毛玻璃肝細胞為帶有B型肝炎病毒大表面蛋白的肝細胞，在肝硬化病人中被認為是肝癌發生的前兆。在慢性B型肝炎病毒感染晚期中，第二型毛玻璃肝細胞與肝癌的產生有極大的關係，其帶有preS2突變大表面蛋白(preS2-LHBs)並分佈在細胞的邊緣。在此研究中，主要探討造成preS2-LHBs形成邊緣分佈的原因以及其生理意義。我們發現帶有preS2-LHBs的細胞中內質網會被徵召到細胞膜附近。此徵召是由鈣池調控鈣離子進入(store-operated calcium entry, SOCE)機制中的兩個元件所調節：分別位於內質網和細胞膜的蛋白質STIM1與Orai1。藉由preS2-LHBs或其他化學誘導物所造成內質網壓力就可以促使位於內質網上的STIM1向細胞膜移動而與其上的Orai1結合。STIM1-Orai1的連接促使鈣離子經由SOCE機制的調控而進入細胞內，進而造成preS2-LHBs細胞中的鈣離子濃度增加。然而我們亦發現SOCE會活化鈣離子依賴性鈣蛋白酶(calpain)，進而促使中心體過度生成。帶有額外中心體的細胞會誘導多極性分裂，最後造成染色體不穩定。總結，我們發現B型肝炎病毒大表面蛋白的邊緣分佈代表著在第二型毛玻璃肝細胞中SOCE的活化，進而藉由中心體過度生成以及多極性分裂所誘發的染色體不穩定促進腫瘤生成。

Ground glass hepatocytes (GGHs) are characterized by hepatocytes expressing abundant HBV large surface proteins (LHBs) in the cytoplasm and recognized as preneoplastic lesions in cirrhotic livers. Particularly, the type-II GGHs with marginal distribution of preS2 mutant LHBs are implicated in the pathogenesis of hepatocellular carcinoma. In the present study, the cause and consequences for the marginal recruitment of preS2-LHBs in hepatocytes are explored. We found that endoplasmic reticulum (ER) was recruited towards plasma membrane in hepatocytes carrying preS2-LHBs. This recruitment was mediated by the interaction between two components of the store-operated calcium entry (SOCE) machinery: the ER-resident protein STIM1 and the plasma membrane-resident calcium channel protein Orai1. The activation of ER stress, by preS2-LHBs or chemical inducers, provokes the recruitment of ER-resident STIM1 towards plasma membrane-resident Orai1. The STIM1-Orai1 connection initiated SOCE-mediated calcium influx and thereby increased the subcellular calcium concentration in hepatocytes expressing preS2-LHBs. For the consequence of SOCE activation, we demonstrated that SOCE promoted centrosome overduplication through calcium-dependent calpain proteases. Hepatocytes carrying extra centrosomes underwent multipolar division and hence chromosome instability. We conclude that marginal distribution of LHBs reflects the activation of SOCE machinery in the type-II GGHs. The activation of SOCE machinery may promote tumorigenesis through the induction of chromosome instability via centrosome overduplication and multipolar division.

Abstract.........................................................................................................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 ER stress...................................................................................................5
1.4 Store-operated calcium entry (SOCE) and intracellular calcium homeostasis ......................................................................................................................6
1.5 Centrosome overduplication and multipolar division................................7
1.6 Aneuploidy and micronuclei formation are the evidence of chromosomal instability......................................................................................................8
2. Hypothesis and specific aims..................................................................10
3. Materials and Methods.............................................................................11
4. Results.....................................................................................................16
4.1 PreS2-LHBs displayed marginal distribution similar to HBsAg in type-II GGHs..........................................................................................................16
4.2 ER recruitment to the cell periphery in hepatocytes expressing preS2-LHBs ..................................................................................................................16
4.3 PreS2-LHBs promotes ER-plasma membrane connections through ER stress ..................................................................................................................17
4.4 Calcium homeostasis is disrupted in preS2-LHBs cells..........................18
4.5 STIM1-Orai1 interaction leads to marginal recruitment of preS2-LHBs ..................................................................................................................18
4.6 Constitutive activation of SOCE machinery results from [Ca2+]ER insufficiency..................................................................................................19
4.7 SOCE promotes centrosome overduplication and multipolar division.......20
4.8 Abnormalities of centrosome number and cell division lead to chromosome instability.....................................................................................................21
4.9 The inhibition of SOCE machinery suppresses chromosome instability in LHBs-positive cells.......................................................................................22
5. Discussion................................................................................................23
6. Figures.....................................................................................................26
Figure 1. Different HBsAg staining pattern in cirrhotic livers.........................26
Figure 2. Subcellular distribution of WT-LHBs and preS2-LHBs in NeHepLxHT cells.............................................................................................................27
Figure 3. Relative distribution of plasma membrane (PM), endoplasmic reticulum (ER) and LHBs...............................................................................28
Figure 4. Increased ER-PM connections under ER stress...............................29
Figure 5. Using GRP78 as ER marker to further confirm ER-PM connection...................................................................................................30
Figure 6. Intracellular calcium concentration of ctrl, WT-LHBs, preS2-LHBs cells.............................................................................................................31
Figure 7. The activation of SOCE and marginal recruitment of STIM1 under ER stress..........................................................................................................32
Figure 8. Marginal distribution of preS2-LHBs cells was disrupted upon STIM1 depletion....................................................................................................33
Figure 9. The marginal recruitment of STIM1 and activation of SOCE machinery in type-II GGHs..........................................................................................34
Figure 10A. STIM1-Orai1 interaction is regulated by ER calcium storage in preS2-LHBs cells.......................................................................................35
Figure 10B. STIM1-Orai1 interaction is regulated by ER calcium storage in WT-LHBs cells..........................................................................................36
Figure 11. LHBs promotes centrosome overduplication.............................37
Figure 12. Inhibition of calcium, ER stress, SOCE, calpain protease and STIM1 depletion suppress centrosome overduplication in preS2-LHBs cells ........38
Figure 13. Multipolar division results from ER stress-mediated centrosome overduplication ........................................................................................39
Figure 14. Inhibition of calcium, ER stress, SOCE and calpain protease reduce multipolar division in preS2-LHBs cells.....................................................40
Figure 15. Aneuploidy serves as the evidence of chromosome instability...41
Figure 16. Micronuclei formation works as another evidence of chromosome instability..................................................................................................42
Figure 17. Aneuploidy was relieved upon long-term inhibition of SOCE.....43
Figure 18. Micronuclei formation was suppressed upon persistent inhibition of SOCE..........................................................................................................44
Figure 19. Illustration of cause and consequence of marginal pattern in type-II GGHs..........................................................................................................45
7. Reference...............................................................................................46
8. Tables....................................................................................................51

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