Effects of miR-1280 expression on the cell cycle and proliferation of bladder cancer by activating p21 gene expression / 国际肿瘤学杂志

ABSTRACT

Objective To investigate the activation effect of microRNA-1280 (miR-1280) on the expression of p21 gene in bladder cancer cell line BIU-87 and its effect on cell cycle and proliferation of bladder cancer cell line.Methods Real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) was used to detect the expressions of miR-1280 in bladder cancer cell lines T24,5637,J82,BIU-87 and normal bladder epithelial cells SV-HUC-1.miR-1280 mimics (experimental group) and miR-NC (control group) were transfected into the bladder cancer cells with the lowest expression of miR-1280.The expressions of miR-1280 and p21 mRNA were detected by qRT-PCR.Chromatin immunoprecipitation (ChIP) was used to verify the targeting effect of miR-1280 and p21 gene promoter.Western blotting was used to detect the expressions of p21,cell cycle-dependent kinase 1 (CDK1),Cyclin A2 mRNA and protein in the two groups.Cell cycle was detected by flow cytometry,and cell proliferation was detected by methyl thiazolyl tetrazclium (MTT) assay.Results The results of qRT-PCR indicated that the expression levels of miR-1280 in bladder cancer cell lines T24,5637,J82 and BIU-87 and normal urothelium cell line SV-HUC-1 were 0.503 ±0.094,0.611 ±0.054,0.567 ± 0.077,0.257 ± 0.032 and 1.014 ± 0.090 respectively,with a significant difference (F =1.880,P ＜0.001).Compared with bladder cancer cell lines T24,5637 and J82 cells,the expression of miR-1280 in BIU-87 cell was the lowest (P =0.026,P =0.003,P =0.008).Compared with the control group,the expression of miR-1280 in BIU-87 cell was significantly increased (1 041.000 ± 157.500 vs.1.023 ± 0.118,t =6.606,P ＜0.001),and the expression of p21 mRNA was also significantly increased (5.280 ± 0.660 vs.1.007 ± 0.070,t =6.440,P ＜ 0.001).Western blotting showed that p21 protein expression was up-regulated,CDK1 and Cyclin A2 protein expressions were down-regulated.ChIP experiments showed that compared with the miR-NC transfection group,the concentration of biotin modified miR-1280 in the p21 gene promoter region was significantly increased (1.246 ±0.171 vs.0.519 ± 0.087,t =3.787,P =0.009).The proportion of G0-G1 cells in the experimental group BIU-87 cells was significantly higher than that in the control group (68.360％ ±3.064％ vs.46.970％ ±3.971％,t =4.263,P =0.005).The results of MTT showed that compared with the control group,the cell proliferation ability of BIU-87 cells after being transfected miR-1280 was significantly decreased starting from day 3 (0.826 ± 0.099 vs.1.224 ± 0.057,t =3.505,P =0.013).Conclusion miR-1280 can activate the expression of p21 gene in bladder cancer cell line BIU-87 by binding the promoter region of p21 gene,blocking the progression of cell cycle and inhibiting cell proliferation,which provides a new direction for bladder cancer targeted therapy theory.