Abstract
Non-alcoholic fatty liver disease (NAFLD) is a leading cause of chronic liver disease. Although genetic predisposition and epigenetic factors contribute to the development of NAFLD, our understanding of the molecular mechanism involved in the pathogenesis of the disease is still emerging. Here we investigated a possible role of a microRNAs-STAT3 pathway in the induction of hepatic steatosis. Differentiated HepaRG cells treated with the fatty acid sodium oleate (fatty dHepaRG) recapitulated features of liver vesicular steatosis and activated a cell-autonomous inflammatory response, inducing STAT3-Tyrosine-phosphorylation. With a genome-wide approach (Chromatin Immunoprecipitation Sequencing), many phospho-STAT3 binding sites were identified in fatty dHepaRG cells and several STAT3 and/or NAFLD-regulated microRNAs showed increased expression levels, including miR-21. Innovative CARS (Coherent Anti-Stokes Raman Scattering) microscopy revealed that chemical inhibition of STAT3 activity decreased lipid accumulation and deregulated STAT3-responsive microRNAs, including miR-21, in lipid overloaded dHepaRG cells. We were able to show in vivo that reducing phospho-STAT3-miR-21 levels in C57/BL6 mice liver, by long-term treatment with metformin, protected mice from aging-dependent hepatic vesicular steatosis. Our results identified a microRNAs-phosphoSTAT3 pathway involved in the development of hepatic steatosis, which may represent a molecular marker for both diagnosis and therapeutic targeting.
Highlights
Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in developed countries[1]
Since accumulation of triglycerides and reactive oxygen species (ROS) plays an important role in inducing hepatocyte injury associated with NAFLD13, we decided to investigate whether the effect of lipid droplets and ROS generation were able to trigger gene expression modulation in differentiated HepaRG cells (dHepaRG) cells
We performed a qPCR analysis of sodium oleate 250 μM treated dHepaRG transcripts using a Human Fatty Liver Array (Sabiosciences), that profiles the expression of 84 key genes involved in the mechanisms of NAFLD and hepatic insulin resistance (Supplementary Table 1)
Summary
Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in developed countries[1]. Intrahepatic activation of pro-inflammatory signaling, such as the IL6/STAT3 pathway, plays a significant role in the development of chronic liver diseases. The present study investigates the role of a miRNAs-phospho-STAT3 pathway in the induction of liver vesicular steatosis in an in vitro model, represented by hepatic differentiated HepaRG cells treated with sodium oleate, fatty dHepaRG. We identified by Chromatin Immunoprecipitation Sequencing that phospho-STAT3, which is activated in fatty dHepaRG, is able to bind and regulate several miRNAs after lipid overload. Our results showed for the first time that suppression of the miRNAs-phospho-STAT3 pathway by S3I-201, a STAT3 specific inhibitor, decreased hepatic lipid accumulation. Activation of a new miRNAs-phospho-STAT3 pathway that is involved in the development of hepatic steatosis may represent a target for novel therapeutic strategies and might have potential as a biomarker for diagnosis
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