性激素調控B型肝炎病毒mRNA轉錄過程之分子機制研究

Abstract

在B型肝炎相關聯之肝癌發生率上，有著男性顯著高於女性的盛行率。男性帶原者通常具有較高的血清病毒量，而較高的血清病毒量已被證實為導致肝癌的風險因子之一。在此論文研究中，將探討性激素訊息傳遞路徑是否藉由調控病毒轉錄及複製過程為導致男性體內較高血清病毒量之一可能機制。我們利用B肝病毒基因體轉殖鼠的動物模式來評估血清中S表面抗原與病毒量是否有著性別歧異，並且針對性激素參與其中的重要性以切除性腺的方式來加以探討。公鼠血清中的S表面抗原與病毒量皆高於母鼠，這兩項病毒指標在公鼠去勢睪丸之後會顯著降低；相反地，血清病毒量在母鼠切除卵巢之後會明顯上升，而公鼠在接受雌激素外來補充之後則會有所下降。這結果顯示出雄激素、雌激素在B肝病毒生活史中，扮演著正、負向調控的角色。 對於雄激素部份的研究，我們發現受到雄激素活化的雄激素受器，可透過其轉活化功能結構域來增強病毒整體的轉錄核醣核酸量。在B肝病毒的一號增強子序列中，我們發現到特定片段專門負責媒介雄激素受器對於轉錄活化的效果。染色質免疫沉澱法與體外核酸蛋白結合測定的實驗結果也進一步地證實，受到雄激素活化的雄激素受器會直接黏合至此特定片段上。兩套雄激素反應元在此片段中被我們確切定位出來，若是以點突變的方式破壞它們，將會嚴重影響到雄激素受器增進B肝病毒轉錄生成量的效果。 對於雌激素部份的研究，我們發現即使在沒有雌激素的情況下，雌激素受器本身就能夠造成病毒整體核醣核酸量的降低。藉由螢光酶報告檢測的協助，B肝病毒基因體中負責媒介轉錄抑制效果的區域亦位於一號增強子序列中。經由實驗證實，雌激素受器並非主動地經由直接黏合至目標序列，或透過組織蛋白去乙醯化酶的活性來發揮效果；相反地，它是藉由困鎖肝细胞核因子4α此一正向轉錄調控因子的被動機制，來阻止其結合至一號增強子序列上而達到抑制病毒轉錄核醣核酸的效果。因此超量外表現肝细胞核因子4α能夠克服雌激素受器對於B肝病毒的轉錄抑制。而且若是一號增強子序列上的肝细胞核因子4α結合位置被破壞，雌激素受器對於病毒轉錄的影響將會完全消失。協同免疫沉澱法的分析也指出，雌激素受器可透過其蛋白結構上的鍊橋功能域來與肝细胞核因子4α結合。目前的實驗結果顯示，雌激素受器抑制B肝病毒轉錄的可能機制是毋需雌激素的參與；然而接受雌激素活化的雌激素受器，是否也是經由困鎖肝细胞核因子4α的機制來抑制B肝病毒的轉錄生合成，需要更進一步地研究驗證。 總結而論，這篇研究論文已證實：雄激素、雌激素這兩種性激素的訊息傳遞路徑，分別以正、負向的角度來調控B肝病毒的轉錄核醣核酸量。本研究成果所發現的此一現象，將可能造成血清病毒量上的性別差異，並且對其提出一項可能的分子機制來說明男性B肝帶原者體內較高的血清病毒量，以及後續較高的肝癌發生風險。

Hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) occurs more often in men than in women. Male HBV carriers usually have higher viral loads, which was a well-known risk factor for HCC. Whether the sex hormone pathways are involved in regulating HBV life cycle as one possible mechanism for the higher titer in male patients was investigated in this thesis. The HBV transgenic mice were used for evaluating any sex difference of serum viral antigens and titers, as well as the gonadectomy effect on this difference. The serum Hepatitis B surface antigen (HBsAg) levels and viral titers were higher in male than in female HBV-transgenic mice, which could be lessened by castration of the males. In contrast, the HBV titers were elevated by ovariectomy in the females and were decreased by supplementation of estrogen in males. The results suggested the positive role of androgen and negative role of estrogen pathways in regulating the HBV life cycle. For the androgen part of study, we found that the ligand-stimulated AR could increase the amounts of overall HBV transcripts through its transactivation domain. A genomic region within HBV enhancer I was identified to be responsible for the transcriptional activation of AR. The results from chromatin immunoprecipitation and in vitro binding assays further demonstrated a direct binding of AR to this region, in a ligand-dependent manner. Two androgen response elements (ARE) in this region were identified, and mutations of either site can significantly abolish the AR effects. For the estrogen part of study, we found that ERα could decrease the HBV mRNA levels, even in the absence of ligand. Aided by reporter assay, a region within HBV enhancer I was identified responsible for the repression. The active repression mechanism, either by direct binding of ERα or through the HDAC activity, was not required for ERα to repress the transcription of HBV. In contrast, a passive mechanism by ERα to squelch a positive transcriptional factor, HNF4α, from binding to the enhancer I region was identified. Overexpression of HNF4α could overcome the repressive effect of ERα on HBV transcription. Moreover, the ERα effects on the transcription of HBV could be abolished by disrupting the HNF4α-binding site within HBV enhancer I. Co-immunoprecipitation analysis further indicated that ERα can bind to HNF4α through its hinge region. The current results thus illustrate a possible mechanism for ER to repress the transcription of HBV, in a ligand independent manner. Whether the ligand stimulated ERα can also through chelating the HNF4α for repression of HBV transcription warrants further investigation. In conclusion, this thesis demonstrated that both androgen and estrogen pathways can regulate the transcription of HBV mRNAs, but in an opposite manner. It could contribute to the gender difference of viral titers and provide one molecular mechanism for the higher HBV titer and increased risk of HCC in male HBV carriers.