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標題: | 乙型肝炎病毒DNA嵌入對肝細胞基因體對肝癌性別差異之調控及其臨床應用 Hepatitis B virus DNA integration in contributing to the gender difference of liver cancers and its clinical application |
作者: | 李巧玲 Chiao-Ling Li |
指導教授: | 葉秀慧 Shiou-Hwei Yeh |
關鍵字: | 乙型肝炎病毒,肝癌,性別差異,生物標記, hepatitis B virus (HBV),hepatocellular carcinoma (HCC),gender difference,biomarker, |
出版年 : | 2019 |
學位: | 博士 |
摘要: | 肝癌之性別差異於乙型肝癌病毒相關肝癌中最為顯著,此類肝癌通常會發生乙型病毒之基因嵌入現象。其中位於telomerase (TERT) 啟動子之基因嵌入頻率最高,且可增加TERT基因表現並可能參與致癌機制。乙型肝炎病毒中包含雄激素反應序列及雌激素反應序列,因此我們提出乙型肝炎病毒相關肝癌之病毒嵌入可能會使得被嵌入之宿主基因受到性激素路徑之調控,而使得乙型肝炎病毒相關肝癌較好發於男性。為測試此假說,我們利用Capture-NGS平台辨識101個乙型肝炎病毒相關肝癌的乙型肝炎病毒嵌入序列以及TERT啟動子與TP53基因中常見於肝癌之體突變。結果顯示於TERT啟動子之乙型肝炎病毒嵌入及點突變為互斥事件,且皆較顯著發生於男性。(乙型肝炎病毒嵌入 P = 0.0285*; 點突變, P = 0.0201*; 兩者合計, P < .0001***). 我們進一步利用reporter assay研究性激素路徑藉由乙型肝炎病毒嵌入及點突變影響TERT表現之機制。嵌入的HBV DNA使TERT受性激素路徑調控,藉由HNF4α此轉錄因子,雄激素路徑可促進但雌激素可抑制TERT之轉錄。此外,雄激素路徑亦可藉由參與轉錄因子GABPA於TERT啟動子點突變之調控而促進TERT轉錄。此二機制皆可促進乙型肝炎病毒相關肝癌好發於男性之現象。
由NGS分析得知高於九成之乙型肝炎病毒相關肝癌中皆可發現乙型肝炎病毒DNA隨機嵌入所產生的乙型肝炎病毒-宿主嵌合DNA (vh-chimera DNA),此病毒感染產生之極具特異性之基因變異有潛力作為腫瘤之新穎指標。此部分研究之目的即為調查病毒-宿主嵌合DNA可否作為腫瘤切除後殘存腫瘤細胞之血液指標。因此我們蒐集50位乙型肝炎病毒相關肝癌病患之腫瘤組織,及其術前與術後兩個月之血液檢體。我們利用caputure-NGS平台可於88% (44/50) 之乙型肝炎病毒相關肝癌中偵測到HBV DNA之嵌入。結果顯示目前之偵測敏感度為1-2公分大小之腫瘤,且術前血之vh-chimera DNA含量與腫瘤大小呈正相關。而於26.2% (11/42) 之術後兩個月血漿中可偵測到與術前血相同之vh-chimera DNA,顯示這些個案可能有殘餘腫瘤細胞。實際上,其中82% (9/11) 的個案的確於一年內發生腫瘤復發。因此目前結果支持vh-chimera DNA可作為一新穎之乙型肝炎病毒相關肝癌生物指標。且顯示vh-chimera DNA對於偵測術後之殘存腫瘤及一年內復發極具潛力,但仍需對未來臨床應用性進一步研究。 The gender disparity of hepatocellular carcinoma (HCC) is most striking in hepatitis B virus (HBV)‐related HCC. The majority of such HCC cases contain integrated HBV. Some hotspot integrations, such as those in the telomerase (TERT) promoter may activate gene expression to drive carcinogenesis. As the HBV genome contains both androgen‐responsive and estrogen‐responsive motifs, we hypothesized that the integrated HBV DNA renders a similar regulation for downstream gene expression and thus contributes to male susceptibility to HCC. To test this hypothesis, the HBV integration sites and the common mutations in the TERT promoter and TP53 coding region were analyzed in 101 HBV‐related HCC cases using a capture‐next‐generation sequencing (Capture-NGS) platform. The results showed that both HBV integration and –124G>A mutation in the TERT promoter region, occurring in a mutually exclusive manner, were more frequent in male than in female patients with HCC. (HBV integration, P = 0.0285*; promoter mutation, P = 0.0201*; in combination, P < .0001***). The effects of sex hormone pathways on the expression of TERT with both genetic changes were investigated using a reporter assay. HBV integration in the TERT promoter rendered the TERT transcription responsive to sex hormones, with enhancement by androgen receptor (AR) but suppression by estrogen receptor, both of which were dependent on hepatocyte nuclear factor 4 alpha (HNF4α). Besides, AR also increased TERT expression by targeting TERT promoter mutations in a GA binding protein transcription factor subunit alpha (GABPA)–dependent manner. Therefore, TERT elevation by AR through integrated HBV and point mutation at the TERT promoter region was identified as a mechanism for the male dominance of HBV‐related HCCs. As revealed by the NGS analysis, more than 90% of HBV-related HCC contain integrated HBV randomly distributing in chromosomes, which generates a unique HBV-human chimera DNA (vh-chimera DNA) for individual HCC as potential signature tumor marker. This study aims to investigate if the vh-chimera DNA could be a cell free circulating signature DNA biomarker for HBV related HCC, in monitoring the presence of residual tumor cells after tumor resection. Fifty HBV related HCC cases were recruited. In addition to the tumor tissues, the plasma samples were collected just before and 2 months after surgery. The HBV integration sites were identified in 44 cases (88%) by capture-NGS platform. The results showed that the copy number of vh-chimera DNA in plasma at surgery well correlated with the tumor size, with the detection limit at 1-2 cm. Among the plasma collected at 2 months after surgery, 26.2% (11 out of 42) of samples contained the same signature vh-chimera DNA as baseline plasma, indicating the presence of residual HCC. Consistently, 82% (9 out of 11) of these cases suffered HCC recurrence in one year. Therefore, this part of study well supports the vh-DNA as a new circulating DNA marker for detecting HCC in HBV-related HCC patients. Moreover, vh-chimera DNA shows great potential for detecting residual HCC in HBV-related HCC after surgery and for monitoring recurrence within one year of surgical resection, which warrants more investigations for future clinical use. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/78808 |
DOI: | 10.6342/NTU201900566 |
全文授權: | 未授權 |
電子全文公開日期: | 2024-03-11 |
顯示於系所單位: | 微生物學科所 |
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