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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

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)

http://en.citizendium.org/wiki/File:HCV_structure.png

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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)

Chronic Hepatitis C

Cancer

DLC-1Induce

Infected cellapoptosis

MiRNA

HCV

HCV

?

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Materials and MethodsMaterials and Methods

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

NATURE METHODS | VOL.1 NO.2 | NOVEMBER 2004

Luciferase Reporter Assay

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ResultsResults

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).

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.

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.

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.

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Summary- 3

HCV replication in infected hepatocytes relies on miR-141–mediated depletion of tumor suppressor DLC-1.

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

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

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

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