studies

238A AASLD ABSTRACTS HEPATOLOGY, October, 2015
60
FoxO3 increases miR-34a to cause palmitate-induced
cholangiocytes lipoapoptosis
Sathish Kumar Natarajan, Cody J. Wehrkamp, Ashley M. Mohr,
Mary A. Smith, Sizhao Lu, Duygu Dee Harrison-Findik, Justin L.
Mott; UNIVERSITY OF NEBRASKA MEDICAL CENTER, Omaha,
NE
Abstract: Non-alcoholic fatty liver disease (NAFLD) patients
have elevated plasma saturated free fatty acid (FFA) levels.
We had recently demonstrated the critical role of FoxO3 and
its downstream target, PUMA in FFA-induced cholangiocyte
lipoapoptosis and provided insights for biliary damage in a
subset of NAFLD patients. Here, we explored whether miR-34a
have a role in cholangiocyte lipoapoptosis. Methods: Apoptosis
was assessed by TUNEL, characteristic nuclear morphology
staining with DAPI, and caspase 3/7 activity assay. miR-34a
levels were measured using quantitative real time-PCR. FoxO3,
cleaved PARP, cleaved caspase 3 and miR-34a target expression
was examined by Western blot. Results: Treatment of cholangiocytes
with palmitate (PA) showed 7-20 fold increase in
caspase3/7 activity in normal immortalized cholangiocytes,
H69 and cell lines derived from cholangiocarcinoma, KMCH,
Mz-ChA-1 and HuCCT cells. PA treatment also resulted in a dramatic
increase in the levels of caspase cleaved products such
as cleaved PARP and cleaved caspase 3 suggesting cholangiocyte
lipoapoptosis. Interestingly, treatment with PA significantly
increased levels of miR-34a in cholangiocytes and hepatocyte
cell line, Huh7. PA-induced increase in miR-34a levels were
abolished in cholangiocytes transduced with FoxO3 shRNA,
implicating that miR-34a is a transcriptional target of FoxO3.
Further, cholangiocytes transfected with anti-miR-34a were protected
from PA-induced lipoapoptosis suggesting a major role
for miR-34a. Further, direct and indirect targets of miR34a such
as Sirt1, receptor tyrosine kinase (cMET), Kruppel like factor 4
(KLF4), fibroblast growth factor receptor (FGFR1 and 4) were
all decreased in PA-treated cholangiocytes. In addition, our
data also shows that cholangiocyte apoptosis and miR-34a are
dramatically increased in the liver from mice fed with high-fat
high-sucrose for 3 months compared to control fed animals.In
conclusion, PA induces the expression of proapoptotic miR-34a
via FoxO3 and increased miR-34a levels results in targeting
protein deacetylase Sirt1 and anti-apoptotic proteins like cMET
and KLF4 resulting in cholangiocyte lipoapoptosis.
Disclosures:
The following authors have nothing to disclose: Sathish Kumar Natarajan, Cody
J. Wehrkamp, Ashley M. Mohr, Mary A. Smith, Sizhao Lu, Duygu Dee Harrison-Findik,
Justin L. Mott
61
Prior Blockade of TNF-α Signaling Promotes Liver
Regeneration and Offers Strategies for Improving Outcomes
after Liver Resection Surgery
Fadi L. Jaber; GASTROENTEROLOGY AND LIVER DISEASES,
ALBERT EINSTEIN COLLEGE OF MEDICINE, Bronx, NY
The presumed beneficial role in liver regeneration (LR) of
pro-inflammatory cytokines, such as TNF-α or IL6, contrasts
with deleterious effects of these cytokines in other settings,
e.g., after ischemia/reperfusion, inflammation, etc. Recently,
cell transplantation-induced liver injury was shown to activate
multiple chemokines/cytokines/receptors, which were essentially
normalized after prior blockade of TNF-α by the drug,
Etanercept (ETN), with superior transplanted cell engraftment
and proliferation during liver repopulation in retrorsine (Ret)/
partial hepatectomy (PH) rat model. To resolve what role TNF-α
may serve in LR, we hypothesized that administering ETN to
block TNF-α before PH will allow determination of whether LR
will be impaired or if it would still proceed. Groups of F344
rats were established for 2/3 PH alone or ETN followed by
2/3 PH along with untreated controls. Time-course analysis
at 6, 32 or 72 h and 7 d after these procedures showed ETN
was well tolerated and liver/body weight ratios were actually
superior rather than inferior in ETN-pretreated rats with PH,
p