Antagonizing circRNA_002581–miR-122–CPEB1 axis alleviates NASH through restoring PTEN–AMPK–mTOR pathway regulated autophagy

Abstract

AbstractCircular RNAs (circRNAs) have been shown to play critical roles in cancer biology, but their functions in nonalcoholic steatohepatitis (NASH) remain unexplored. Full length of circRNA_002581 was amplified and sequenced, followed by RNA immunoprecipitation, RNA-Fluorescence in Situ Hybridization and dual luciferase reporter gene analysis to confirm the existence of the circRNA_002581–miR-122–CPEB1 regulatory axis in vitro. CircRNA_002581 knockdown was used to study its roles in high concentration of free fatty acids-induced NASH-like cell model and a methionine and choline deficiency (MCD) diet-induced NASH mice model. Autophagy flux and related potential PTEN–AMPK–mTOR pathway were tested by western blot. CircRNA_002581 overexpression significantly relieved the inhibitory role of miR-122 on its target CPEB1 by sponging miR-122. CircRNA_002581 knockdown markedly attenuated lipid droplet accumulation, reduced the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), pro-inflammatory cytokines, apoptosis, H2O2, and increased ATP level in both mice and cellular models of NASH. Mechanistically, circRNA_002581 interference significantly rescue the defective autophagy evidenced by increased autophagosome number, upregulated LC3-II/I level, and decreased p62 level. Further chloroquine-mediated total autophagy inhibition antagonizes the protective effect of circRNA_002581 knockdown. Finally, CPEB1–PTEN–AMPK–mTOR pathway is shown to link the autophagy and circRNA_002581 knockdown-mediated NASH alleviation. CircRNA_002581–miR-122–CPEB1 axis actively participates in the pathogenesis of NASH through PTEN–AMPK–mTOR pathway-related autophagy suppression. Targeting circRNA_002581 is a potential therapeutic strategy for NASH through partial autophagy restoration.