B 형 간염 바이러스 단백질인 HBx와 HBxAP/RSF1에 의한 암화과정 조절
Regulation of cancer progression by hepatitis B virus X protein (HBx) and HBxAP/RSF1
- 주제(키워드) Hepatitis B virus X (HBx) , catalase , cysteine , liver cancer , HBxAP/RSF1
- 발행기관 아주대학교
- 지도교수 조혜성
- 발행년도 2016
- 학위수여년월 2016. 2
- 학위명 박사
- 학과 및 전공 일반대학원 의생명과학과
- 실제URI http://www.dcollection.net/handler/ajou/000000021592
- 본문언어 한국어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Hepatitis B virus X protein (HBx), encoded by hepatitis B virus (HBV) genome, plays a crucial role in the pathogenesis of liver cancer, and we previously showed that reactive oxygen species (ROS) significantly elevated the HBx protein levels. First, I herein investigated the role of antioxidants in regulation of HBx protein expression and their clinical relevance. Overexpression of catalase or superoxide dismutase 2 (MnSOD) induced a significant decrease in HBx expression level. Complete disruption for cysteine residues in HBx protein resulted in a dramatically reduced HBx protein level and this HBx Cys-null (Cys-) mutant no longer responded to catalase, suggesting that disulfide bonds in HBx are important for its protein stability. Moreover, Huh7-Cys- cells failed to generate colonies in clonogenic survival assays, while Huh7 cells expressing wild-type HBx (Huh7-X) yielded a significant number of colonies. Next, I analyzed 50 human HBV-induced hepatocellular carcinoma (HCC) samples. Seventy-eight percent of HCC samples contained lower catalase levels than surrounding tissues. Importantly, patients with a high T/N (tumor/non-tumor tissue) ratio for catalase showed significantly longer survival than those with a low T/N ratio. Interestingly, there was a significant inverse relationship between catalase and HBx expression levels in stage IV HCCs. Thus, catalase expression in HCC patients can be clinically useful for prediction of patient survival and restoration. Second, HBx in the host cells was found to be tightly associated with protein called HBxAP (HBx-associated protein), which was identified as a subunit of chromatin remodeler RSF complex. I found that RSF1 protein was overexpressed in HBV-related HCC specimens along with elevated HBx protein compared to those in surrounding liver tissues. Notably, depletion of RSF1 induced the premature separation of sister chromatids and fluorescence resonance energy transfer (FRET) analysis revealed that centromeric H3K9me3 levels were reduced in mitotic RSF1 depleted cells. Moreover, in histone peptide array RSF1 widely bound to modified histone H3 peptides except that it no longer interacted with the phosphorylated histone H3 peptide at the Ser28. Further investigation is needed to reveal underlying mechanisms by which RSF1 regulates chromosomal instability. Taken together, these findings suggest that HBx and RSF1 contribute to liver cancer development.
more목차
I. Regulation of catalase on hepatitis B virus X protein (HBx) in HBV-related advanced hepatocellular carcinomas 1
A. INTRODUCTION 2
B. MATERIALS AND METHODS. 4
1. Cell cultures and plasmids 4
2. RNA extraction, reverse transcription-polymerase chain reaction 4
3. Western blotting . 5
4. Luciferase assay 5
5. Clonogenic cell proliferation assays 5
6. Patients characteristics 6
7. Statistics 6
C. RESULTS 7
1. The protein levels of HBx are decreased by catalase and MnSOD 7
2. Catalase downregulates HBx protein levels at the translational level 7
3. Cysteine residues of HBx are essential to maintain protein stability . 9
4. Cysteine null mutant of HBx is insufficient for transactivation activities 12
5. Cysteine null mutant is not affected by catalase or N-acetyl cysteine 12
6. Cysteine residues are important for HBx-mediated clonogenic cell proliferation 14
7. Catalase expression level is lower in tumor tissues than in non-tumor tissues in HBV-related HCC. 17
8. Catalase expression level is significantly lower in HBV-related advanced HCC 17
9. HBx protein levels are negatively correlated with catalase expression in HBV-related advanced HCC. 20
10. HBx protein levels have no correlation with MnSOD expression in HBV-related advanced HCC 23
11. Prognostic significance of catalase expression in HBV-related advanced HCC 23
D. DISCUSSION 30
II. HBxAP/RSF1 as a chromatin remodeller and HBx-associated protein 32
A. INTRODUCTION 33
B. MATERIALS AND METHODS . 36
1. Cell cultures and antibody. 36
2. Plasmid. 36
3. Chromosome spreading and Giemsa staining. 36
4. FRET (Fluorescence Resonance Energy Transfer) assay. 37
5. Modified histone peptide array. 37
6. Purification of RSF1. 37
7. Protein microchip array. . 37
C. RESULTS 39
1. RSF1 is overexpressed in HBV-related advanced HCC. 39
2. RSF1 depletion induces premature separation of sister chromatids. 39
3. The centromeric expression level of H3K9me3 is downregulated at metaphase in RSF1 knock-down cells 43
4. RSF1 depletion enhances the expression levels of H3K9me3 and H3K27me3 in mitosis 46
5. RSF1 dissociates from histone peptides with phosphorylated H3S28 modification 46
6. RSF1 directly interacts with diverse targets. 48
D. DISCUSSION 51
III. CONCLUSION 53
IV. REFFERENCE 54
국문요약 59
