Effect of recombinant adenovirus Ad-mir-22 on glucose uptake in HepG2 cells / 生物工程学报

ABSTRACT

The recombinant adenoviruses expressing miR-22 (Ad-miR-22) was constructed and the effect of Ad-miR-22 on insulin signal pathway and glucose uptake in HepG2 cells was analyzed. MiR-22 gene was amplified by PCR from human hepatocytes and cloned into the pAdTrack-CMV vector to generate the shuttle plasmid pAdT-22. The positive colonies were confirmed by PCR and sequencing. The resultant shuttle plasmid was linearized with Pme I, followed by co-transformation into competent BJ5183 cells containing an adenoviral backbone plasmid (pAdEasy-1) to create the recombinant plasmid pAd-miR-22. After digested with Pac I, the linearized pAd-miR-22 was transfected into 293A packaging cell line to generate recombinant adenoviruses Ad-miR-22. HepG2 cells were infected with Ad-miR-22 or control Ad-GFP (adenoviruses expressing green fluorescent protein), and then the miR-22 expression levels were analyzed by qPCR. The result shows that adenovirus-mediated overexpression of miR-22 significantly decreased insulin-induced glucose uptake in HepG2 cells. Moreover, overexpression of miR-22 markedly decreased insulin-induced phosphorylation of GSK-3β. miR-22 also increased the mRNA levels of gluconeogenic genes in HepG2 cells. Furthermore, Western blotting results indicate that the protein expression of SIRT1 decreased in Ad-miR-22 infected HepG2 cells as compared with Ad-GFP infected HepG2 cells. In summary, overexpressing of miR-22 significantly increased gluconeogenesis while decreased glucose uptake in HepG2 cells. The effect of miR-22 on glucose metabolism may be mediated by SIRT1.