inducing-characterizing-vesicular-steatosis-differentiated-heparg

Inducing and Characterizing Vesicular Steatosis in Differentiated HepaRG Cells

Hepatic steatosis represents a metabolic dysfunction that results from an accumulation of triglyceride-containing lipid droplets in hepatocytes. Excessive ...

Istituto Italiano di Tecnologia

The Hepatitis B Virus (HBV) HBx regulatory protein is required for HBV replication and involved in HBV-related carcinogenesis. HBx interacts with chromatin modifying enzymes and transcription factors to modulate histone post-translational modifications and to regulate viral cccDNA transcription and cellular gene expression. Aiming to identify genes and non-coding RNAs (ncRNAs) directly targeted by HBx, we performed a chromatin immunoprecipitation sequencing (ChIP-Seq) to analyse HBV recruitment on host cell chromatin in cells replicating HBV.

Genome-wide identification of direct HBx genomic targets.

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.

Targeting a phospho-STAT3-miRNAs pathway improves vesicular hepatic steatosis in an in vitro and in vivo model.

Modification of histones by lysine methylation plays a role in many biological processes, and it is dynamically regulated by several histone methyltransferases and demethylases. The polycomb repressive complex contains the H3K27 methyltransferase EZH2 and controls dimethylation and trimethylation of H3K27 (H3K27me2/3), which trigger gene suppression. JMJD3 and UTX have been identified as H3K27 demethylases that catalyze the demethylation of H3K27me2/3, which in turns lead to gene transcriptional activation. EZH2, JMJD3 and UTX have been extensively studied for their involvement in development, immune system, neurodegenerative disease, and cancer. However, their role in molecular mechanisms underlying the differentiation process of hepatic cells is yet to be elucidated. Here, we show that EZH2 methyltransferase and JMJD3/UTX demethylases were deregulated during hepatic differentiation of human HepaRG cells resulting in a strong reduction of H3K27 methylation levels. Inhibition of JMJD3 and UTX H3K27 demethylase activity by GSK-J4 epi-drug reverted phenotype of HepaRG DMSO-differentiated cells and human primary hepatocytes, drastically decreasing expression of hepatic markers and inducing cell proliferation. In parallel, inhibition of EZH2 H3K27me3 activity by GSK-126 epi-drug induced upregulation of hepatic markers and downregulated the expression of cell cycle inhibitor genes. To conclude, we demonstrated that modulation of H3K27 methylation by inhibiting methyl-transferase and dimethyl-transferase activity influences the differentiation status of hepatic cells, identifying a possible new role of EZH2, JMJD3 and UTX epi-drugs to modulate hepatic cell plasticity.

Article	Year
Genome-wide identification of direct HBx genomic targets. BMC genomics\| PubMed ID: 28212627	2017
Targeting a phospho-STAT3-miRNAs pathway improves vesicular hepatic steatosis in an in vitro and in vivo model. Scientific reports\| PubMed ID: 30206377	2018
EZH2, JMJD3, and UTX epigenetically regulate hepatic plasticity inducing retro-differentiation and proliferation of liver cells. Cell death & disease\| PubMed ID: 31285428	2019

Laura Belloni

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