肝癌是全球常見的癌症之一，形成肝癌許多危險因子中，B型肝炎病毒感染和肝癌的發展有高度相關。粒線體是調控細胞許多功能的重要胞器，包括ATP的生成、細胞代謝、鈣離子濃度穩態、活性氧分子信號途徑、以及細胞生存等。粒線體進行融合和分裂的動態，能夠維持粒線體的數量、生物質量、型態和功能。先前，許多研究已經指出HBx突變和pre-S突變在肝癌形成中扮演重要角色，除此，HBx已經被證實會促使粒線體動態的不平衡，然而，顯少研究致力於LHBs和粒線體之間的關聯。本研究中，我們目的是探討LHBs對於粒線體動態平衡的調控機制，以及其生理意義。我們發現，在肝癌細胞中，HBV或LHBs會透過Drp1使得粒線體的動態平衡偏向分裂(Fission)，然而，在此狀況抑制Drp1則會減少粒線體分裂，此外，Drp1也能調控細胞的自噬作用，並在養分匱乏時，影響細胞增殖。當Drp1被抑制時，會顯著地減少HBV蛋白生成、病毒的分泌、以及cccDNA的表現量。這些結果顯示：在肝癌細胞中，Drp1對於HBV的複製和病毒蛋白的生成扮演相當重要的角色。若瞭解Drp1參與HBV複製的調控機制，將有利於發展出新的治療方式，控制慢性B型肝炎感染的病人其肝癌的發生。

Hepatocellular carcinoma (HCC) is a common cancer worldwide. Among many risk factors, hepatitis B virus (HBV) infection is the major risk for development of HCC. Mitochondria is a critical organelle for cellular functions, including ATP production, metabolism, calcium homeostasis, reactive oxygen species (ROS) signaling pathway and cell survival. Mitochondrial dynamics, including fusion and fission, is essential to maintain the number, biomass, morphology, and function of mitochondria. Previous studies have suggested HBx mutant and pre-S mutant large surface antigen (LHBs) play important roles in HCC. HBx have been demonstrated that promote aberrant mitochondrial dynamics. Researches focusing on the association between LHBs and mitochondria are limited. In this study, we aim to clarify the underlying mechanism and biological implication of LHBs on mitochondrial dynamics. We found that HBV or LHBs switched mitochondrial dynamic balanced towards fission through dynamic-related protein 1 (Drp1). Overexpression of a dominant-negative Drp1 suppressed mitochondria fission in hepatocytes. Besides, Drp1 also regulated autophagy and cell growth, in particularly under nutrient deprivation. Surprisingly, in the absence of Drp1, HBV production, viral secretion, and even cccDNA expression were all declined significantly. These results indicate that Drp1 plays an important role in HBV replication and virion production in hepatocytes. Understanding the mechanism of Drp1-involved HBV replication will be beneficial to develop a novel therapeutic strategy to control HBV replication in patients with chronic HBV infection.

Abstracts 1
中文摘要 2
1. Introduction 3
1.1 Hepatitis B virus (HBV) and hepatocellular carcinoma (HCC) 3
1.2 Ground glass hepatocyte (GGH) and pre-S mutant 4
1.3 Mitochondria 5
1.3.1 Structure of mitochondria 5
1.3.2 Function of mitochondria 5
1.4 Mitochondrial dynamics 6
1.5 Autophagy and mitophagy 8
2. Hypothesis and specific aims 10
3. Material and methods 11
4. Results 18
4.1 HBV promote mitochondrial fragmentation in hepatocytes 18
4.2 HBV does not influence expressions of Mfn2 and Drp1 in HepG2 cell 18
4.3 HBV LHBs promote mitochondrial fragmentation in hTERT-immortalized hepatocytes 18
4.4 Drp1 is upregulated in WT-LHBs cells 19
4.5 Inhibitions of CDK1 and CDK5 increase elongated mitochondria and decrease cell viability in WT-LHBs cells 20
4.6 LHBs-induced mitochondrial fragmentation is dependent on Drp1 20
4.7 LHBs induces autophagy in hepatocytes 21
4.8 Mitochondrial membrane potential is decreased in WT-LHBs cells 22
4.9 Knockdown Drp1 reduces cell proliferation after recovery from serum starvation 23
4.10 Knockdown Drp1 attenuates HBV production and virus secretion in hepatocytes 23
5. Discussion 25
6. Figures 28
Figure 1. HBV promote mitochondrial fragmentation in hepatocytes. 28
Figure 2. HBV does not affect expressions of Mfn2 and Drp1 in HepG2 cells. 29
Figure 3. Both wild type and PreS2-mutant LHBs increases mitochondrial fragmentation in hTERT-immortalized hepatocytes. 30
Figure 4. Depletion of LHBs reduces mitochondrial fragmentation in WT-LHBs cells. 31
Figure 5. Mitochondrial fragmentation in WT-LHBs cells is mediated by increased fission. 32
Figure 6. Drp1 is upregulated in WT-LHBs cells but the depletion of LHBs does not restore Drp1 expression. 33
Figure 7. Inhibitions of CDK1 and CDK5 increase elongated mitochondria and decrease cell viability in WT-LHBs cells. 34
Figure 8. LHBs-induced mitochondrial fragmentation is dependent on Drp1. 35
Figure 9. Dominant negative Drp1 expression reduces mitochondrial fragmentation in WT-LHBs cells. 36
Figure 10. LHBs induces autophagy in hTERT-immortalized cells. 37
Figure 11. Knockdown LHBs attenuates autophagy in WT-LHBs cells. 38
Figure 12. Drp1 has no impact on autophagy in WT-LHBs cells. 39
Figure 13. Drp1 depletion reduces autophagy in HepG2 carrying HBV. 40
Figure 14. Drp1 depletion reduces autophagy in HuH7 carrying HBV. 41
Figure 15. Mitochondrial membrane potential is decreased in WT-LHBs cells. 42
Figure 16. Oxidative phosphorylation is declined in WT-LHBs cells upon Drp1 depletion. 43
Figure 17. Knockdown Drp1 reduces cell proliferation in WT-LHBs cells after recovery from serum starvation. 44
Figure 18. Knockdown Drp1 attenuates expression of HBV production in hepatocytes. 45
Figure 19. Depletion of Drp1 reduces the release of HBV viral particles and reduces the expression of cccDNA in hepatocytes. 46
Figure 20. Current model. 47
7. Reference 48
8. Tables 52
Table 1. Reagents 52
Table 2. Sequences for siRNA and shRNA targeting gene 53
Table 3. List of the drugs 53
Table 4. List of antibodies 54
Table 5. Buffer and solution preparation 55

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