MicroRNA-122 regulates inflammatory and autophagic proteins by downregulating pyruvate kinase M2 in non-alcoholic fatty liver disease

Abstract

Non-alcoholic fatty liver disease (NAFLD) is one of the serious global health concerns, leading to non-alcoholic steatohepatitis (NASH), and to hepatocellular carcinoma (HCC). Despite its prevalence, the molecular mechanisms regulating NAFLD progression remain elusive. The present study aims to determine role of microRNA-122-mediated regulation of pyruvate kinase M2 (PKM2) on regulating inflammatory and autophagic proteins during the pathogenesis of NAFLD. Huh7 cells were incubated with free fatty acids (FFAs) or transfected with single guide RNA to PKM2 containing CRISPR-Cas9 system or miR-122 for up to 72 h. C57BL/6 mice were fed with sham-operated control, choline sufficient L-amino acid defined (CSAA) or choline-deficient L-amino acid defined (CDAA) diet for 6, 18, 32 and 54 weeks. The RNA or protein was isolated from the Huh7 cells and the liver tissue of the mice. RT-PCR was performed for miR-122 expression and Western blots were performed for PKM2, iNOS, COX2, Beclin-1, Atg7 and LC3-II. FFAs induced the expression of PKM2, iNOS and COX2, while decreased the expression of miR-122, Beclin-1, Atg7 and LC3-II. Overexpression of miR-122 resulted in decreased PKM2, iNOS and COX2 and increased Beclin-1, Atg7 and LC3-II. Silencing of PKM2 led to decreased iNOS and COX2 and increased Beclin-1, Atg7 and LC3-II. In CDAA fed-mice, there was a significant increase in PKM2, iNOS and COX2 and decreased miR-122, Beclin-1, Atg7 and LC3-II. The data showed that FFAs downregulated miR-122 expression, which resulted in the upregulation of PKM2, which in turn upregulated inflammatory proteins and downregulated autophagic proteins during the pathogenesis of NAFLD.

Keywords: Autophagy; HCC; Inflammation; MiR-122; Non-alcoholic fatty liver disease (NAFLD); PKM2.