Impact of AMPKgamma silencing on AMPK activation and intracellular lipids regulation / 药学学报

ABSTRACT

The study is aimed to confirm the silencing efficiency of the vector in human hepatocellular liver carcinoma cell line (HepG2), and observe effects of AMPKgamma 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 AMPKgamma 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 AMPKgamma was selected by Western blotting, and the regulation of CCS on protein expression of AMPKgamma and p-AMPK in AMPKgamma 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 AMPKgamma gene was successfully constructed. Western blotting analysis confirmed that GR085 had the highest interfering 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 AMPKgamma 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 AMPKgamma protein. In conclusion, the AMPKgamma gene may be related to AMPK activation and intracellular lipids regulation by CCS.