微小RNA-373靶向调控LATS2的表达及其对肝癌细胞HepG2增殖与凋亡的影响

The influence of miR-373 on the proliferation and apoptosis of hepatocellular carcinoma HepG2 cells by targeting LATS2

目的探讨微小RNA-373(miR-373)靶向调控大肿瘤抑制因子2(LATS2)的表达及其对肝癌细胞HepG2增殖与凋亡的影响。方法采用实时荧光定量PCR(QPCR)法检测HepG2细胞及正常肝细胞LO2中miR-373的表达水平,采用Lipofectamine脂质体法将miR-373抑制剂及阴性对照转染至HepG2细胞并分为抑制剂组和对照组,采用QPCR法检测两组的miR-373水平以评价转染效果,MTT法和流式细胞术分别检测各组细胞的增殖及凋亡情况,Western blotting检测各组凋亡相关基因(Bax和caspase-3)及LATS2的表达情况,采用双荧光素酶报告实验验证miR-373与LATS2间的靶标关系。结果与LO2细胞相比,HepG2细胞中miR-373的表达水平升高,差异有统计学意义(P<0.05);转染后抑制剂组的miR-373水平低于对照组(P<0.05),提示抑制HepG2细胞miR-373表达成功;与对照组比较,抑制组的细胞增殖率降低,凋亡率及Bax、caspase-3和LATS2蛋白水平均升高,差异有统计学意义(P<0.05);双荧光素酶报告基因实验表明miR-373可显著抑制野生型LATS2-3’UTR质粒转染细胞的荧光素酶活性,而对突变型LATS2-3’UTR质粒转染细胞的荧光素酶活性并无影响。结论 miR-373可靶向调控LATS2的表达,且抑制miR-373表达可抑制HepG2细胞增殖并诱导凋亡,为肝癌的靶向治疗提供一定参考。

Objective To explore the effect of microRNA-373 （miR-373） on the expression of large tumor suppressor 2 （LATS2） and its influence on the proliferation and apoptosis of hepatocellular carcinoma cell line HepG2. Methods The miR-373 level was detected in HepG2 cells and normal hepatocytes LO2 by fluorescence real-time quantitative PCR（QPCR）. HepG2 cells were assigned into inhibitor group and control group, and transfected with miR-373 inhibitors or negative control （NC） by Lipofectamine li- posome method. The miR-373 levels of both groups were detected by QPCR in order to evaluate the transfection efficiency. MTT method and flow cytometry were used to detect the cell proliferation and apoptosis. The expressions of apoptosis related genes （ Bax and caspase- 3 ） and LATS2 in each group were detected by Western blotting. The relationship between miR-373 and LATS2 was verified using doub- le luciferase reporter assay. Results Compared with LO2 cells, the level of miR-373 in HepG2 cells was increased, and the difference was statistically significant （P〈0. 05 ）. The level of miR-373 in the inhibition group was lower than that of control group （P〈0. 05 ）, indicating that the miR-373 expression of HepG2 cells was inhibited successfully. Compared with control group, there were decreased proliferative rate, and increased apoptotic rate and protein levels of Bax, caspase-3 and LATS2 in inhibitor group （P〈0. 05）. Dual lu- ciferase reporter assay showed that miR-373 could significantly inhibit the luciferase activity of cells transfected with wild-type LATS2- 3＇ UTR, and had no effect on lueiferase activity of ceils transfected with mutant LATS2-3＇ UTR. Conclusion MiR-373 can regulate the expression of LATS2, and inhibiting the expression of miR-373 can inhibit the proliferation and induce apoptosis of HepG2 cells, providing some reference for the targeted therapy of hepatocellular carcinoma.