利用果蠅作為生物模式動物尋找B型肝炎病毒X蛋白之修飾基因

Abstract

肝癌(Hepatocellular carcinoma, HCC)為台灣地區主要癌症死亡原因之一，肝癌的發生與B型肝炎病毒慢性感染具有密切相關。B型肝炎病毒X蛋白(Hepatitis B virus X protein, HBx)是一種B型肝炎病毒製造的調節蛋白，HBx除了影響病毒複製之外，許多研究指出HBx也參與肝癌的發生。但是HBx究竟如何影響病毒複製及肝癌之癌化過程，以及細胞中參與調控之關鍵分子基因，仍有待進一步釐清。尤其是目前許多研究利用cell culture system所指出參與調控HBx功能之細胞因子及訊息傳遞路徑眾多且多有爭議，此問題將有可能藉由利用果蠅單純之genetic 分析工具達到釐清關鍵分子機制之目的。因此本研究計劃以果蠅作為實驗動物，利用果蠅眼睛發育時，表現HBx蛋白所造成的rough eye表現型作為篩選的依據，分別進行「Non-biased」的Dose-sensitive genetic screen，以及針對特定的訊息傳遞路徑進行 genetic screen。 實驗設計乃利用p-element mediated transgenesis技術製造HBx的轉殖果蠅，並利用UAS/GAL4系統在果蠅眼睛表達HBx蛋白，結果發現30℃培養下，果蠅眼睛具有清楚的可供基因篩選的不正常表徵，表示HBx的確對細胞中特定調控機制產生影響，可提供後續進行whole genome dose-sensitive genetic screen之研究。在之後接續進行果蠅染色體缺失篩選時，利用Bloomington Deficiency Kits (Bloomington Stock Center)，一共有270株基因缺陷的果蠅作為篩選材料(包含了95%以上的果蠅基因組)，目前已完成50個染色體 (約1/6 基因組)之缺失篩選。完成的染色體位置分別為第二條染色體左臂的21A1到31C1，以及第二條染色體右臂的44D1到53A1。實驗結果發現在這兩區域間，分別存在兩個會增強HBx轉殖果蠅所造的眼睛不規則表徵之基因缺失果蠅株，分別是Df(2L)ed1、Df(2L)BSC143、Df(2R)en-A、Df(2R)BSC134。後續將可以相同的實驗方法完成果蠅染色體缺失篩選，並再利用基因區域中的每一基因的RNAi果蠅株，藉此找出HBx相關的修飾基因。 至於HBx和特定訊息傳遞路徑的基因互動實驗，結果發現HBx與EGFR、Notch、Hippo、JAK/STAT、 Autophagy等訊息路徑之間不存在明顯之交互作用。反之HBx 與PI3K、Wnt、mTOR訊息路徑之間可偵測到正向交互作用之存在，而與Hedgehog訊息路徑間則存在負向的交互作用。進一步分析HBx和Wnt訊息路徑間的關係時，採用constitutively active 之 GSK-3 kinase作為實驗材料時，發現HBx和其存在明顯的正向基因互動。於是假設HBx所造成的複眼發育不正常表徵可能透過GSK-3 kinase，這樣的推測在利用Dominant negative form GSK-3與HBx基因轉殖果蠅進行cross 時，發現DN-GSK-3會減弱HBx所造成的複眼發育不正常表徵，間接說明HBx所造成的複眼發育不正常表徵應透過GSK-3所調控的路徑。實驗結果顯示，在果蠅的實驗系統中GSK-3 kinase 可能為HBx的修飾基因，然而HBx如何影響此路徑，對病毒複製造成什麼影響，以及最終導致癌化的過程是未來可進一步研究的方向。

Hepatitis B virus (HBV) infection is hyperendemic in Taiwan and is a major risk factor for HCC. The HBV X protein (HBx) not only can regulate the viral replication, it is also implicated as a carcinogenic factor for hepatocellular carcinoma (HCC). But currently the molecular mechanisms by which HBx contributes to viral replication and HCC carcinogenesis remain largely inconclusive. We propose to dissect the complicated HBx regulated cellular events by using the drosophila genetic approaches. Two approaches are conducted in this thesis, one is the non-biased whole genome genetic screen to find out the HBx modifier gene(s), and the other is the genetic screening for specific signaling pathways interacting with HBx. We first try to generate the HBx transgenic fly and study the phenotypes caused by HBx expression in Drosophila compound eyes. The full length HBx was cloned into pUAST and the resulting pUAST-HBx was used to generate UAS-HBx transgenic flies by microinjection. HBx mRNA is detectable when ectopic expressed UAS-HBx under the control of eye specific driver, GMR-GAL4 . We found that at 30 ℃, the HBx expressing ommatidia displays a rough eye phenotype, which can be used as a screen platform for subsequent genetic screening analysis. To do the non-biased whole genome genetic screen, we have included a set of chromosome deficient fly stocks, which cover more than 95% of fly genome, to explore the novel factors which can modify the HBx-mediated rough eye phenotype. Currenly, 50 chromosome deficient lines have been screened (~1/6 of the fly genome), spanning the chromosome regions of 21A1 to 31C1 and 44D1 to 53A1 on the second chromosome. Four deficiency lines were found with ability to enhance the severity of HBx-mediated rough eye, including Df(2L)ed1, Df(2L)BSC143, Df(2R)en-A, and Df(2R)BSC134. The same experiments will be applied to the other 5/6 genome and the putative modifier gene(s) within each chromosome region will be identified by RNAi approach. For the genetic screening of specific signaling pathways interacting with HBx, no genetic interactions between HBx and the signaling pathways of EGFR, Notch, Hippo, JAK/STAT, and Autophagy were identified. In constrast, there exists positive genetic interactions between HBx and PI3K、Wnt、mTOR signaling pathways and a negative genetic interaction between HBx and Hedgehog signaling pathway. To focus on the interaction between HBx and Wnt signaling pathway, our results further deomonstrated a positive genetic interaction between HBx and the constitutively active form of GSK-3 kinase. It suggested that HBx might increase the activity of GSK-3 kinase pathway. Accordingly, we propose that HBx-induced rough eye might be caused by regulating the GSK-3 kinase pathway. Indeed, the HBx-mediated rough eye is diminished in the dominant negative GSK-3 genetic background. Our current results suggested that GSK-3 kinase might function as a genetic modifier of HBx in this fly system. How does HBx affect this pathway and what is the resulting effect on viral replication and carcinogenic process warrants further investigation.