miR-140-5p Aggravates Insulin Resistance via Directly Targeting GYS1 and PPP1CC in Insulin-Resistant HepG2 Cells

Background: Much attention has been paid to the regulatory role of microRNA (miRNA) in insulin resistance. Nevertheless, how miR-140-5p regulates insulin resistance remains unclear. In this research, we aim to investigate the roles of miR-140-5p in insulin resistance. Methods: qRT-PCR is used to analyze the expression level of miR-140-5p in insulin-resistant HepG2 cells. Glucose consumption and glucose uptake are detected to study the effect of miR-140-5p knockdown in insulin-resistant HepG2 cells and miR-140-5p overexpression in HepG2 cells. Bioinformatic analysis, luciferase reporter assay and confirmatory experiments are applied to identify the target gene bound with miR-140-5p and study the effect of miR-140-5p on the downstream substrates of target genes. Rescue experiments have verified the roles of miR-140-5p and target gene in glucose metabolism. Results: The expression level of miR-140-5p was upregulated in insulin-resistant HepG2 cells and was significantly correlated with cellular glucose metabolism. Functionally, miR-140-5p overexpression induced impairment of glucose consumption and glucose uptake. Besides, bioinformatics analysis indicated that glycogen synthetase (GYS1) and protein phosphatase 1 catalytic subunit gamma (PPP1CC) were the target genes of miR-140-5p. Western blotting and qRT-PCR results revealed a negative correlation between GYS1, PPP1CC and miR-140-5p. The glycogen detection results showed that miR140-5p inhibited the production of the downstream substrates of the target gene. Rescue experiments showed that inhibition of GYS1 or PPP1CC partially enhanced the insulin-resistant effects of miR-140-5p knockdown in insulin-resistant HepG2 cells. Conclusion: miR-140-5p overexpression augments the development of insulin resistance and miR-140-5p may be served as a therapeutic target of metabolic diseases.