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Differential MicroRNA Expression Between Hepatitis B and Hepatitis C Leading Disease Progression to Hepatocellular Carcinoma HEPATOLOGY 2009;49:000-000 Yin-Hong Zhu

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Overview of Presentation Backgroud -why Protocol -how Discussion-what Instruction-learn

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1 HBV,HCV,HCC HEPATOLOGY 2009;49:000-000. HCV HBV epidemiologic findings 1998 Disease progression HCC promotion Genes involved in inflammation Genes responsible for Anti-inflammation Pro-apoptotic and DNA repair responses inammatory and anti-apoptotic phenotypes Factors inducing those differences ? Signal pathwaysGenes expression Signal pathways virologic featureshistologic findings 2001 2006Different oncogenic factors

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2 Regulation of gene expression microRNA 30% of human genes

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3 Gene expression regulated by microRNAs.

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microRNA Development Proliferation (Stem cells) diseases such as infection and cancer Differentiation Apoptosis 4 Function of microRNA in animals and plants

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microRNA host or virus Avian influenza virus EBV,KSHV,MHV-68, HCMV,HSV-1 HBV,HCV HIV-1 SV40(Simian virus 40) hAV(adenovir) 5 microRNA and viral infection HEPATOLOGY 2009;49:000-000.

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6 microRNAs, HBV,HCV,HCC HBV or HCV Normal HBV or HCV,HCC HBV between HCV,HCC mirRNA-122SPC3649 LNA-antimiRTM-122 Clinic trial phase I on health person Blank

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7 Scientific supposition of this research HCV HBV microRNAs Genes expression Signal pathways Disease progression HCC promotion Dignosis,Therapy

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Overview of Presentation Backgroud-why Protocol -how Discussion -what Instruction-learn

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1 Basic problems for miRNA researches What miRNAs are expressed miRNA library selection Isolation,clone,sequence miRNA miRNA profiles analysis Which gene is the target of the miRNAs Which important miRNA influence Some certain bio-process When and where the miRNAs are expressed Functional analysis Therapy Diagnosis

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2 Researches of microRNA and diseases miRNA Compare miRNA profiles of diseases and normal samples miRNA Identify miRNAs that affect cell division,apoptosis,angiogenesis,cell proliferation,telomerrase activity miRNAs miRNAs Target genes

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3 Protocol for this research RTD-PCR Microarray Analysis. Identification of microRNA Target genes Verication in HBV/ HCV Infection Model Functional analysis miRNAs expressing profiles analysis Verification of Regulation of genes by microRNA

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Normal RTD-PCR 3 Protocol for this research HCV HBV 12 patients 14 patients Control 9 normal liver tissue from patients with metastatic liver cancer Hierarchical cluster analaysis Class Prediction Binary tree calssification 13significantly differential microRNAs 188 known miRNAs

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3 Protocol for this research Microarray Analysis. Global analysis of mRNA expression in the liver tissues

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3 Protocol for this research Identification of microRNA Target genes Predicted by MIRANDA Pro3.0 Step 1Step 2

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3 Protocol for this research Verification of Regulation of genes by microRNA 2 Lost of funtcion transfect with Small interfering RNAs specific to 13 candidate miRNAs 1 Gain of function transfect Huh7.5 cells with Pre-miRNA miRNAs and Target genes mRNA were detected by RTD-PCR

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3 Protocol for this research Verication in HBV/ HCV Infection Model Huh 7.5 cellsJFH1-RNApHBV Virus proteins and DNA/RNA were deteced miRNA expression

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Overview of Presentation Backgroud-why Discussion -What Protocol -how Instruction -learn

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1 CH and HCC miRNAs in the same patient HEPATOLOGY 2009;49:000-000. HCV HBV No dependence Paired conditionDisease Normal

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2 HBV and HCV infection- specific miRNAs HEPATOLOGY 2009;49:000-000. HCV HBV 6 miRNAs Cell death,DNA damage and recombination, trancription signals 13 miRNAs Immune response,antigen presentation, Cell cycle,proteasome,and lipid metabolism Endogennous miRNA involved in defense mechanisms,mainly RNA viruses Normal

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HEPATOLOGY 2009;49:000-000. 3 Tumor-associated miRNAs HCV HBV has-miR-191 Worse survival in acute myeloid leukemia p53 tumor supressor pathwayhas-miR-34 has-let-7a ras and c-myc genes has-miR-133bSequamous cell carcinoma has-miR-142-5pHematopoietic specific Normal

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HEPATOLOGY 2009;49:000-000. 4 HCC-specific miRNAs HCV HBV Cytoskelton Cell cycle Ubiqutin proteasome Transiation Immune response Pathway Cancer-associated Inflammation-mediated miRNAs Normal

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HEPATOLOGY 2009;49:000-000. 5 miRNAs in HBV/HCV disease progression and HCC promotion

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Overview of Presentation Backgroud-why Instruction -learn Protocol -how Discussion - What

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1 The same problem in differ- ent aspects Disease progression HCC promotion HCV HBV microRNAs Genes expression Signal pathways 200120062009 Researches are still in need

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2 The same aspect with diffe- rent protocols Northern Blot miRNarray qRT-PCR Luciferase reporter EMS A CHIP Western Blot 5RACE MicroRNA Sensor reporter Bioinformatics prediction microarray Cellular model Cellular invasion Cellular apoptosis Cellular proliferation Cellular cycle qRT-PCR

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3 The same protocol for the similar researchs miRNA Compare miRNA profiles of cancer and normal samples miRNA Identify miRNAs that affect cell division,apoptosis,angiogenesis,cell proliferation,telomerrase activity miRNAs miRNAs Target genes

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miRNA HEPATOLOGY 2009;49:000-000.

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qRT-PCR---miRNAs HEPATOLOGY 2009;49:000-000.

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miRNA siRNA

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MicroRNA

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miRNA HEPATOLOGY 2009;49:000-000.

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Q-rt-pcr HEPATOLOGY 2009;49:000-000.

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HEPATOLOGY 2009;49:000-000.

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microRNA HEPATOLOGY 2009;49:000-000.

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RTD-PCR 3 Protocol for this research HCV HBV 12 patients 14 patients Control 9 normal liver tissue from patients with metastatic liver cancer Nomal

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spc3649 Santaris Pharma Santaris Pharma 2003 LNA LNA RNA RNA Santaris Pharma mRNA microRNA mRNA microRNA Santaris Pharma 2006 5 2007 12 Santaris Pharma Enzon Santaris Pharma RNA GlaxoSmithKline SPC3649 48 6 8 6 SPC3649 ( ) SPC3649 6