Ddx3x在小鼠胚胎發育、器官形成及腫瘤生成的基因功能研究

Abstract

DEAD box RNA 解螺旋酶DDX3（亦稱為CAP-Rf, DDX3X及DBX）為一可與C型肝炎核心蛋白 發生交互作用的細胞因子。多年來，本實驗室持續致力於研究此一解螺旋酶在生物學上所扮演的角 色。近年來的研究結果顯示，DDX3不僅能扮演轉錄活化因子，同時也具有抑制cap-dependent 轉譯起 始作用，亦在腫瘤形成的過程中，扮演調控細胞之生長之抑癌基因。而近年來也陸續有許多文獻報導， DDX3對於調控mRNA從細胞核的輸出以及RNA的剪接上扮演重要的角色。儘管過去已證實DDX3在細 胞生物學或分子生物學上所扮演的多種重要的角色，然而研究的基礎大多以活體外細胞系統為主，在 生物體內的生理調控機制則有待進一步地釐清。因此，為了能進一步探討DDX3在活體內所調控的生 理機轉，我們成功建立Ddx3x條件式基因剔除小鼠（conditional knockout mice），並與不同組織特異表 現的Cre recombinase小鼠進行交配，在不同器官組織中剔除Ddx3x並進行分析。在三週齡子代的基因型 分析中，我們無法得到全身性基因剔除（conventional knockout mice）Ddx3x-/Y的公鼠，此結果暗示著 當細胞缺乏Ddx3x基因時，對小鼠造成致死的影響。為了證實此一結果，我們進一步利用上胚層 （epiblast）專一性表現的Sox2-Cre對Ddx3x進行基因剔除。初步的結果亦顯示，Sox2-Cre/+; Ddx3xflox/Y 的小鼠在胚胎發育第9.5天時便出現生長遲緩的情形，在胚胎時期第11.5天就已經死亡。此外，我們也 利用神經脊細胞（neural crest cells, NCCs）特異表現的Wnt1-Cre，在具有移行能力（migration）、分化 多能性（multipotent）並可在發育過程具有特異分化成不同細胞形式的細胞族群，對Ddx3x基因進行剔 除，初步的研究結果顯示，神經脊細胞缺乏Ddx3x情況下，同樣也會造成基因剔除公鼠在出生前後 （perinatal）致死的現象。綜合上述的研究結果顯示，Ddx3x在胚胎發育及器官形成的過程扮演了重要 的角色。因此，在未來三年，本計畫將以探討Ddx3x對於胚胎發育及器官形成上所扮演的生理功能作 為研究主軸。首先，希望釐清全身性Ddx3x及外胚層專一性Sox2-Cre/+; Ddx3xflox/Y基因剔除小鼠發生胚 胎致死的確切時間及原因；另外，以Wnt1-Cre/+; Ddx3xflox/Y 基因剔除小鼠，探討Ddx3x在這群多潛能 的神經脊細胞之特異分化過程中對不同器官組織所造成的缺陷及致死原因，深入分析Ddx3x在胚胎發 育及神經脊細胞所衍生顱顏形成（craniofacial development）的相關器官中所扮演的角色，並針對其所 參與調控的分子機制及訊息傳導作進一步的研究。此外，過去在我們及他人的研究中，都曾報導過C 型肝炎病毒(HCV)和B型肝炎病毒(HBV)會將DDX3作為宿主標的蛋白。而實驗室先前發表的文獻，也 進一步指出DDX3的表現量下降與HCV及HBV所引發的肝癌有密切的關聯性。為了能明確地研究 Ddx3x在肝臟生長、分化及功能所扮演的角色，探討其與肝癌形成的關聯性，在此計畫裡，我們也將 利用Alb-Cre來剔除肝臟中的Ddx3x，進一步分析Ddx3x在肝臟細胞中的生物功能及在腫瘤的形成所扮 演的角色。也希望透過本計畫的研究，能夠釐清Ddx3x在肝臟中的確切生物功能，評估其作為腫瘤的 形成早期診斷指標的可行性。

The DEAD-box RNA helicase DDX3 was originally isolated as hepatitis C virus (HCV) core-interacting protein by several laboratories including ours. Over the past years, my lab has investigated the biological function of DDX3. Our recent findings indicated that DDX3 possesses pleiotropic functions, such as acting as a transcriptional activator, a negative regulatory role in the cap-dependent translational initiation, and a cell growth regulator involved in tumorigenesis. Additionally, a growing number of reports have shown that DDX3 can exert a multiplicity of effects on cellular processes including mRNA export and RNA splicing. Despite DDX3 has been implicated in a variety of biological functions by molecular and cellular biological approaches, characterization of its broadly diverse functions in vivo awaits further studies. To gain insight into the physiological function of DDX3, we inactivate the Ddx3x locus in mice by targeted gene disruption. Upon analyzing the genotype of the offspring in conventional knockout mice, we can’t obtain Ddx3x-/Y mutant males. Lack of male offspring suggested that targeted inactivation of Ddx3x leads to lethality. To further confirm the observation that loss of Ddx3x resulted in lethality in male mice, epiblast-specific deletion of Ddx3x using Sox2-Cre was carried out. The preliminary results showed that Sox2-Cre/+; Ddx3xflox/Y embryos were growth retarded from E9.5 and found dead by E11.5. In parallel, we targeted inactivated Ddx3x in neural crest cells (NCCs) lineage by Wnt1-Cre, which resulted in the perinatal lethality of the mutants as well. Taken together, these preliminary findings implied that Ddx3x is crucial for embryogenesis and organs development. In view of these, the objective of this project in the next three years will access the physiological roles of Ddx3x during embryonic and organs development. The time and causes of this embryonic lethality will be characterized in Ddx3x null and Sox2-Cre/+; Ddx3xflox/Y conditional mutants. The phenotypic abnormalities of Wnt1-Cre/+; Ddx3xflox/Y mutants and the essential roles of Ddx3x in the subset of peripheral neurons, as well as vascular structures and craniofacial skeleton, which originally derived from the unique population of NCCs, will be characterized. The molecular mechanism underlying the lethal phenotype and the genetic pathways through which Ddx3x acts to influence during organogenesis will be explored. Moreover, we and others studies showed that DDX3 is a host target by human hepatitis C virus (HCV) and hepatitis B virus (HBV). Our previous study suggested that deregulation of DDX3 may involve in HCV- and HBV-associated hepatocarcinogenesis. To explore the possible link between Ddx3x gene expression and the development of hepatocellular carcinoma (HCC), we also plan to substantiate cellular functions of Ddx3x in liver by targeted inactivation of Ddx3x using Alb-Cre. The novel insights from the functions of host target Ddx3x in liver might be valuable to the development of early histological indicator for biopsy diagnosis of HCC.