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  • 學位論文

D型肝炎病毒轉活化能力及病毒基因體序列參與RNA複製之相關證據

Evidences of the hepatitis delta virus (HDV) transactivation and the association of multiple genomic sequences with RNA replication

指導教授 : 許國堂

摘要


研究目的: D型肝炎病毒 (HDV) 引起嚴重肝臟疾病,是造成肝癌以及肝硬化的重要危險因子,而其獨特的複製生活史更加引起研究者對它的興趣。可能因為HDV RNA的序列結構,使得轉錄因子透過D型肝炎病毒抗原 (hepatitis delta antigen, HDAgs) ,能夠轉而辨識HDV RNA來進行RNA之轉錄及複製作用。因為HDV所產生的HDAgs,Small-HDAg (S-HDAg) 以及Large-HDAg (L-HDAg),具有基因轉活化能力。因此,本研究旨在探討: (1) HDAgs轉活化能力對於細胞基因之調控以及基因與HDV之相關性。(2) 鑑定HDV RNA基因體關鍵性序列片段與HDV RNA複製之相關性。 研究方法: 我們首先建立人類肝癌細胞株Huh7穩定表現HDAg之細胞株,Huh-S和Huh-L,進行HDAgs對細胞基因轉活能力研究。利用人類全基因晶片來篩選受HDAgs所調控之基因。其中部分基因利用real-time PCR以及Western blots確認基因表達量,利用luciferase reporter gene assay以及chromatin immunoprecipitation assay (ChIP assay) 鑑定HDAgs對此基因之調控方式以及確認此基因是否接受HDV調控。此外,本研究將HDV RNA基因體反轉錄成HDV cDNA序列,並搜尋含有啟動子活性之片段,接著共同轉染HDV cDNA啟動子片段以及HDAgs質體觀察HDAgs對此啟動子活性之影響。之後利用in vitro transcription合成野生型和突變型HDV RNA,將in vitro RNA轉染至Huh-S細胞中,利用Northern blots分析序列之複製功能以及利用Western blots分析HDAgs合成能力。 研究結果: 在HDAgs對細胞基因轉活化能力研究中發現: (1) HDAgs能轉活化細胞基因,以全人類基因晶片篩選出近兩萬個受HDAgs影響之基因,當中我們挑選11個基因進行分析。(2) Clusterin (CLU) 受HDAgs誘導表現,且CLU表現與HDV複製成正相關性。(3) S-HDAg以及L-HDAg皆可增強CLU啟動子histone H3乙醯化程度及CLU表現,降低細胞對於化學治療藥物之感受性。在HDV RNA基因體序列的研究中發現: (1) HDV cDNA序列含有兩段雙向啟動子活性區 (TR-P1, nt 1582~1683和TR-I5, nt 1223~1363),且HDAgs不同程度地正向調控此啟動子活性。(2) HDAg-ORF上的AUG密碼 (nt 1599~1601) 突變成UAG (Amber) 使得單套體RNA能夠合成但是雙套體RNA卻無法合成。(3) TR-P1以及TR-I5進行突變分析會影響HDV RNA複製的能力。(4) 也發現HDV單套體RNA即可編輯合成L-HDAg且獨立於double rolling-circle複製機轉之外。 研究結論: HDAgs基因轉活化能力的研究中發現,CLU是能夠被HDV所調控的基因之一,S-以及L-HDAg能夠增強CLU基因轉錄活性。透過CLU,本研究提供的證據顯示,HDAgs有能力藉由基因體學上 (epigenetic) 乙醯化程度的改變來調控基因表達。然而,細胞內有多少基因透過此方式受HDAgs調控目前仍不清楚。根據基因篩選結果,細胞內受HDAgs影響之基因數量甚為龐大,當中可能部分基因與HDV RNA複製作用相關,另一部分則與CLU功能相似,具有保護細胞承受外界壓力之功能。因此,HDV帶原者可能因為HDAgs對細胞基因的轉活化能力,促使細胞存活能力增強,進而提升細胞致癌性的可能。在鑑定HDV RNA基因體序列的研究中發現,TR-P1可能是合成HDV mRNA、genomic和antigenomic RNA的RNA啟動子。此外,我們發現TR-I5序列與HDAg-ORF序列的正確性與RNA double rolling-circle replication的機制密不可分。藉由本研究對於HDAgs的基因轉活化能力以及HDV RNA-dependent RNA replication過程 相關證據能夠對HDV的探討提供新的想法。

並列摘要


Objective: Hepatitis delta virus (HDV) is a major cause of severe liver disease. HDV is a negative single-stranded RNA with a circular conformation and has the ability to fold as a unique double-stranded, rod-like structure on itself. There are two different forms of hepatitis delta antigens (HDAgs) are produced during life cycle of HDV: small HDAg (S-HDAg) and large HDAg (L-HDAg). HDAgs are indispensible for HDV life cycle. Both HDAgs are transcriptional inducers and capable of regulating cellular gene expression. However, replication of HDV RNA is dependent of cellular RNA polymerases and does not involve any DNA intermediates. How cellular polymerases change the template from DNA to RNA and synthesize HDV RNA is a very important issue and requires further investigation. Therefore, the two objectives of this study are: (1) to analyse the cellular genes regulated by HDAgs and to characterize the gene function in HDV infection, (2) to investigate the cis-elements in HDV genome that are critical for RNA-dependent RNA replication. Materials and Methods: The S- or L-HDAg-expressing Huh7 cells (Huh-S and Huh-L) were established to identify genes activated by S-HDAg or L-HDAg using Agilent Human Whole Genome Oligo 4×44 K Microarrays. Several up-regulated genes were further examined by real-time PCR and Western blots. The luciferase reporter gene assay and chromatin immunoprecipitation assay (ChIP assay) were used to examine how the genes regulated by HDAgs and by HDV. In addition, we screened various fragments of putative HDV promoter by luciferase reporter gene assay. We also examined the promoter activities regulated by HDAgs. The WT and mutated HDV RNAs were synthesized by in vitro transcription and transfected into Huh-S cells to characterize the HDV de novo RNA synthesis on Northern blots and Western blots. Results: We found: (1) Nearly 20,000 genes are affected in Huh7 cells that stably expressing HDAgs. We selected 11 genes and further identified by real-time PCR and Western blots. (2) CLU expression is upregulated by HDAgs and positively associated with HDV replication. HDAgs enhance histone H3 acetylation of clusterin promoter to increase gene expression and decrease drug sensitivity to adriamycin treatment. We also found several evidences of the cis-elements in HDV genome associated with HDV RNA replication: (1) Two HDV cDNA fragments (TR-P1, nt 1582~1683 and TR-I5, nt 1223~1363) containing bidirectional promoter activity and enhanced by HDAgs to differing degrees. (2) The AUG codon at HDAg-ORF of HDV RNA (nt 1599~1601) that mutates to UAG (Amber) results in loss of dimeric but not monomeric HDV RNA synthesis. (3) Mutagenesis of TR-P1 and TR-I5 affect HDV RNA replication. (4) RNA editing event occurs in the monomeric HDV RNA and independent of double rolling-circle RNA synthesis. Conclusion: We demonstrated that clusterin is an authentic HDV-regulated gene, which is specifically transcriptional induced by S-HDAg and L-HDAg via histone hyperacetylation in Huh7 cells. However, how many genes are regulated by HDAgs via epigenetic-mechanism remains unclear. Cytoprotective genes, like CLU, are induced in HDV infected cells may increase cell survival and further increase oncogenic potential in the liver of HDV carrier. We also provide evidences that TR-P1 region is most likely the authentic HDV RNA promoter for mRNA, genomic, and antigenomic RNA synthesis. Although TR-I5 is not an RNA promoter but it is associated with RNA double rolling-circle replication. The integrity of HDAg-ORF is also essential for HDV RNA double rolling-circle replication. These results provide evidences to unmask the mechanisms of transactivation and RNA-dependent RNA replication with hepatitis delta virus.

參考文獻


曾昭榮 (2007). 研究clusterin在人類肺癌H1299細胞的表現及功能 碩士論文
尤信超 (2005). 利用二維膠電泳分型肝炎抗原與宿主細胞之關係 碩士論文
張瑞玶 (2005). 使用FISH-PCR尋找D型肝炎病毒抗原所誘導的基因 碩士論文
蔡居政 (2007). 研究Huh7細胞株中D型肝炎病毒抗原所誘導的基因及其相關蛋白 碩士論文
陳德合 (2005). 研究HDV cDNA promoter活性和HDV antigen之關係 碩士論文

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