Effects of miR-1280 expression on the cell cycle and proliferation of bladder cancer by activating p21 gene expression

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 expre-ssion 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. Key words: MicroRNAs; Urinary bladder neoplasms; Proto-oncogene proteins p21 (ras); RNA activation