Effects of miR-185 on proliferation and apoptosis of hepatocellular carcinoma cells

LI Manni; LIU Jianping; TAO Yongsheng; CHEN Jingbo; CHEN Xinxin; TAN Zhiqin

doi:10.3969/j.issn.1002-266X.2016.41.003

Abstract：Objective To investigate the effects of miR-185 on proliferation and apoptosis of hepatocellular carcinoma cells and its mechanism.Methods The online prediction software was employed to predict the target genes of miR-185. The effect of miR-185 on the activity of pyruvate kinase isozyme type M2 (PKM2)3′UTR was examined by luciferase re-porter vector system.The effect of miR-185 on PKM2 protein expression was observed by Western blotting.HepG2 cells were divided into the observation group 1,control group 1,observation group 2,control group 2,and observation group 3. The observation group 1 was transfected by miR-185 mimics,the control group 1 was transfected with scramble,the obser-vation group 2 was transfected by PKM2-siRNA,the control group 2 was transfected by control-siRNA,and the observation group 3 was transfected by miR-185 inhibitors and PKM2-siRNA.MTT assay was used to observe the changes of cell prolif-eration in the five groups,and Annexin V-FITC and PI staining were used to measure the apoptotic rate of the five groups. Results The online prediction software confirmed that miR-185 and 3'UTR of PKM2 had the common binding sites.Lucif-erase reporter vector system showed that miR-185 inhibited the luciferase activity of Wild-PKM2 reporter plasmid.Western blotting showed that the protein level of PKM2 was decreased by miR-185,which showed that PKM2 was a target gene di-rectly regulated by miR-185.MTT assay showed that the OD values of HepG2 cells in the observation group 1 and observa-tion 2 were 0.446 ±0.034 and 0.472 ±0.028,respectively,which were significantly lower than those in control group 1 (0.649 ±0.041)and control group 2 (0.610 ±0.023)(all P <0.05).The OD value of the observation group 3 was 0.606 ±0.016,which was not significantly different as compared with that of the control group 1 or the control group 2. The apoptotic rates of HepG2 cells in the observation group 1 and observation group were 29.13% ±2.04% and 27.46%±1.95%,which were significantly higher than those in control group 1 (8.76% ±0.53%)and control group 2 (8.51%±0.47%),and the difference was statistically significant between the observation group 1,2 and the control group 1,2 (all P <0.05).The apoptotic rate of the observation group 3 was 9.47% ±0.61%,which was not significantly different from that of the control group 1 or 2.Conclusion miR-185 inhibits cell proliferation and induces apoptosis by down-regula-ting PKM2 expression in hepatocellular carcinoma.