Effect of recombinant adenovirus Ad-mir-22 on glucose uptake in HepG2 cells.

Lihong Liao; Wenbin Yuan; Yong Chen; Jichao Liang

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Effect of recombinant adenovirus Ad-mir-22 on glucose uptake in HepG2 cells.

Author: Lihong LIAO ¹ ; Wenbin YUAN ² ; Yong CHEN ² ; Jichao LIANG ²
Author Information

1. Department of Pediatrics, Zhongnan Hospital of Wuhan University, Wuhan 430071, Hubei, China.
2. Hubei Province Key Laboratory of Biotechnology of Chinese Traditional Medicine, National and Local Joint Engineering Research Center of High-throughput Drug Screening Technology, Hubei University, Wuhan 430062, Hubei, China.
Publication Type:Journal Article
Keywords: SIRT1; gene therapy; gluconeogenesis; glucose uptake; miR-22; recombinant adenoviruses
MeSH: Adenoviridae; genetics; Glucose; metabolism; Glycogen Synthase Kinase 3 beta; metabolism; Hep G2 Cells; Humans; MicroRNAs; genetics; metabolism; Signal Transduction; genetics; Transfection
From: Chinese Journal of Biotechnology 2020;36(4):763-771
CountryChina
Language:Chinese
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.