Abstract
Nonalcoholic fatty liver disease (NAFLD) is one of the most prevalent chronic liver disorders that is featured by the extensive deposition of fat in the hepatocytes. Current treatments are very limited due to its unclear pathogenesis. Here, we investigated the function of circ_0057558 and miR-206 in NAFLD. High-fat diet (HFD) feeding mouse was used as an in vivo NAFLD model and long-chain-free fatty acid (FFA)-treated liver cells were used as an in vitro NAFLD model. qRT-PCR was used to measure levels of miR-206, ROCK1 mRNA, and circ_0057558, while Western blotting was employed to determine protein levels of ROCK1, p-AMPK, AMPK, and lipogenesis-related proteins. Immunohistochemistry were performed to examine ROCK1 level. Oil-Red O staining was used to assess the lipid deposition in cells. ELISA was performed to examine secreted triglyceride (TG) level. Dual-luciferase assay was used to validate interactions of miR-206/ROCK1 and circ_0057558/miR-206. RNA immunoprecipitation was employed to confirm the binding of circ_0057558 with miR-206. Circ_0057558 was elevated while miR-206 was reduced in both in vivo and in vitro NAFLD models. miR-206 directly bound with ROCK1 3’-UTR and suppressed lipogenesis and TG secretion through targeting ROCK1/AMPK signaling. Circ_0057558 directly interacted with miR-206 to disinhibit ROCK1/AMPK signaling. Knockdown of circ_0057558 or overexpression of miR-206 inhibited lipogenesis, TG secretion and expression of lipogenesis-related proteins. ROCK1 knockdown reversed the effects of circ_0057558 overexpression. Injection of miR-206 mimics significantly ameliorated NAFLD progression in vivo. Circ_0057558 acts as a miR-206 sponge to de-repress the ROCK1/AMPK signaling and facilitates lipogenesis and TG secretion, which greatly contributes to NAFLD development and progression.
Highlights
Nonalcoholic fatty liver disease (NAFLD) is a chronic disorder with the hallmark of accumulation of excessive fat in the hepatocytes, which does not result from alcohol [1, 2]
We found that high-fat diet (HFD) upregulated the was associated with hepatic steatosis and circRNA_021412/miR- expression levels of ROCK1 and lipogenesis-related proteins, 1972 signaling was a key regulator of lipid metabolism [24]. including SREBP1, FAS, SCD1, ACC1, and CD36, but downregulated
We found that circ_0057558 was miR-206 directly targeted ROCK1 to reduce its expression increased while miR-206 was decreased in NAFLD using both the It is well known that miRNAs exert functions by regulating gene in vivo mouse model and in vitro cell model. miR-206 directly expressions of downstream targets [12]
Summary
Nonalcoholic fatty liver disease (NAFLD) is a chronic disorder with the hallmark of accumulation of excessive fat in the hepatocytes, which does not result from alcohol [1, 2]. Overexpression of miR-206 reversed the circRNAs, circ_0057558, has been shown to promote those changes, recovering the level of p-AMPK and AMPK activity triglyceride (TG) production and the expression of circ_0057558 and decreased the levels of ROCK1 and lipogenesis-related positively correlates with the level of TG [23] This implies that proteins and ROCK1 activity (Fig. 2E–H). We found that circ_0057558 was miR-206 directly targeted ROCK1 to reduce its expression increased while miR-206 was decreased in NAFLD using both the It is well known that miRNAs exert functions by regulating gene in vivo mouse model and in vitro cell model. Our study reveals an significantly diminished the relative luciferase activity of the essential role of circ_0057558/miR-206/ROCK1/AMPK axis in ROCK1-WT reporter but not the ROCK1-Mut reporter wherein the NAFLD and provides novel molecular targets for the development binding sites with miR-206 were mutated (Fig. 3C), suggesting that of therapeutic strategies for NAFLD.
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