Impact of AMPKγ silencing on AMPK activation and intracellular lipids regulation

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