studies

HEPATOLOGY, VOLUME 62, NUMBER 1 (SUPPL) AASLD ABSTRACTS 293A
165
The HBx-DLEU2 lncRNA complex regulates transcription
from cellular genes and the HBV cccDNA
Francesca Guerrieri 1,2 , Letizia Chiodo 1 , Debora Salerno 1,2 , Safaa
Jeddari 2 , Giancarlo Ruocco 1 , Massimo Levrero 2,1 ; 1 CLNS@SAPI-
ENZA, Istituto Italiano di Tecnologia (IIT), Rome, Italy; 2 Dept of
Internal Medicine, La Sapienza University, Rome, Italy
Background: HBx regulatory protein is required for HBV
cccDNA transcription/viral replication and contributes to HBV
oncogenicity. HBx affects the epigenetic control of HBV viral
chromatin, by preventing HDACs and PMRT1 recruitment onto
the cccDNA, as well as of cellular chromatin, by modulating
the recruitment of chromatin modifying enzymes. We previously
showed that HBx binds to the regulatory regions of several
ncRNAs (75 miRNAs and 34 lncRNAs). DLEU2 lncRNA
overlaps with the autophagy TRIM13 gene in the opposite
orientation. Upregulation of specific DLEU2 splicing variants
correlates with the development of solid and onco-hematolocic
malignancies. Objectives: Aim of this study is to clarify the role
of HBx in the regulation of the DLEU2/TRIM13 transcriptional
unit and their functions. Methods: The anti-HBx ChIPSeq was
performed in mock and HBV-replicating HepG2 cells using an
Illumina NGS platform. Independent ChIPs were analyzed by
TaqMan real-time PCR using lncRNA and gene specific primers.
The nCounter Nanostring technology and real-time RT-PCR
were used to evaluate lncRNAs and target gene expression,
respectively. Specific LNA longRNA GapmeRs (Exiqon) were
used for highly efficient inhibition of DLEU2 lncRNA function.
Results: ChIPSeq analysis of HBx global chromatin recruitment
revealed a specific binding to 34 lncRNAs regulatory
regions. Using a custom Nanostring panel we found that all
putative lncRNAs targets were modulated in HBV-replicating
HepG2 cells. Focusing on DLEU2 lncRNA, we demonstrated
that HBx is able to deregulate both its expression and the overlapping
gene TRIM13. HBx binding to the DLEU2 promoter
affects its epigenetic control, changes DLEU2 splicing profile
and up-regulates the antisense gene TRIM13. Selective degradation
of DLEU2 RNA resulted in a reduced H4 acetylation
on the TRIM13 promoter and a ~50% reduction of TRIM13
expression in HBV replicating HepG2 cells. In silico analysis of
a model HBx protein tertiary structure and DLEU2 tertiary structure
suggested a direct interaction. Using a RIP (RNA Immune
Precipitation) approach we confirmed HBx-DLEU2 interaction
in vivo. Finally, we found that DLEU2 inactivation inhibits the
expression of the HBx targets SREBP2 and miR138 as well as
HBV cccDNA transcription, with a sharp decrease of pgRNA
levels, thus suggesting a functional relevance of the DLEU2-
HBx interaction of HBV replication. Conclusion: HBx binds to
the DLEU2 promoter region to modify its epigenetic regulation
change the DLEU2 splicing profile. HBx forms a functional complex
with DLEU2 and regulates HBV replication.
Disclosures:
Massimo Levrero - Advisory Committees or Review Panels: BMS, Jansen, Gilead,
Tekmira, Galapagos, Medimmune; Speaking and Teaching: MSD, Roche
The following authors have nothing to disclose: Francesca Guerrieri, Letizia Chiodo,
Debora Salerno, Safaa Jeddari, Giancarlo Ruocco
166
Viral Expression and Molecular Profiling of Liver Tissue
and Microdissected Hepatocytes in Hepatitis D Virus
(HDV) - Associated Hepatocellular Carcinoma (HCC)
Patrizia Farci 1 , Ashley B. Tice 1 , Ronald E. Engle 1 , Fausto Zamboni
2 , Marta Melis 1 , Zhifeng Long 3 , Jaime Rodriguez-Canales 4 ,
Jeffrey Hanson 4 , Michael R. Emmert-Buck 4 , David E. Kleiner 4 ,
Giacomo Diaz 5 ; 1 Laboratory of Infectious Diseases, National Inst
of Health, Bethesda, MD; 2 Liver Transplantation Center, Brotzu
Hospital, Cagliari, Italy; 3 Personal Diagnostix, Gaithersburg, MD;
4 Laboratory of Pathology, National Cancer Institute, Bethesda,
MD; 5 Department of Biomedical Sciences, University of Cagliari,
Cagliari, Italy
Although HCC develops in a high proportion of patients
with chronic hepatitis D, there are no data on the molecular
mechanisms of HDV-induced hepatocarcinogenesis. Because
of the vital dependence of HDV on HBV, the role of HDV in
promoting HCC is unknown. We used genomic techniques
to dissect the role of HDV and HBV. Microarray (Affymetrix
Human U133 Plus2) was performed on 33 specimens of
whole liver tissue (WLT, tumor vs. non-tumor) and selected
laser capture-microdissected hepatocytes (LCM, malignant vs.
non-malignant hepatocytes) obtained at liver transplant (LT)
from 5 patients with HDV-HCC; 29 WLT specimens from 7
patients with non-HCC HDV cirrhosis served as controls. A
parallel analysis was performed on WLT and LCM from 11
patients with HBV-HCC without HDV. Microarray data were
analyzed using BRB-Array Tools, with multivariate permutation
F-tests (FDR