studies

898A AASLD ABSTRACTS HEPATOLOGY, October, 2015
Kevin Moore - Advisory Committees or Review Panels: Servier
The following authors have nothing to disclose: Lisa Longato, Fausto Andreola,
Sean S. Davies, Jackson L. Roberts, Giuseppe Fusai, Krista Rombouts
1408
Sequence Profiling of miRNAs Reveals mir-150-5p as a
Regulator of Transdifferentiation
Luigi Locatelli, Pier P. Paoli, Timothy Hardy, Rachel M. Howarth,
Fiona Oakley, Derek Mann; Institute of Cellular Medicine, Newcastle
University, Newcastle upon Tyne, United Kingdom
Cellular transdifferentiation is important in many physiological
processes (e.g. wound healing), and is implicated in pathologies
such as cancer and tissue fibrosis. To-date the role of
miRNAs, a class of small noncoding RNAs that regulate gene
expression by binding to target mRNAs suppressing its translation
or initiating its degradation, in transdifferentiation has not
been fully addressed. We use a cell culture model for transdifferentiation
of human and rat hepatic stellate cells (HSC) into
myofibroblasts to determine functional changes in miRNAs. The
aims of this study were: a) perform Next Generation Sequencing
(NGS) to generate a miRNA profile in human and rat
activated HSCs to identify potential miRNA-mRNA regulatory
networks in common with both; b) replicate NGS results based
on gene profile and biological validation of specific miRNA
target genes. The most critical step in miRNA biology lies in the
definition of the rules for miRNA target recognition. Data generated
by sequencing were analysed via the Seq-Imp pipeline
(http://www.ebi.ac.uk/research/enright/software/kraken), to
identify individual miRNA and an initial statistical analysis.
Further analysis was performed with a custom R script to generate
data correlation and heatmap matrices. Finally, targets for
each miRNA were identified from TargetScan, microCOSM,
and miRTar, by picking genes that were in at least two out of
three databases, revealing 11 significantly upregulated and 11
downregulated miRNAs that were identical in both activated
human and rat HSC. From the list of differentially regulated
miRNAs, we focused our attention on mir-150-5p, which regulates
a series of target proteins such as the Zinc finger E-box
binding homeobox (ZEB1) involved in the cell cycle, cellular
development, and apoptosis. Particularly mir-150-5p has been
found to be downregulated in both human and rat activated
HSCs. A significant inverse correlation between ZEB1 and mir-
150-5p was found at the RNA and protein level. In vitro we
showed that the overexpression of mir-150-5p in human and
rat HSCs by a mir-150-5p mimic was able to downregulate
the expression of ZEB1 with an observed reduction of extracellular
matrix proteins collagen type I (COL1A1) and α-smooth
muscle actin (α-SMA). Moreover, the level of mir-150-5p in the
serum of 10 Alcoholic Liver Disease (ALD) patients has been
found to be significantly downregulated compared to healthy
controls. In conclusion, HSC transdifferentiation is associated
with discrete alterations in the expression of miRNAs, and in
particular we have identified mir-150-5p as a regulator of the
myofibroblast phenotype and potential biomarker of fibrosis in
ALD patients.
Disclosures:
The following authors have nothing to disclose: Luigi Locatelli, Pier P. Paoli, Timothy
Hardy, Rachel M. Howarth, Fiona Oakley, Derek Mann
1409
Human precision Cut Liver Slices model for testing
anti-fibrotic drugs
Lynda Aoudjehane 1,2 , Margaux Legrand 1 , Grégoire Bisch 1 , Rolland
Delelo 2 , Chantal Housset 2 , Pierre Balladur 3 , Claire Goumard
4 , Jérôme Becquart 1 , Yvon Calmus 2,5 , Filomena Conti 2,5 ;
1 Human HepCell, Faculté de Médecine Pierre et Marie Curie,
Paris, France; 2 UMPC UNIV Paris 6 & INSERM, UMR_S 938,
CDR Saint-Antoine, Paris, France; 3 AP-HP, Hôpital Saint Antoine,
Service de Chirurgie Digestive, Paris, France; 4 AP-HP, Hôpital Pitié-
Salpêtrière, Service de Chirurgie Digestive, Paris, France; 5 AP-
HP, Hôpital Pitié-Salpêtrière, Unité de Transplantation Hépatique,
Paris, France
Background: Fibrosis is the common end stage of all chronic
liver diseases. Understanding the molecular mechanisms underlying
liver fibrogenesis is mandatory to develop new antifibrotic
therapies. Current methods for evaluating the efficacy
of anti-fibrotic drugs rely mostly on immortalized cell lines and
animal models. Human precision-cut liver slices (PCLS) preserve
the cell types and proportions, and particularly the cell-cell
interactions. Several studies have recently suggested that PCLS
are potentially useful as in vitro model to study liver fibrosis.
The current study aimed to test the efficacy of human PCLS to
evaluate the effect of anti-fibrotic drugs. Methods: Human PCLS
of 250 μm thickness were obtained from normal or cirrhotic
liver samples. PCLS were incubated for 1-48h at 37°C under
85% O2 and, after 24h, were treated with different anti-fibrotic
drugs: ciclosporin A (CsA), pirfenidone (PFD) or SMAD3
inhibitor (SIS3). The viability of PCLS was assessed by ATP production.
Hepatic metabolism and fibrosis markers (α-SMA and
Coll1, HSP47, TIMP-1, MMP-2 and TGF-beta) were determined
by RT-PCR. Results: PCLS from normal and cirrhotic human liver
samples could be cultured with an excellent viability over 48h
and this culture induced a spontaneous fibrosis. The expression
of fibrotic markers (α-SMA and Coll1, HSP47, TIMP-1,
MMP-2 and TGF-beta) significantly increased at 24 and 48h
when compared to 1h. Fibrosis markers significant decreased
(α-SMA and Coll1 and TIMP1 mRNA expression) when normal
or cirrhotic human PCLS were treated for 24h with PFD
and SIS3. However, CsA had no significant effect on fibrosis
marker expression on normal or cirrhotic PCLS. Conclusion:
These preliminary results suggest that human PCLS can be
maintained in culture for 48 hours without significant modification
of viability. The culture of PCLS induces a spontaneous
activation of the fibrotic process. This feature can be used to
study the effect of antifibrotic molecules and thus constitutes
an interesting physiological human model for the screening of
antifibrotic drugs.
Disclosures:
Jérôme Becquart - Management Position: Human Hepcell
The following authors have nothing to disclose: Lynda Aoudjehane, Margaux Legrand,
Grégoire Bisch, Rolland Delelo, Chantal Housset, Pierre Balladur, Claire
Goumard, Yvon Calmus, Filomena Conti
1410
The AMPK-related kinase NUAK2 interacts with TGF-β
and regulates the activation of hepatic stellate cells
Cristina Tosti Guerra 1 , Alessandra Caligiuri 1 , Angela Provenzano 1 ,
Krista Rombouts 2 , Massimo Pinzani 2 , Fabio Marra 1 ; 1 University
of Florence, Florence, Italy; 2 University College London, London,
United Kingdom
Background and Aims: Nuak2 is a member of AMPK related
kinases (ARKs), that act as energy sensors and controllers of
cellular structure, with different effects on cell motility and cytoskeletal
organization, depending on the cell types. AMPK pos-