migration and invasion of bladder cancer cells / 中华泌尿外科杂志

ABSTRACT

Objective:To investigate the expression of miR-527 in bladder cancer (BC) and its effect on the proliferation, migration and invasion of bladder cancer cells.Methods:From February 2018 to June 2019, the immortalized human bladder epithelial cell line SV-HUC-1 and human bladder cancer cell lines T24, UM-UC-3, 5637 and RT-112 were cultured in vitro. Real time quantitative PCR (qRT-PCR) was used to detect the expression of miR-527 in BC bladder cancer tissues and adjacent normal bladder tissues, human bladder cancer cell lines and human bladder epithelial immortalized cell lines. MiR-527 mimics, miR-527 inhibitor, ENO1 overexpression plasmid, ENO1 siRNA and corresponding negative control were transfected into bladder cancer cell line. CCK8 test, clone formation test and Transwell test were used to study the cell proliferation, migration and invasion. Luciferase reporter gene assay was used to verify the target gene of miR-527. Western blotting was used to analyze the regulation of miR-527 on target gene expression.Result:Compared with normal bladder tissue, the expression of miR-527 in bladder cancer was significantly lower (1.723±1.070 vs. 1.148±0.760, P<0.05). The relative expression of miR-527 in T24 (0.540±0.082), UM-UC-3 (0.230±0.053), 5637(0.463±0.085) and RT-112 (0.310±0.056) were significantly lower than those in SV-HUC-1 cells (0.987±0.111) with statistical significance ( P<0.05). Compared with the negative control (NC) group, CCK8 assay results showed that the cell viability was significantly decreased after transfection of miR-527 mimics into UM-UC-3 cells ( P<0.05). The clone formation test showed that the number of cell clones in UM-UC-3 cells transfected with miR-527 mimics was significantly lower than that in the control group (157.00±15.52 vs 57.33±15.50, P<0.05). Compared with the control group, the cell activity of T24 cells transfected with miR-527 inhibitor was significantly increased ( P<0.05). Compared with the control group, the number of cell clone formation was significantly increased (76.67±9.07 vs. 141.70±10.50, P<0.05). According to the prediction of targetscan database, ENO1 was the target gene of mir-527. Luciferase reporter gene experiment showed that the luciferase activity of mir-527 mimics group was significantly lower than that of control group (0.99±0.02 vs. 0.47±0.10, P<0.05), while the luciferase activity of miR-527 mimics group was significantly lower than that of control group (0.99 ± 0.02 vs. 0.47 ± 0.10, P<0.05), without statistical significance (1.03±0.04 vs. 0.96±0.05, P>0.05). Western blot analysis showed that the expression of ENO1 in miR-527 mimics group was significantly lower than that in NC mimics group (1.09±0.17 vs. 0.31±0.13, P<0.05), and the expression of ENO1 in miR-527 inhibitor group and NC inhibitor group were significantly increased (0.97±0.09 vs. 2.17±0.15, P<0.05). Compared with miR-527 mimics group, transfection of miR-527 mimics+ ENO1 overexpression plasmid could reduce the inhibitory effect of miR-527 mimics on proliferation, migration and invasion of bladder cancer cell line ( P<0.05). Compared with miR-527 inhibitor group, transfection of miR-527 inhibitor+ ENO1 siRNA could weaken the inhibit effect of miR-527 on the proliferation, migration and invasion of bladder cancer cell lines ( P<0.05). Conclusion:miR-527 is low expressed in BC and can be used as a tumor suppressor gene to inhibit the proliferation, migration and invasion of BC cells.