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MicroRNA Silencing of Tumor Suppressor DLC-1 Promotes Efficient Hepatitis C Virus Replication in Primary Human Hepatocytes
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
HEPATOLOGY, 2011;53:53-61
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IntroductionIntroduction
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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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5Development, 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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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
Deleted in Liver Cancer-1 (DLC-1)
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Chronic Hepatitis C
Cancer
DLC-1Induce
Infected cellapoptosis
MiRNA
HCV
HCV
?
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Materials and MethodsMaterials and Methods
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Primary Hepatocyte Coculture
RNA-Primed, Array-Based Klenow Enzyme Assay
Luciferase Reporter Assay
Western Blot Analysis
Reverse-Transcription, and QuantitativePCR
Flow Cytometry of Ki67 Nuclear Antigen FluorescenceStained
HCV 1a 、 1b 、 2a infection
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NATURE METHODS | VOL.1 NO.2 | NOVEMBER 2004
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Luciferase Reporter Assay
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ResultsResults
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Fig. 1. Changes in miRNAs in HCV-infected primary hepatocytes. Array-based 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).
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Fig. 2. Changes in miR-141 and miR-200a upon HCV infection of hepatocytes.
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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.
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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.
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Fig. 5. Changes in DLC1 protein levels in response to miR-141 in uninfected and HCV1a-infected cells.
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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.
+--
+-+
++-
+--
+-+
++-
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Fig. 6.(C) Effects of miR-141 on DLC-1 mRNA.
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Summary- 3
HCV replication in infected hepatocytes relies on miR-141–mediated depletion of tumor suppressor DLC-1.
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Fig. 7. (A) Immunostaining of Ki67 nuclear antigen with fluorescein isothiocyanate. (B) Quantitation of Ki67-positive cells.
Uninfected control HCV 1a infection DLC-1+HCV 1a infection
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Fig. 7.(C) Quantitation of Ki67-positive cells by flow cytometry.
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Summary- 4
The results showed an approximately 40% increase in cell proliferation of HCV1a-infected 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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ConclusionConclusion
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Chronic Hepatitis C
Cancer
InduceInfected cellapoptosis
MiRNA
HCV
HCV
DLC-1
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Thanks for your attention!!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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49Nature 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 units1.Intronic miRNA in non-coding transcription units.2.Intronic miRNA in protein-coding transcription units