miR-26a mimics转染人肝癌细胞株HepG2的表达蛋白质组分析

Analysis of expressive proteome in human hepatocarcinoma cell HepG2 transfected with miR-26a mimics

目的:通过分析microRNA-26a(miR-26a)mimics转染对人肝癌细胞株HepG2表达蛋白质组的影响,以确定miR-26a与肝癌发生发展的相关性。方法:常规培养人肝癌细胞株HepG2,经miR-26a mimics转染48 h后进行细胞周期分析,并裂解转染72 h的HepG2细胞提取蛋白,双向电泳分离,匹配对比各蛋白斑点的表达量,筛选主要差异表达蛋白进行质谱鉴定。结果:HepG2细胞经miR-26a mimics转染后细胞增殖受到抑制;其蛋白2-DE图谱与对照组比较,差异表达超过2倍的蛋白斑点有11个。其中,有3个蛋白斑点为表达上调,有8个蛋白斑点为表达下调。质谱鉴定为:膜联蛋白A1、过氧化物酶4、增殖细胞核抗原、载脂蛋白A1、细胞色素C氧化酶5a、细胞周期蛋白E2、磷酸核糖焦磷酸激酶3、周期素依赖性蛋白激酶1和磷脂酰乙醇胺结合蛋白。结论:miR-26a可能通过影响上述蛋白分子的表达,直接或间接地调控HepG2肝癌细胞的增殖、分化和死亡,以发挥其抗癌作用。

AIM： To determine whether microRNA -26a （miR- 26a） is involved in development of liver cancer by analysis of proteomic expression profile of human hepatocarcinoma cell HepG2 transfected with miR - 26a mimics. METHODS ： HepG2 cells were cultured by a routine method and transfected with miR - 26a mimics for 48 h for cell cycle analysis. The expressive proteome profiles of HepG2 cells with or without miR -26a mimics treatment were established by the methods of two - dimensional electrophoresis separation following lysis of the cells and extraction of the proteins. The proteomic expression profiles were analyzed by comparative proteomics technique to discover the important protein spots with differential expression. The identification of the proteins was conducted by matrix - assisted laser desorption/ ionization time - of - flight mass spectrometry（ MALDI - TOF - MS）. RESULTS ： miR - 26a brought down the proliferation of HepG2 cells. Total 11 protein spots with alteration of expressive amounts more than 2 times were successfully identi- fied in the proteomic expression profile of HepG2 cells treated with miR - 26a mimics, including annexin A1, peroxiredoxin 4, proliferating cell nuclear antigen, apolipoprotein A1, cytochrome C oxidase subunit 5A, cyclin E2, ribose -phosphate pyrophosphokinase 3, cyclin - dependent kinase 1 and phosphatidylethanolamine - binding protein 1. Among these, the expression of 3 protein spots was up - regulated and 8 of them was down - regulated. CONCLUSION ： miR - 26a contributes to the anti - cancer effect by expressive regulation of the proteins mentioned above, or directly or indirectly controls the proliferation, differentiation and death of hepatocarcinoma cells.