96. Jahrestagung der Deutschen Gesellschaft für Pathologie e. V ...

Abstracts
Methods. LDL-receptor knockout mice were fed with a fatty diet. Subsequently,
the miR-101 level was detected by Real-Time PCR. Primary
hepatocytes of mice as well as human hepatoma cells were stimulated
with free fatty acids or with proinflammatory factors TNF-a or IL-6.
Following, the cellular and extra-cellular miR-101 levels were analyzed
by PCR. Additionally, miR-101 was quantified in histological characterized
biopsies (NASH-score 1–8) and serum samples of 55 patients with
NAFLD (Ntissue, Nserum=55) and correlated to clinical data as well as
liver apoptosis determined by M30/M65 measurement.
Results. The expression of miR-101 was increased in fatty livers of LDLreceptor
mouse model as well as in human patients with steatosis. In
contrast, the miRNA level was diminished in livers with inflammatory
changes. Both, the miR-101 enhancement in fatty liver as well as repression
of miR-101 upon inflammation could be mimicked in vitro in
a hepatoma cells stimulated with free fatty acids or proinflammatory
mediators, respectively. Interestingly, miR-101 was also detected in human
serum. These circulating miR-101 levels were associated with the
M30 apoptosis values and with grades of steatosis and inflammation. In
addition, circulating miR-101 levels inversely correlated to the expression
of miR-101 in liver tissues.
Conclusions. In NAFLD, the expression of miR-101 in liver tissues is contrarily
influenced by free fatty acids and proinflammatory mediators.
Alterations of miR-101 serum levels are suggested to indicate NAFLD
progression into steatohepatitis.
DO-002a
Regulation and function of the nuclear transport factor CAS in
hepatocarcinogenesis
J . Winkler1 , J . Samarin1 , V . Ehemann1 , K . Breuhahn1 , P . Schirmacher1 , S . Singer1 1Institute of Pathology/University Hospital Heidelberg, Heidelberg
Aims. There is rising evidence that deregulation of nuclear transport
factors contributes to cancer formation. An important member of the
nucleocytoplasmic transport machinery is the RanGTPase dependent
exporter CAS (Cellular Apoptosis Susceptibility) that recycles importinalpha
from the nucleus to the cytoplasm. CAS was also shown to bind to
p53 target gene promoters (e.g. PIG-3) and to be involved in apoptosome
formation. Considering these pro-apoptotic properties high expression
levels of CAS observed in different malignant tumors suggest additional
protumorigenic functions. Here, we analyzed the expression, function,
and regulation of CAS in hepatocarcinogenesis.
Methods. CAS expression analyses in 188 HCCs, 9 dysplastic nodules
and 20 normal liver samples were performed by using immunhistochemistry
and in a subset of samples by semiquantitative real-time PCR
(qRT-PCR). The impact of siRNA mediated CAS knockdown on cell viability,
cell cycle, and apoptosis was analyzed in different HCC cell lines
by using MTT-assays and FACS. The role of p53 and mTOR (the mammalian
target of rapamycin) in regulating CAS expression in HCC cell lines
was investigated by Westernblot (WB) and qRT-PCR upon treatment
with appropriate compounds.
Results. CAS was overexpressed on mRNA and protein level in up to
~70% of the HCCs analyzed and its expression was positively correlated
with tumor dedifferentiation, proliferation (Ki-67) and nuclear accumulation
of p53. A significantly decreased cell viability and increased
apoptosis was observed upon CAS knockdown. Induction of wild-type
p53 by Nutlin-3 led to reduced CAS protein and mRNA expression levels.
Lowered levels of CAS protein were also observed after Rapamycin
(mTOR inhibitor) treatment.
Conclusions. Our data suggest a protumorigenic role of CAS in hepatocarcinogenesis
apparently linked to a pro-survival function. We also
conclude that CAS is a target of p53 mediated repression and identified
mTOR as a positive regulator of CAS expression. Future studies in vitro
and in vivo are required to gain further mechanistic insights into CAS
dependent functions and to examine if CAS is a potential therapeutic
target in HCC.
16 | Der Pathologe · Supplement 1 · 2012
DO-003
Molecular and functional analysis of long non-coding RNAs in
hepatocellular carcinoma
M . Hämmerle1 , T . Gutschner1 , M . Polycarpou-Schwarz 1 , C . Hildenbrand1 ,
K . Breuhahn2 , T . Longerich2 , P . Schirmacher2 , S . Diederichs1 1Institute of Pathology, University Hospital & German Cancer Research
Center (DKFZ), Heidelberg, 2Institute of Pathology, University Hospital,
Heidelberg
Aims. The vast majority of the human genome is represented by nonprotein-coding
RNAs (ncRNAs), which are ribonucleic acids of different
lengths without an open reading frame. Recently, different functions
have been attributed to the few well-characterized ncRNAs, e.g. in epigenetics
and cancer. However, the function of most of the newly discovered
long ncRNAs is still unknown and a detailed analysis is lacking.
Since cancer research has focused on protein-coding genes for the last
decades, the potential of involvement of ncRNAs in the pathogenesis and
prognosis of hepatocellular carcinoma (HCC) is not known so far. Therefore,
our study aimed at identifying differentially expressed ncRNAs
in HCC compared to control liver samples and at elucidating their role
on the cellular and molecular level in order to draw conclusions about
their contribution to the development of HCC.
Methods. We screened for the expression of 17,000 ncRNAs in 32 cases of
HCC and 7 control tissue samples. After identifying tumor-specific candidates,
their expression was validated in HepG2 and Huh7 cells. Their
impact on cell viability was uncovered after siRNA-mediated ncRNA
knockdown in liver cancer cell lines. By using RNA affinity purification
(RNA-AP), protein interaction partners were identified.
Results. Statistical analysis unravelled 187 upregulated and 278 downregulated
ncRNAs in HCC. One ncRNA, LOHC (Long non-coding RNA
Overexpressed in Hepatocellular Carcinoma), was highly expressed in
liver cancer compared to normal liver patient samples. Knockdown of
LOHC expression significantly reduced cell viability and influenced
cell cycle progression of HepG2 and Huh7 cells. Using RNA-AP, IGF2
mRNA binding proteins (IMPs) were identified as LOHC interaction
partners. Moreover, interaction of LOHC and IMPs was largely different
in diverse stages of cell cycle, which additionally influenced LOHC expression
and stability over time.
Conclusions. LOHC is an important ncRNA in HCC, which regulates cell
viability and cell cycle. It was found to be an interaction partner of IMPs,
which can regulate LOHC stability and have a major role in the pathogenesis
of liver cancer. These data show that besides protein-coding genes,
the expression of ncRNAs could be highly and specifically regulated in
HCC, which will allow conclusions about the use of ncRNAs as potential
diagnostic and prognostic markers. Most importantly, ncRNA expression
profiling in cancer has identified functionally important players in
liver tumorigenesis.
DO-004
miR-125b regulates the lin28/IGF-II axis during hepatocellular
carcinogenesis
N . Elfimova1 , K .S . Ommer1 , N . Winkler2 , M . Quasdorff2 , I . Strack1 , J . Riemer1 ,
A . Noetel1 , H .-P . Dienes1 , M . Odenthal1 1 2 University Hospital of Cologne, Institute for Pathology, Köln, University
Hospital of Cologne, Department of Gastroenterology and Hepatology, Köln
Aims. MicroRNA (miRNA), involved in posttranscriptional regulation
of gene expression, play an important role in cell proliferation and differentiation.
miR-125b expression was shown to be divergently expressed
in liver carcinogenesis. Here, we focused on the role of miR-125b in development
of hepatocellular carcinoma (HCC).
Methods. A Cre-expressing adenoviral vector was applied to Alb-SV40
T-Ag transgenic mice in order to induce liver carcinogenesis. Expression
levels of miR-125b were determined at different time points of tumorgenesis
and in human hepatoma cell lines. Additionally, from 52 human