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

Abstract

Circular 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, H 2 O 2 , 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.

NRBP2 Functions as a Tumor Suppressor and Inhibits Epithelial-to-Mesenchymal Transition in Breast Cancer

“…Most tumors are often accompanied by abnormal activation or inhibition of various molecules and signaling pathways during their development and metastasis ( 22 , 23 ), and AMPK/mTOR signaling is one of the irreplaceable pathways in tumor cells ( 24 ) because it not only regulates the level of intracellular autophagy ( 25 ) but also regulates biological processes such as the proliferation, apoptosis and invasion of tumor cells ( 25 ). AMPK signaling plays a critical role in the development of almost all cancer cells, and its mechanism is to regulate tumor progression through a variety of downstream effector molecules or pathways, such as the mTOR complex ( 26 ). AMPK inhibits the phosphorylation of the mTOR complex and its downstream molecules through various molecular mechanisms, thereby reducing the progression and metastasis of tumor cells ( 27 ).…”

Circular RNAs in metabolism and metabolic disorders

“…CircRNA_002581 depletion significantly abolished lipid droplet accumulation and reduced proinflammatory cytokines in mouse NASH models. Further study found that perturbation of circRNA_002581 markedly alleviated the impaired autophagy, manifested by increased LC3‐II/I levels and autophagosome numbers 100 . Moreover, CPEB1‐PTEN‐AMPK‐mTOR signaling has been demonstrated to be associated with autophagy 101 .…”

“…A recent study implicated increased levels of circRNA_002581 and autophagy induced by FFAs in NASH 100 . CircRNA_002581 upregulation markedly alleviated the repressive effect of miR‐122 on the activity of CPEB1, which may cause pathologic angiogenesis in NASH.…”

MicroRNAs in the Pathogenesis of Nonalcoholic Fatty Liver Disease

“…In addition to their function as upstream regulators of target mRNAs, microRNAs can themselves serve as downstream targets. For example, circRNA_002581 can suppress miR-122 expression, leading to the upregulation of CPEB1 and phosphorylated-mTOR and the concomitant suppression of autophagy, finally resulting in aggravated steatosis, inflammation, hepatocyte apoptosis, and oxidative stress [28]. In contrast, however, Long et al reported that miR-122 could directly bind to the 3'-UTR of Sirt1, thereby inhibiting its expression and promoting lipogenesis [29].…”