转染rnf6 基因对肝癌细胞irs-2 表达的影响 - 生物技术通报.pdf

生物技术通报 ·研究报告· BIOTECHNOLOGY BULLETIN 年第 期 2014 6 转染 RNF6 基因对肝癌细胞 IRS-2 表达的影响 巩健  宋健 （淄博职业学院制药与生物工程系，淄博 255314） 摘 要 ： 构建环指蛋白6 （RNF6 ）真核表达载体，并探讨其对胰岛素受体底物1 （IRS-2 ）表达的影响。以人cDNA 为模板， PCR 扩增RNF6 全长编码基因，并将其克隆至载体pcDNA3.1-CHA 中，将重组质粒转染肝癌细胞株HepG2 ，利用Real time-PCR、 Western blot 检测细胞内IRS-2 mRNA 水平及蛋白表达情况。携带RNF6 目的基因的质粒转染HepG2 细胞48 h 后IRS-2 的mRNA 表 达降低，为对照组的37% ，显著低于对照组，差异有统计学意义 （ 0.0 1 ）。 RNF6 引起IRS-2 的表达下调，这一过程可能由于泛 P 素化导致胰岛素信号转导通路障碍。 关键词 ： 胰岛素受体底物-2 泛素化 环指蛋白6 胰岛素 Effect of Gene Ring Finger Protein 6 on the Expression of Insulin - Receptor Substrate 2 in Hepatoma Cells Gong Jian Song Jian （Dep artment of Pharmaceutical and Biological Engineering ，Zibo Vocational Institute ，Zibo 255314 ） Abstract:  It was to construct an eukaryotic expression vector of ring finger protein 6 （RNF6 ）gene and investigate the effect of RNF6 on the expression of insulin receptor substrate-2 （IRS-2 ）. The coding sequence of hRNF6 gene was amplified by PCR with human cDNA as template. The pcDNA3.1-CHA-RNF6 was constructed and transfected into hepatocarcinoma cells （HepG2 ）by routine mole cular biology technology. The total RNA was extracted from HepG2 cells 72 hours post-transfection, the expression levels of IRS-2 was detected by real-time quantitative PCR. Western blotting was applied to detect the protein levels of IRS-2. Result showed that the mRNA level of IRS-2 gen