Chinese Clinical Oncology

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Effects of miRNA-199b-3p on the targeted regulation of SOX6 and proliferation and apoptosis of colon cancer cell line SW620

LING Binxun, ASIMUGULI Abdukerim, CAI Yun.   

  1. Department of Internal Medicine, Jiangsu Provincial Tumor Hospital, Nanjing 210009, China
  • Received:2017-01-25 Revised:2017-04-17 Online:2017-06-30 Published:2017-06-30

Abstract: Objective To investigate the effects of microRNA-199b-3p (miR-199b-3p) on the expression of SOX6 and its effect on proliferation and apoptosis of colon cancer cell line SW620. Methods The miR-199b-3p inhibitor and negative control (NC) were transfected into SW620 cells by Lipofectamine liposome method and then divided into inhibitor group and NC group. The SW620 cells without transfection were used as control (non-transfection group). Real-time quantitative PCR (QPCR) was used to detect the expression of miR-199b-3p in three groups as to evaluate the transfection efficiency. MTT and Annexin Ⅴ-FITC/PI flow cytometry were used to detect the proliferative activity and apoptotic rate after transfection, respectively. The mRNA and protein levels of apoptosis related genes (Bax and caspase-3) and SOX6 were detected by QPCR and Western blotting. The luciferase reporter gene plasmids containing wild type and mutant SOX6-3’UTR were constructed. The effect of miR-199b-3p on the regulation of SOX6 was verified by double luciferase reporter assay. Results The QPCR results at 48 h after transfection showed that the level of miR-199b-3p in the inhibitor group was 0.526±0.034, lower than 1.009±0.064 of non-transfection group and 0.960±0.057 of NC group, and the difference was statistically significant (P<0.05). Compared with other two groups, the proliferative activity of the inhibitor group was decreased at 24,48 and 72 h after transfection (P<0.05). The apoptotic rate of the inhibitor group was (37.533±1.459)%, higher than (6.101±0.663)% of non-transfection group and (8.753±1.061)% of NC group, and the difference was statistically significant (P<0.05). The mRNA and protein levels of SOX6, Bax and caspase-3 in inhibitor group were higher than those in non-transfection group and the NC group, and the difference was statistically significant (P<0.05). Dual luciferase reporter gene assay showed that miR-199b-3p could significantly inhibit the luciferase activity of wild-type SOX6-3’UTR, but had no effect on the luciferase activity of mutant plasmid transfected cells. Conclusion MiR-199b-3p can regulate the expression of SOX6, and inhibiting the expression of miR-199b-3p can inhibit the proliferation of SW620 cells and induce apoptosis, which provide a potential molecular therapeutic target for the treatment of colon cancer.

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