miR-185对肝癌细胞增殖、凋亡的影响及机制

摘要

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.%目的：探讨 miR-185对肝癌细胞增殖、凋亡的影响及机制。方法用 Target scan human 在线软件预测miR-185调控的靶基因；采用荧光素酶报告载体系统检测 miR-185对 M2型丙酮酸激酶（PKM2）3′UTR 荧光酶活性的影响；蛋白质印迹法检测 miR-185对 PKM2蛋白表达的影响。将肝癌 HepG2细胞分为观察1组、对照1组、观察2组、对照2组、观察3组。观察1组转染 miR-185 mimics，对照1组转染 Scramble；观察2组转染 PKM2-siRNA，对照2组转染 Control-siRNA；观察3组转染 miR-185 inhibitors 与 PKM2-siRNA。采用 MTT 法检测上述5组细胞增殖能力，采用 AnnexinV-FITC 和 PI 染色法检测上述5组细胞凋亡率。结果在线预测软件发现 miR-185与 PKM2的3′UTR 有共同的结合位点，荧光素酶报告载体系统显示在 HepG2细胞中 miR-185能抑制 Wild-PKM2报告质粒组荧光素酶的活性，Western blotting 结果显示 miR-185能下调 HepG2细胞中 PKM2蛋白表达，证实 PKM2是 miR-185直接调控的靶基因；MTT 结果显示，观察1组与观察2组 HepG2细胞 OD 值分别为0．446±0．034、0．472±0．028，与对照1组（0．649±0．041）和对照2组（0．610±0．023）相比，差异均有统计学意义（P 均＜0．05）。观察3组的 OD值为0．606±0．016，与对照1组或对照2组相比，差异均无统计学意义；凋亡实验结果显示，观察1组与观察2组HepG2细胞凋亡率分别为（29．13±2．04）％、（27．46±1．95）％，对照1组、对照2组分别为（8．76±0．53）％、（8．51±0．47）％，观察1组、观察2组与对照1组、对照2组比较，P 均＜0．05。观察3组的凋亡率为（9．47±0．61）％，与对照1组或对照2组相比，差异均无统计学意义。结论 miR-185可抑制人肝癌细胞增殖并诱导细胞凋亡；机制可能是通过下调 PKM2表达实现。