肝細胞癌 (hepatocellular carcinoma, HCC) 是現今全球高致死率的癌症之一，其中B型肝炎病毒 (HBV) 是促成肝細胞癌形成的原因。先前研究中已知B型肝炎病毒在腫瘤組織中活躍度較正常組織低，其中小表面蛋白 (SHBs) 在腫瘤組織中的表現量更是明顯的減少。然而，先前實驗室研究發現，即使小表面蛋白在腫瘤中表現下降，但大表面蛋白 (LHBs) 卻依然穩定表現，這現象指出大、小表面蛋白的蛋白質表現是受到不同的方式調控。本研究目的探討大、小表面蛋白在腫瘤組織中表現的調控機轉。
首先利用5'RACE PCR取得完整的pre-S1 RNA與pre-S2 RNA的5’UTR序列，接著利用5’UTR reporter assay觀察pre-S2/S RNA 在不同細胞株的轉譯活性。於pre-S2 RNA 5’UTR reporter assay實驗結果發現，當pre-S2 5’UTR 存在的組別中，其轉譯活性有下降的現象。利用RT-PCR與西方墨點法確認此現象確實於轉譯過程受到調控而導致，同時我們也在西方墨點法的結果中發現pre-S2 RNA僅表現中表面蛋白，無小表面蛋白。為此，我們探討小表面蛋白是否源自pre-S1 RNA。利用相同的實驗方式，觀察pre-S1 RNA蛋白表現以及pre-S1 ATG與S ATG的轉譯活性，其結果發現當由S ATG進行轉譯時轉譯活性較高，並且在西方墨點實驗中確認pre-S1 RNA能夠轉譯小表面蛋白。在本研究中，我們雖未詳細闡明大、小表面蛋白在腫瘤組織中的調控機制。但發現pre-S1 RNA可轉譯小表面蛋白，而pre-S2 RNA主要表現中表面蛋白。

SUMMARY
In this study, we aimed to investigate the translational regulation of pre-S1 and pre-S2/S RNAs. First, we utilized 5’Rapid amplification of cDNA ends (5’RACE) PCR to find the 5’UTR of pre-S1 RNA and pre-S2/S RNA. Then, we evaluated the translation activities of the 5’UTR of pre-S2/S RNA in different cell lines by 5’UTR reporter assay, and the result showed that translation activity was decreased when pre-S1 or pre-S2 5’UTR exist. We also detected the mRNA expression and protein expression of pre-S1 or pre-S2 5’UTR reporter clone. The results showed that the phenomenon of pre-S2 5’UTR reporter assay was truly regulated in translation. Interestingly, we also found that pre-S2 RNA translated MHBs mainly. Therefore, we further focused on whether pre-S1 RNA could be the template for SHBs. The result showed pre-S1 RNA could translate both LHBs and SHBs observed by Western blot.

Alavian, S. M., & Miri, S. M. (2011). Dilemma of HBsAg seroconversion in chronic hepatitis B infection: Dilemma of HBsAg in chronic HBV. Hepat Mon, 11(2), 67-68.
Altinel, K., Hashimoto, K., Wei, Y., Neuveut, C., Gupta, I., Suzuki, A. M., . . . Carninci, P. (2016). Single-Nucleotide Resolution Mapping of Hepatitis B Virus Promoters in Infected Human Livers and Hepatocellular Carcinoma. J Virol., 90(23), 10811-10822. doi:10.1128/jvi.01625-16
Belloni, L., Pollicino, T., De Nicola, F., Guerrieri, F., Raffa, G., Fanciulli, M., . . . Levrero, M. (2009). Nuclear HBx binds the HBV minichromosome and modifies the epigenetic regulation of cccDNA function. Proc Natl Acad Sci U S A, 106(47), 19975-19979.
Block, T. M., Guo, H., & Guo, J. T. (2007). Molecular virology of hepatitis B virus for clinicians. Clin Liver Dis, 11(4), 685-706, vii. Retrieved from
Blumberg, B. S., Gerstley, B. J. S., Hungerford, D. A., London, W. T., & Sutnick, A. I. (1967). A Serum Antigen (Australia Antigen) in Down's Syndrome, Leukemia, and Hepatitis. Ann Intern Med, 66(5), 924-931.
Chang, M. H., Chen, C. J., Lai, M. S., Hsu, H. M., Wu, T. C., Kong, M. S., . . . Chen, D. S. (1997). Universal hepatitis B vaccination in Taiwan and the incidence of hepatocellular carcinoma in children. Taiwan Childhood Hepatoma Study Group. N Engl J Med, 336(26), 1855-1859.
Churin, Y., Roderfeld, M., & Roeb, E. (2015). Hepatitis B virus large surface protein: function and fame. Hepatobiliary Surg Nutr, 4(1), 1-10.
Coppola, N., Onorato, L., Minichini, C., Di Caprio, G., Starace, M., Sagnelli, C., & Sagnelli, E. (2015). Clinical significance of hepatitis B surface antigen mutants. World J Hepatol, 7(27), 2729-2739.
Dalla Venezia, N., Vincent, A., Marcel, V., Catez, F., & Diaz, J.-J. (2019). Emerging Role of Eukaryote Ribosomes in Translational Control. Int J Mol Sci., 20(5), 1226. Retrieved
Farza, H., Hadchouel, M., Scotto, J., Tiollais, P., Babinet, C., & Pourcel, C. (1988). Replication and gene expression of hepatitis B virus in a transgenic mouse that contains the complete viral genome. J Virol., 62(11), 4144-4152. doi:10.1128/jvi.62.11.4144-4152.1988
Firth, A. E., & Brierley, I. (2012). Non-canonical translation in RNA viruses. J Gen Virol., 93(Pt 7), 1385-1409.
Gu, W., Gallagher, G. R., Dai, W., Liu, P., Li, R., Trombly, M. I., . . . Finberg, R. W. (2015). Influenza A virus preferentially snatches noncoding RNA caps. Rna, 21(12), 2067-2075.
Gu, W., Lee, H.-C., Chaves, D., Youngman, Elaine M., Pazour, Gregory J., Conte, D., & Mello, Craig C. (2012). CapSeq and CIP-TAP Identify Pol II Start Sites and Reveal Capped Small RNAs as C. elegans piRNA Precursors. Cell, 151(7), 1488-1500.
Hao, R., He, J., Liu, X., Gao, G., Liu, D., Cui, L., . . . Guo, D. (2015). Inhibition of hepatitis B virus gene expression and replication by hepatocyte nuclear factor 6. J Virol, 89(8), 4345-4355.
Hassemer, M., Finkernagel, M., Peiffer, K.-H., Glebe, D., Akhras, S., Reuter, A., . . . Hildt, E. (2017). Comparative characterization of hepatitis B virus surface antigen derived from different hepatitis B virus genotypes. Virology, 502, 1-12. Retrieved from
Hellen, C. U. T. (2018). Translation Termination and Ribosome Recycling in Eukaryotes. Cold Spring Harb Perspect Biol, 10(10).
Hildt, E., Saher, G., Bruss, V., & Hofschneider, P. H. (1996). The hepatitis B virus large surface protein (LHBs) is a transcriptional activator. Virology, 225(1), 235-239. doi:10.1006/viro.1996.0594
Hildt, E., Urban, S., & Hofschneider, P. H. (1995). Characterization of essential domains for the functionality of the MHBst transcriptional activator and identification of a minimal MHBst activator. Oncogene, 11(10), 2055-2066.
Hu, J., & Liu, K. (2017). Complete and Incomplete Hepatitis B Virus Particles: Formation, Function, and Application. Viruses, 9(3).
Idilman, R., Cinar, K., Seven, G., Bozkus, Y., Elhan, A., Bozdayi, M., . . . Bahar, K. (2012). Hepatitis B surface antigen seroconversion is associated with favourable long-term clinical outcomes during lamivudine treatment in HBeAg-negative chronic hepatitis B patients. J Viral Hepat, 19(3), 220-226.
İnan, N., & Tabak, F. (2015). Hepatitis B Virus: Biology and Life Cycle. Viral Hepatit Dergisi, 21(1), 1-7. doi:10.4274/vhd.36036
Jeong, J. K., Yoon, G. S., & Ryu, W. S. (2000). Evidence that the 5'-end cap structure is essential for encapsidation of hepatitis B virus pregenomic RNA. J Virol., 74(12), 5502-5508.
Kang, J., Wang, J., Cheng, J., Cao, Z., Chen, R., Li, H., . . . Lu, F. (2017). Down-regulation of NTCP expression by cyclin D1 in hepatitis B virus-related hepatocellular carcinoma has clinical significance. Oncotarget, 8(34), 56041-56050.
Koukouraki, P., & Doxakis, E. (2016). Constitutive translation of human α-synuclein is mediated by the 5'-untranslated region. Open Biol, 6(4), 160022.
Koumbi, L., Pollicino, T., Raimondo, G., Kumar, N., Karayiannis, P., & Khakoo, S. I. (2016). Hepatitis B viral replication influences the expression of natural killer cell ligands. Ann Gastroenterol, 29(3), 348-357.
Li, J., Liu, Y., Wang, Z., Liu, K., Wang, Y., Liu, J., . . . Yuan, Z. (2011). Subversion of cellular autophagy machinery by hepatitis B virus for viral envelopment. J Virol., 85(13), 6319-6333.
Lok, A. S., Zoulim, F., Dusheiko, G., & Ghany, M. G. (2017). Hepatitis B cure: From discovery to regulatory approval. J Hepatol, 67(4), 847-861.
Loncarević, I. F., Schranz, P., Zentgraf, H., Liang, X. H., Herrmann, G., Tang, Z. Y., & Schröder, C. H. (1990). Replication of hepatitis B virus in a hepatocellular carcinoma. Virology, 174(1), 158-168.
MacLachlan, J. H., & Cowie, B. C. (2015). Hepatitis B virus epidemiology. Cold Spring Harb Perspect Med, 5(5), a021410.
Mahoney, F. J. (1999). Update on diagnosis, management, and prevention of hepatitis B virus infection. Clin Microbiol Rev, 12(2), 351-366.
Mani, S. K. K., & Andrisani, O. (2018). Hepatitis B Virus-Associated Hepatocellular Carcinoma and Hepatic Cancer Stem Cells. Genes (Basel), 9(3). doi:10.3390/genes9030137
Moucari, R., Mackiewicz, V., Lada, O., Ripault, M. P., Castelnau, C., Martinot-Peignoux, M., . . . Marcellin, P. (2009). Early serum HBsAg drop: a strong predictor of sustained virological response to pegylated interferon alfa-2a in HBeAg-negative patients. Hepatology, 49(4), 1151-1157.
Murakami, Y., Saigo, K., Takashima, H., Minami, M., Okanoue, T., Bréchot, C., & Paterlini-Bréchot, P. (2005). Large scaled analysis of hepatitis B virus (HBV) DNA integration in HBV related hepatocellular carcinomas. Gut, 54(8), 1162-1168.
Ng, S. A., & Lee, C. (2011). Hepatitis B virus X gene and hepatocarcinogenesis. J Gastroenterol, 46(8), 974-990.
Ni, Y., Sonnabend, J., Seitz, S., & Urban, S. (2010). The pre-s2 domain of the hepatitis B virus is dispensable for infectivity but serves a spacer function for L-protein-connected virus assembly. J Virol., 84(8), 3879-3888.
Niu, B., & Hann, H.-W. (2017). Hepatitis B Virus–Related Hepatocellular Carcinoma: Carcinogenesis, Prevention, and Treatment. In Updates in Liver Cancer.
Pasquinelli, C., Melegari, M., Villa, E., Seidenari, M., Scaglioni, P. P., Tiribelli, C., . . . Manenti, F. (1990). Detection of hepatitis B virus transcripts in patients with chronic liver disease. J Hepatol, 10(2), 180-185.
Pfefferkorn, M., Böhm, S., Schott, T., Deichsel, D., Bremer, C., Schröder, K., . . . Bömmel, F. (2017). Quantification of large and middle proteins of hepatitis B virus surface antigen (HBsAg) as a novel tool for the identification of inactive HBV carriers. Gut, 67, gutjnl-2017.
Schlüter, V., Rabe, C., Meyer, M., Koshy, R., & Caselmann, W. H. (2001). Intracellular accumulation of middle hepatitis B surface protein activates gene transcription. Dig Dis, 19(4), 352-363.
Sheu, S. Y., & Lo, S. J. (1992). Preferential ribosomal scanning is involved in the differential synthesis of the hepatitis B viral surface antigens from subgenomic transcripts. Virology, 188(1), 353-357.
Sriram, A., Bohlen, J., & Teleman, A. A. (2018). Translation acrobatics: how cancer cells exploit alternate modes of translational initiation. EMBO Rep, 19(10).
Su, I.-J., Wang, L. H.-C., Hsieh, W.-C., Wu, H.-C., Teng, C.-F., Tsai, H.-W., & Huang, W. (2014). The emerging role of hepatitis B virus Pre-S2 deletion mutant proteins in HBV tumorigenesis. J Biomed Sci., 21(1), 98.
Sunbul, M. (2014). Hepatitis B virus genotypes: global distribution and clinical importance. World J Gastroenterol, 20(18), 5427-5434.
Tong, S., & Revill, P. (2016). Overview of hepatitis B viral replication and genetic variability. J Hepatol, 64(1 Suppl), S4-s16.
Tu, T., Budzinska, M. A., Shackel, N. A., & Urban, S. (2017). HBV DNA Integration: Molecular Mechanisms and Clinical Implications. Viruses, 9(4), 75.
Turton, K. L., Meier-Stephenson, V., Badmalia, M. D., Coffin, C. S., & Patel, T. R. (2020). Host Transcription Factors in Hepatitis B Virus RNA Synthesis. Viruses, 12(2).
Walsh, D., & Mohr, I. (2011). Viral subversion of the host protein synthesis machinery. Nat Rev Microbiol, 9(12), 860-875.
Wang, Y., Wu, M. C., Sham, J. S., Tai, L. S., Fang, Y., Wu, W. Q., . . . Guan, X. Y. (2002). Different expression of hepatitis B surface antigen between hepatocellular carcinoma and its surrounding liver tissue, studied using a tissue microarray. J Pathol, 197(5), 610-616.
Wu, C. C., Chen, Y. S., Cao, L., Chen, X. W., & Lu, M. J. (2018). Hepatitis B virus infection: Defective surface antigen expression and pathogenesis. World J Gastroenterol, 24(31), 3488-3499.
Wu, Y., Wen, J., Xiao, W., & Zhang, B. (2019). Pregenomic RNA: How to assist the management of chronic hepatitis B? Rev Med Virol, 29(4), e2051.
Xuanyong Lu, Y. L., Ryan Geschwindt, Raymond A. Dwek, and Timothy M. Block. (2001). Hepatitis B Virus MHBs Antigen Is Selectively Sensitive to Glucosidase-Mediated Processing in the Endoplasmic Reticulum. DNA and Cell Biology, 20(10), 647-656.
Yaginuma, K., Shirakata, Y., Kobayashi, M., & Koike, K. (1987). Hepatitis B virus (HBV) particles are produced in a cell culture system by transient expression of transfected HBV DNA. Proc Natl Acad Sci U S A, 84(9), 2678-2682.
Yan, H., Zhong, G., Xu, G., He, W., Jing, Z., Gao, Z., . . . Li, W. (2012). Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus. eLife, 1, e00049-e00049.
Yan, R., Cai, D., Liu, Y., & Guo, H. (2017). Detection of Hepatitis B Virus Particles Released from Cultured Cells by Particle Gel Assay. Methods Mol Biol, 1540, 193-202.
Yen, C. J., Ai, Y. L., Tsai, H. W., Chan, S. H., Yen, C. S., Cheng, K. H., . . . Huang, W. (2018). Hepatitis B virus surface gene pre-S2 mutant as a high-risk serum marker for hepatoma recurrence after curative hepatic resection. Hepatology. 68 (3), 815-826
Zajakina, A., Kozlovska, T., Bruvere, R., Aleksejeva, J., Pumpens, P., & Garoff, H. (2004). Translation of hepatitis B virus (HBV) surface proteins from the HBV pregenome and precore RNAs in Semliki Forest virus-driven expression. Journal of General Virology, 85(11), 3343-3351.
Zhu, H. L., Li, X., Li, J., & Zhang, Z. H. (2016). Genetic variation of occult hepatitis B virus infection. World J Gastroenterol, 22(13), 3531-3546.
Zucman-Rossi, J., Villanueva, A., Nault, J. C., & Llovet, J. M. (2015). Genetic Landscape and Biomarkers of Hepatocellular Carcinoma. Gastroenterology, 149(5), 1226-1239.e1224.