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Molecular Genetics Laboratory

Molecular Genetics Laboratory. Professor Guhung Jung E-mail : drjung@snu.ac.kr Tel : 02-880-7773 Room # : 504-420 Homepage: mgl.snu.ac.kr. HBV HCC. Our interests. 분자유전학 실험실. 1. HBV life cycle mechanism. Journal of Biological Chemistry 2003. Virology , 2005. Biochemical Journal , 2006.

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Molecular Genetics Laboratory

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  1. Molecular Genetics Laboratory Professor Guhung Jung E-mail : drjung@snu.ac.kr Tel : 02-880-7773 Room # : 504-420 Homepage: mgl.snu.ac.kr HBV HCC

  2. Our interests 분자유전학 실험실 1. HBV life cycle mechanism Journal of Biological Chemistry 2003 Virology, 2005 Biochemical Journal, 2006 이승근 강항 심희연 민지영 2. HBV and Host interaction Gastroenterology 2005 Journal of Biological Chemistry 2006 FEBS Letters 2006 Cancer science 2007 이스마엘 김혜림 3. Mechanism of Hepatocarcinogenesis Clinical Cancer Research 2003 구진모 김현수 임승외

  3. 우리 나라의 B형 간염 실태 • 우리나라 인구의 약 5-8%가 감염되어 있음. • 만성 B형 간염 환자 중 20년 후 48%가 간경변증 발병. • 가장 사회적으로 활동력과 영향력이 높은 4, 50대 남성의 경우는 간질환이 사망 원인 2위. • 전체 간질환의 66.6%가 B형 간염바이러스가 원인임. • 한국인 호발성 바이러스 질환에 대한 선택과 집중이 절실함.

  4. 국내 만성 간질환의 질환별 점유율 (대한 소화기학회 총서2, 간염, 18개 대학병원:20,964명 2000년)

  5. 만성 B형 간염과 간암의 관련성 • 만성 간염 분포 지역과 간암 발생 지역의 일치 • 간암 환자의 상당수가 HBsAg 양성(70%) • HBV 감염에 의한 간암 발달 증가 • HBV 보균자의 높은 간암 발생율(비보균자에 비해 223배의 위험성을 가짐) • HBV 백신에 의한 소아간암 발생율 감소 • 감염자 중 약 10%가 간암으로 발병(우리나라 암 사망률 중 3위) Lancet. 1981; Gastroenterology. 2004.

  6. Hepatocellular carcinoma (HCC) • 1년에 간암으로 백만명 이상(전세계) 만 명(국내) 이상이 사망. • 암 종류별 사망 원인 중 간암이 3위. • 초기에는 임상 증상이 거의 없어 조기 발견이 힘들고 효과적인 치료제가 없어 아직까지는 수술에 의한 제거가 가장 효율적임. • 유방암이나 대장암에 비해 연구가 부족하여 간암 발달 기작은 자세히 밝혀져있지 않음. 간암

  7. 만성 간염 단계에서의 간암 예방 • 조기 발견이 힘들고, 수술 이외의 효과적인 치료제가 없는 간암은 예방이 중요함. • 따라서 높은 간암 발생 위험성을 가진 만성 간염 단계에서의 치료를 통한 간암 예방이 필요함. • ‘만성 간염-> 간경화 -> 간암’에 이르는 간암 발달 기작에 대한 연구가 절실함. • 간암 발달 기작에 근거한 만성 간염 단계에서의 치료를 통해 간암 발생을 예방할 수 있음.

  8. Research HBV 1. HBV and host interaction 2. HBV life cycle Hepatitis Cirrhosis HCC HBV replication 3. Hepatocarcinogenesis Hepatocyte

  9. Approach <Molecular Biology> Protein-protein interaction Part. 1 HBV replication & encapsidation <Cell Biology> Immunofluorescent Apoptosis Part. 2 <Biochemical approach> Protein purification Enzyme assay HBV & Host interaction & signaling <2DE> Part. 3 <DNA chip> Mechanism of Hepatocarcinogenesis <ROS analysis> <Clinical case study> <Bioinformatics> System biology

  10. Hepatitis B virus(HBV) Life Cycle HBV gene products Polymerase: P Core : C Surface protein: S X protein : X N Engl J Med 2004;350:1118-29.

  11. HBV Core Assembly Part. 1 Core Dimer-Dimer interaction

  12. The role of phosphorylation in HBV life cycle Part. 1 Core assembly increased by PKA and PKC

  13. HBV core protein and host factors interaction Part. 3 Identify unknown factors Through MS/MS

  14. Protein interaction analysis Part. 3 M-RT M-TP Hsp90 B23.1 Core, BSA, MBP, M-RH Biomolecular Interaction Analysis

  15. Identify protein specific binding site Part. 1 Interesting protein C-terminal Deletion protein

  16. NF-kB siganaling& HBV Part. 2 IFN-r induce NIK localization NIK NIK NF-kB activity inhibition Nucleus 후속 연구 주제 선행 연구결과 밝혀진 mechanism Phenomenon TRAF2,3 expression and localization specific to HBV Problems Relation of HBV with NF-kB activity? • Hypothesis • HBV up- regulate TRAF-2 • HBV down-regulate TRAF-3 • Inhibition of NF-kB activity • Experiments • TRAF-2,3 localization and expression? • TRAF-2,3 localization and NF-kB activity?

  17. IFN-g inhibits HBV-induced NF-kB Activation HBV • HBV-induced TRAF2 increased NF-B activity by interaction with NIK. • IFN-g는 HBV 감염 시 TRAF3를 세포질로 이동시키고 그 결과 NIK를 핵으로 이동시켜 세포질에서의 NIK에 의한NF-kB 활성화를 억제함. TRAF2 TRAF3 localization NIK IFN-g TRAF3 NIK nuclear localization NF-kB activation Inhibition of NF-kB activation

  18. HepG2.2.15 HepG2 HepG2.2.15 HepG2 TRAF2 GAPDH TRAF2 b-actin HBV-induced TRAF2 increased NF-B activity by interaction with NIK • HBV는 TRAF2 transcription, Translation Expression을 증가시킴. 증가된 TRAF2는 NF-kB activity 증가를 야기시킴 • HBV는 TRAF2와 NIK의 binding을 증가시킴. NIK와 binding site가 deletion된 TRAF2의 경우 NF-kB증가 효과가 나타나지않음 IP : TRAF2 IB : NIK

  19. Effect of HBV on TRAF3 localizationHepG2 & HepG2.2.15 cells TRAF3 DAPI Merged HepG2 HepG2.2.15

  20. Effect of IFN-r and LMV on TRAF3 localization in HepG2.2.15 cells TRAF3 Dapi Merged Control IFN-γ LMV

  21. Effect of HBV-1.2x(Chang & Chang-1.2x) TRAF3 DAPI Merged Chang Chang-1.2x

  22. Effect of HBx-1.2x(Huh7 & Huh7-1.2x) TRAF3 DAPI Merged Huh7 Huh7-1.2x

  23. 간암 발생 과정 Part. 3 ROS stress Chemical Selective Clonal expansion Genetic change Genetic change Genetic change Genetic change Normal cell Initiated cell Invasion metastasis Malignant Tumor Clinical liver cancer Pre-neoplastic lesion Virus HBV HCV Inflammatory / Reactive oxygen/ NO species (Toxicology 2002; 181–182: 43–47) 여러 단계(multi-step)를 거쳐 일어나며, 각 step에서 ROS stress가 관여함.

  24. 암 진행 과정 Part. 3 Normal epithelium Dysplasia Carcinoma EMT vascularinvasion EMT Metastasis (EMT: epithelial-mesenchymal transition) (Nature Review cancer 2002; 2: 442) 암세포는 EMT를 통해 invasion 및 metastasis가 가능해짐.

  25. 임상 조직을 대상으로 한 실험 Part. 3 간암 분화도별 조직 Proteome analysis (2-DE & MS) Transcripteome analysis (Microarray) 간암 관련 유전자 발굴 Antioxidant enzyme, Iron metabolic protein, cytoskeleton protein, heat shock protein … Case study (Immunoblot, IHC) ROS 관련 유전자 및EMT 관련 유전자의 발현 변화가 크게 나타남

  26. Proteome analysis of HCC Part. 3 (Cirrhosis) (HCC G2) We have observed down-regulation of antioxidant enzymes (AOEs) in HCC by proteome analysis.

  27. Human tissue 연구 결과 요약 Human tissue study Vascular Invasion ROS stress Frequency E-cad AOE HCC GIII Cirrhosis LGDN HGDN HCC GI HCC GII Histological grade Antioxidant enzymes↓ → ROS stress ↑→ E-cad ↓→ Vascular invasion ↑

  28. The effect of ROS on E-cadherin expression Menadione and PMS down-regulate E-cadherin expression.

  29. ROS에 의한 EMT 유발 Part. 3 B. Hep3B A. Huh7 No treatment H2O2 4days No treatment H2O2 4days E-cad DAPI H2O2 down-regulate E-cadherin expression at day 4.

  30. The effect of ROS on MMP activity Hep3B Huh7 PLC H: H2O2 N: NAC (Gelatin zymography) * P<0.05 H2O2 induce MMP-2 at day 4

  31. The effect of ROS on tumor cell invasion Part. 3 HCC cells Matrigel PET memb. (8um pore) Matrigel invasion assay No treatment H2O2 treatment Invaded cells (Hep3B) ROS level Invasion assay: Hep3B Invasion assay: Huh7 (N=3) (N=3) (N=3) H: H2O2 H2O2 enhances tumor cell invasion.

  32. ROS level of AOE overexpressed cell lines ROS level (H2DCFDA) A. Huh7 cell lines B. Hep3B cell lines ROS level decreased in catalase and SOD1 overexpressd cell lines

  33. The effect of ROS on E-cadherin expression in AOE overexpressed cell lines E-cad immunoblot (H2O2 treatment) A. Huh7 cell lines B. Hep3B cell lines • H2O2 can not down-regulate E-cadherin expression in catalase • and SOD1 overexpressd cell lines.

  34. The effect of ROS on tumor cell invasion in AOE overexpressed cell lines Invasion assay (H2O2 treatment) A. Huh7 cell lines B. Hep3B cell lines Catalase or SOD1 overexpressed cells less invasive than control cells

  35. ROS에 의한 Invasion Invasion ↑ • ROS↑ • E-cadherin↓ • MMP ↑ • Invasion↑ (H2O2, PMS, Mena) Antioxidant enzyme ↑ (catalase, SOD1) : ROS ↓ • Huh7+CAT • Huh7+SOD1 • Huh7+SOD2 • Hep3B+CAT • Hep3B+SOD1 • Hep3B+SOD2 • ROS에 의해 HCC cell line의 Invasion이 증가했음. • NAC(antioxidant) 처리 및 SOD1, catalase overexpression에 의해 ROS에 의한 invasion 증가를 막을 수 있음.

  36. ROS up-regulate Snail expression Part. 3 (RT-PCR) O: untreated, N: NAC, H: H2O2

  37. ROS and Snail induced DNA methlyation of E-cadherin promoter Part. 3 IS1 IS2 IS3 IS1 IS2 IS3 C S C S C S O H O H O H U M U M U M U M U M U M U M U M U M U M U M U M IS: CpG island O: untreated H: H2O2 U: unmethyl M: methyl C: control S: Snail

  38. Snail overexpression induces methylation of the E-cadherin promoter MS-PCR Snail shRNA also blocked H2O2-induced methylation of the E-cadherin promoter.

  39. ROS induce binding of HDAC1 and DNMT1 ChIP assay H2O2 treatment increased Snail, HDAC1, DNMT1, and MeCP2 recruitment to the E-cadherin promoter H2O2 treatment increased repressive heterochromatin

  40. Snail up-regulation and promoter methylation are correlated with down-regulation of E-cadherin in HCC Immunoblot (HCC tissue)

  41. ROS induce tumor cell invasion in HCC via snail mediated epigenetic change Part. 3 • Snail ↑ • DNA methylation • E-cadherin ↓ Invasion ROS • Antioxidant (NAC) • Antioxidant enzyme ↑ • (catalase, SOD1) ROS HDAC1 DNMT1 Snail Promoter Promoter E-cad E-cad

  42. Goal of Research HBV infection Inhibition of HBV Replication Liver disease Discovery of drug target HCC therapy Hepatocellular Carcinoma

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