F. Guerrieri

Genome-wide identification of direct HBx genomic targets

BackgroundThe 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.ResultsChIP-Seq high throughput sequencing of HBx-bound fragments was used to obtain a high-resolution, unbiased, mapping of HBx binding sites across the genome in HBV replicating cells. Protein-coding genes and ncRNAs involved in cell metabolism, chromatin dynamics and cancer were enriched among HBx targets together with genes/ncRNAs known to modulate HBV replication. The direct transcriptional activation of genes/miRNAs that potentiate endocytosis (Ras-related in brain (RAB) GTPase family) and autophagy (autophagy related (ATG) genes, beclin-1, miR-33a) and the transcriptional repression of microRNAs (miR-138, miR-224, miR-576, miR-596) that directly target the HBV pgRNA and would inhibit HBV replication, contribute to HBx-mediated increase of HBV replication.ConclusionsOur ChIP-Seq analysis of HBx genome wide chromatin recruitment defined the repertoire of genes and ncRNAs directly targeted by HBx and led to the identification of new mechanisms by which HBx positively regulates cccDNA transcription and HBV replication.

Combining amplicon sequencing and metabolomics in cirrhotic patients highlights distinctive microbiota features involved in bacterial translocation, systemic inflammation and hepatic encephalopathy

In liver cirrhosis (LC), impaired intestinal functions lead to dysbiosis and possible bacterial translocation (BT). Bacteria or their byproducts within the bloodstream can thus play a role in systemic inflammation and hepatic encephalopathy (HE). We combined 16S sequencing, NMR metabolomics and network analysis to describe the interrelationships of members of the microbiota in LC biopsies, faeces, peripheral/portal blood and faecal metabolites with clinical parameters. LC faeces and biopsies showed marked dysbiosis with a heightened proportion of Enterobacteriaceae. Our approach showed impaired faecal bacterial metabolism of short-chain fatty acids (SCFAs) and carbon/methane sources in LC, along with an enhanced stress-related response. Sixteen species, mainly belonging to the Proteobacteria phylum, were shared between LC peripheral and portal blood and were functionally linked to iron metabolism. Faecal Enterobacteriaceae and trimethylamine were positively correlated with blood proinflammatory cytokines, while Ruminococcaceae and SCFAs played a protective role. Within the peripheral blood and faeces, certain species (Stenotrophomonas pavanii, Methylobacterium extorquens) and metabolites (methanol, threonine) were positively related to HE. Cirrhotic patients thus harbour a ‘functional dysbiosis’ in the faeces and peripheral/portal blood, with specific keystone species and metabolites related to clinical markers of systemic inflammation and HE.

1121 MIR-224 IS A DIRECT TARGET OF HBX AND MODULATES HBV REPLICATION

Background: miR224 is frequently up-regulated in human HCCs, peri-tumoral cirrhotic tissues and cirrhotic livers without HCCs. We have recently shown that miR224 expression in the liver is induced by the NFkB-dependent inflammatory pathways (i.e. LTa, and TNFa) that are activated in a large proportion of chronic viral hepatitis, cirrhosis and HCC patients. Differently from other HCCrelated miRNAs that are over-expressed in several other cancers and are considered as bona fide onco-miRs (i.e. miR-21, miR-221, miR-222), miR-224 is apparently more HCC specific, suggesting a possible role of miR224 in liver physiopathology and/or chronic hepatitis infection before HCC development. Aim: To characterize the transcriptional regulation of miR224 by HBx and its effects on HBV replication. Methods: In vivo recruitment of HBx, transcription factors and chromatin remodeling enzymes was analyzed in ChIP-Seq and conventional ChIP experiments. miR224(pr) luciferase reporter constructs were generated and used to assess the ability of HBx to modulate miR224 expression. Endogenous miR224 expression and the impact of miR224 on HBV replication were assessed in HBV replicating HepG2 cells. Results: In a genome wide search of HBx cellular targets by ChIP-Seq we found that HBx protein binds in vivo to the miR224 regulatory region. HBx binding is accompanied by the co-recruitment of p65/NFkB, a loss of Pol II occupancy, the recruitment of the DNMT3a methyltransferase and reduced H4 histone acetylation. Accordingly, miR224 levels were reduced in HBV replicating cells and HBx repressed the miR224(pr) in luciferase reporter assays. pre-miR224 overexpression resulted in reduced HBV pgRNA levels and a 50% reduction in HBV replication. In silico analysis revealed the presence of several miR224 seed sequences on the HBV genome that were conserved across HBV genotypes, suggesting a direct effect of miR224 on the HBV pgRNA. Conclusions: Our results identify a functional regulatory loop between HBx, miR224 and HBV replication where HBx repression of miR224 expression relieves the negative effects of miR224 on HBV replication. These results are compatible with the low HBV replication observed in HCC tissues and support the hypothesis that the loss of the described regulatory loop might occur at the time of transformation or HCC progression.

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

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.