MicroRNA Silencing of Tumor Suppressor
DLC-1 Promotes Efficient Hepatitis C Virus
Replication in Primary Human Hepatocytes
HEPATOLOGY, 2011;53:53-61
指導老師：鄭伯智老師、林宏榮老師
學生：林怡君(N99H0005)
報告日期:2011/05/01
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Introduction
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MicroRNA (miRNAs)

The first miRNA, lin-4, was identified in 1993
in a genetic screen for mutants that disrupt the
timing of post-embryonic development in
Caenorhabditis elegans (Lee et al., 1993).

miRNAs are approximately 22-nucleotide
noncoding RNAs that constitute silencers of
target gene expression.
Development 132, 4645-4652
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Development, 2005
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
Deregulated cell proliferation and apoptosis
play a major role in hepatocellular carcinoma
(HCC). MicroRNAs participate in the
modulation of key molecules linked to
hepatocarcinogenesis.
Clin Cancer Res 2009;15(16):5073–81
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Hepatitis C Virus (HCV)
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Pathology of HCV

Acute Hepatitis C:

Generally benign:
No jaundice (80%)
 Usually asymptomatic


Can be severe, but liver failure rare
Only real threat of acute Hepatitis C is its ability
to reach chronic stages undetected and
untreated.
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Pathology of HCV

Chronic Hepatitis C:


70% of patients become chronic
Possible results:



Cirrhosis
End-stage liver disease
Hepatocellular carcinoma
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Deleted in Liver Cancer-1 (DLC-1)

The gene deleted in liver cancer-1 (DLC-1) is
located on human chromosome 8p21–22, a
region thought to harbor tumor suppressor
genes on the basis of its frequent deletion or
loss of heterozygosity in a variety of human
cancers, including hepatocellular carcinoma
(HCC).
Oncogene (2004) 23, 1308–1313
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Chronic Hepatitis C
HCV
DLC-1
Induce
Infected cell
apoptosis
MiRNA
HCV
Cancer
Materials and Methods
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Primary Hepatocyte Coculture
HCV 1a、1b、2a infection
RNA-Primed, Array-Based Klenow Enzyme Assay
Luciferase
Reporter Assay
Western Blot
Analysis
ReverseTranscription,
and Quantitative
PCR
Flow Cytometry
of Ki67 Nuclear
Antigen
Fluorescence
Stained
NATURE METHODS | VOL.1 NO.2 | NOVEMBER 2004
Luciferase Reporter Assay
Results
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Fig. 1. Changes in miRNAs in HCV-infected primary hepatocytes. Arraybased Klenow extension profiles are shown for pairwise comparison of
uninfected hepatocyte miRNAs with hepatocytes infected with HCV
genotype 1a (A), genotype 1b (B), or genotype 2a (C).
Fig. 2. Changes in miR-141 and miR-200a upon HCV infection of hepatocytes.
Summary-1

cells infected with the HCV strains showed
induced expression of miR-141, miR-200a,
and miR-200b and miR-200c.
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Fig. 3. Inhibition of DLC-1 expression by miR-141 induced in
HCV infected hepatocytes.
Summary-2

The results suggest that DLC-1 expression
in HCV1a-infected cells is regulated by
intracellular miR-141.
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Fig. 4. DLC-1 protein level is reduced in cells with replicating HCV.
Fig. 5. Changes in DLC1 protein levels in response to miR-141
in uninfected and HCV1a-infected cells.
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+
+
+
+
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+
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+
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+
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Fig. 6. (A) Effects of miR-141 on HCV RNA replication.
(B) Effects of miR-141 on the release of mature HCV particles from
infected cells.
Fig. 6.(C) Effects of miR-141 on DLC-1 mRNA.
Summary- 3

HCV replication in infected hepatocytes relies
on miR-141–mediated depletion of tumor
suppressor DLC-1.
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Uninfected control
HCV 1a infection
DLC-1+HCV 1a infection
Fig. 7. (A) Immunostaining of Ki67 nuclear antigen with fluorescein isothiocyanate.
(B) Quantitation of Ki67-positive cells.
Fig. 7.(C) Quantitation of Ki67-positive cells by flow cytometry.
Summary- 4

The results showed an approximately 40%
increase in cell proliferation of HCV1ainfected hepatocytes.

The increased cell proliferation is neutralized
when the DLC-1 level was artificially
increased through transfection with a DLC-1
expression vector.
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Discussion
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
Efficient HCV replication is correlated with
miR-141–mediated reduction of DLC-1
protein in virus infected cells.

The exact mechanism by which miR-141 or
DLC-1 modulate virus replication is not clear.
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Conclusion
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Chronic Hepatitis C
HCV
DLC-1
Induce
Infected cell
apoptosis
MiRNA
HCV
Cancer
Thanks for your attention!!
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Examples of miRNA Functions & Relevance of miRNA to Human Biology
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Development 132, 4645-4652
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Nature Biotechnology 25, 631 - 638 (2007)
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HCV Structure

RNA virus



Single-stranded
Positive-sense
9400 nucleotide

Virion:



Enveloped
Icosahedral capsid
40-60 nm in
diameter
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
Part of Flaviviridae family of viruses



Associated with both human and animal disease
3 genera: pestiviruses (cattle, pigs), flaviviruses
(dengue, yellow fever), hepaciviruses (HCV)
In hepacivirus family:

6 major clades

>100 different subtypes

Countless quasispecies: mult. seen in each infected individual
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HCV Life Cycle
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The origin of MicroRNA：
1.Extronic miRNA in non-coding transcription units
1.Intronic miRNA in non-coding transcription units.
2.Intronic miRNA in protein-coding transcription units
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