AMPKγ基因沉默对AMPK活化及调血脂作用的影响

Impact of AMPKγ silencing on AMPK activation and intracellular lipids regulation

构建针对AMPKγ基因的siRNA慢病毒载体,检测其介导的RNA干扰对人肝癌细胞株HepG2中AMPKγ基因表达的沉默作用,并考察AMPKγ基因沉默对天然AMPK激动剂虫草素激活AMPK和调节脂质合成的影响。设计并合成了特异针对AMPKγ基因的shRNA Oligo片段,经退火、酶切、连接将目的片段插入到慢病毒干扰载体中,经感受态细胞转化、PCR鉴定挑选阳性克隆测序后,与包装质粒和包膜质粒共转染至293T细胞包装病毒,收集浓缩病毒进行滴度测定。通过荧光观察和Western blotting筛选可有效沉默AMPKγ基因的慢病毒株。在AMPKγ基因稳定沉默细胞株中,利用Western blotting考察AMPKγ基因沉默对虫草素促AMPK磷酸化活性的影响,油红O染色观察虫草素对细胞内脂质堆积情况的影响,并用试剂盒测定细胞内TC、TG含量。结果表明,成功构建了4种特异针对AMPKγ基因的慢病毒干扰载体(GR084、GR085、GR086、GR087),Western blotting检测结果显示重组慢病毒株GR085的基因沉默效率最高;给予虫草素并不会影响AMPKγ蛋白的表达,但能够显著上调正常细胞内AMPK蛋白的磷酸化水平。利用慢病毒稳定沉默AMPKγ基因的表达后,虫草素促AMPK磷酸化作用和下调脂质合成作用被显著抑制。研究表明,AMPKγ亚基可能与虫草素活化AMPK和调节脂质合成作用相关。

The study is aimed to confirm the silencing efficiency of the vector in human hepatocellular liver carcinoma cell line （HepG2）, and observe effects of AMPKy silencing on the AMPK stimulating activity and lipid synthesis of cordycepin （CCS）, a natural product with known AMPK activating function. The downregulating efficacy of siRNAs on AMPKy expression was confirmed in our previous study. The double stranded shRNA Oligo was ligated to lentivirus vector and verified by sequencing. The lentiviral which can effectively inhibited protein expression levels of AMPKy was selected by Western blotting, and the regulation of CCS on protein expression of AMPKy and p-AMPK in AMPKy silence cells were detected by Western blotting analysis. The lipid accumulation in cells was observed by Oil-Red O stain and cells were collected for the estimation of cholesterol （TC）, triglyceride （TG）. The results showed that the lentiviral vector carrying a shRNA targeting the AMPK7 gene was successfully constructed. Western blotting analysis confirmed that GR085 had the highestinterfering efficiency. Treatment with CCS can significantly increase the levels of phospho-AMPK in normal cells, and the level of TC, TG was reduced, but in AMPKy silence cells the effects of CCS on AMPK activation and lipid synthesis were almost completely abolished without changing the expression levels of total AMPK or AMPKy protein. In conclusion, the AMPKy gene may be related to AMPK activation and intracellular lipids regulation by CCS.