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

Abstract

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.