miR-152-5p对肝癌细胞恶性生物学行为的抑制作用及其机制

Inhibitory effect of miR-152-5p on malignant biological behavior of hepatocellular carcinoma cells and its mechanism

目的探讨miR-152-5p对肝癌细胞HepG2增殖和凋亡的影响及其相关机制。方法采用RT-PCR检测正常肝细胞HL-7702和人肝癌细胞HepG2中miR-152-5p差异表达。将HepG2细胞系进行脂质体转染并分为:NC mimics组、miR-152-5p mimics、NC inhibitor组和miR-152-5p inhibitor组,采用RT-PCR检测细胞中miR-152-5p的表达水平,采用CCK-8法和EdU法检测各组细胞的活力及增殖能力,利用流式细胞术及Western blotting检测各组细胞凋亡率以及细胞中凋亡相关蛋白Bcl-2、Bax、cleaved Caspase-3的表达水平。HepG2细胞系进行脂质体转染并分为:NC inhibitor组、miR-152-5p inhibitor组、miR-152-5p inhibitor+LY294002(PI3K/AKT通路抑制剂)组,应用Western blotting、EdU法和流式细胞术检测细胞PI3K/AKT通路相关蛋白PI3K(p-PI3K/PI3K)、AKT(p-AKT/AKT)的磷酸化水平、增殖水平和凋亡率。结果与HL-7702细胞相比,HepG2细胞中miR-152-5p表达显著下降(P<0.01)。与NC mimics组相比较,miR-152-5p mimics组miR-152-5p的表达上调(P<0.05),细胞活力和增殖能力显著下调(P<0.05),凋亡率和促凋亡蛋白cleaved Caspase-3、Bax的表达上调(P<0.05),凋亡抑制蛋白Bcl-2表达均显著下降(P<0.05);与NC inhibitor组相比,miR-152-5p inhibitor组miR-152-5p的表达下调(P<0.05),细胞活力和增殖能力显著升高(P<0.05),凋亡率和促凋亡蛋白cleaved Caspase-3、Bax的表达下调(P<0.05),凋亡抑制蛋白Bcl-2表达均显著上调(P<0.05)。与miR-152-5p inhibitor组相比,miR-152-5p inhibitor+LY294002组细胞PI3K、AKT蛋白的磷酸化水平显著降低(P<0.05),增殖能力降低(P<0.05),而细胞凋亡率显著升高(P<0.05)。结论miR-152-5p能够通过PI3K/AKT信号通路抑制肝癌细胞的增殖活力,并促进细胞凋亡。

Objective To investigate the effect of miR-152-5p on the proliferation and the apoptosis of hepatocellular carcinoma cancer cells HepG2 and its related mechanisms.Methods RT-PCR was used to detect the differential expression of miR-152-5p in HL-7702(normal liver cells)and HepG2(human liver cancer cells).HepG2 cell lines were divided into:NC mimics group,miR-152-5p mimics group,NC inhibitor group and miR-152-5p inhibitor group.The expression levels of miR-152-5p was detected by RT-PCR.The viability and the proliferation ability were detected by CCK-8 and EdU method.Flow cytometry and Western blotting were used to detect the cell apoptosis rate and the expression levels of apoptosis-related proteins Bcl-2,Bax,cleaved Caspase-3.HepG2 cell lines were divided into:NC inhibitor group,miR-152-5p inhibitor group,miR-152-5p inhibitor+LY294002(PI3K/AKT pathway inhibitor)group,and then the phosphorylation levels of PI3K/AKT pathway-related proteins(p-PI3K/PI3K)and AKT(p-AKT/AKT),the proliferation level and the apoptosis rate were detected by Western blotting,EdU and flow cytometry,respectively.Results Compared with HL-7702 cells,the expression of miR-152-5p in HepG2 cells was significantly decreased(P<0.01).Compared with NC mimics group,the expression of miR-152-5p in miR-152-5p mimics group was increased(P<0.05),the cell viability and the proliferation levels were significantly decreased(P<0.05),the apoptosis rate and the expression of pro-apoptotic proteins cleaved Caspase-3 and Bax were increased(P<0.05),and the expression of apoptosis inhibition protein Bcl-2 was significantly decreased(P<0.05).Compared with NC inhibitor group,the expression of miR-152-5p was decreased in miR-152-5p inhibitor group(P<0.05),the cell viability and the proliferation were significantly increased(P<0.05),the apoptosis rate and the expression of pro-apoptotic protein cleaved Caspase-3 and Bax were decreased(P<0.05),and the expression of apoptotic inhibition protein Bcl-2 was significantly increased(P<0.05).Compared with miR-152-5p inhibitor group,the phosphorylation levels of PI3K and AKT proteins in miR-152-5p inhibitor+LY294002 group were significantly decreased(P<0.05),the proliferation ability was decreased(P<0.05),while the apoptosis rate was significantly increased(P<0.05).Conclusion The miR-152-5p can inhibit the proliferation of liver cancer cells and promote the cell apoptosis through the PI3K/AKT signaling pathway.