Roles for RCK/DDX6 in hepatitis C virus pathogenesis and hepatocellular carcinoma

肝细胞癌是癌症相关死亡的主要原因。虽然许多肝细胞癌是由慢性酒精滥用或环境毒素引起的，但大约80%的病例是由乙肝或丙型肝炎病毒(HCV)感染引起的。全球丙型肝炎病毒感染的负担非常大，大部分个人和经济负担还没有到来，因为肝硬变和肝细胞癌需要数年时间才能发展。全世界大约有1.7亿人感染了丙型肝炎病毒。大多数感染者不会清除疾病，但会发展成慢性感染，通常会导致终末期肝病和肝细胞癌。目前的治疗方法仅限于与利巴韦林和干扰素联合治疗，这种治疗方法昂贵，对50%的感染者无效。因此，迫切需要确定新的、可获得的病毒和细胞靶点，用于抗丙型肝炎病毒和肝癌的治疗。最近有研究表明，直接静脉注射修饰的寡核苷酸可以很容易地灭活细胞内的microRNAs，特别是肝脏中的那些。因此，我们的发现是肝脏特异的microRNA miR-122直接与丙型肝炎病毒的5‘非编码区相互作用，这种相互作用对于维持病毒RNA在细胞内的丰度是必不可少的，这为抗病毒治疗确定了一个有希望的靶点。在肝脏中表达microRNA需要哪些宿主蛋白？最近，研究发现宿主细胞蛋白RCK/DDX6是DEAD BOX解旋酶家族的成员之一，在哺乳动物细胞的microRNA介导的基因调控中起重要作用，并且在丙型肝炎病毒诱导的肝细胞癌中miR-122和RCK都有显著的高表达。我们的初步结果表明，RCK是丙型肝炎病毒基因表达的重要正调控因子；RCK缺失会导致丙型肝炎病毒蛋白的丢失。在这项建议中，我们建议研究RCK在明显的转录后步骤上调丙型肝炎病毒基因表达的机制，以及RCK对未感染和丙型肝炎病毒感染的肝细胞中microRNA丰度和细胞内分布的影响。利用新的原位杂交技术，我们将确定RCK表达对microRNA定位到特定的细胞质隔间，即P小体和应激颗粒的影响。最后，我们建议进行实验，以确定其丰度和分布受RCK影响的microRNAs的靶点，这些研究将开发用于丙型肝炎和肝癌的新工具，并可能指出抗病毒治疗的新靶点。

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related deaths. Although many cases of HCC are induced by chronic alcohol abuse or environmental toxins, approximately 80% are caused by infection with either hepatitis B or hepatitis C virus (HCV). The burden of HCV infection worldwide is very large, with most of the personal and economic burden yet to come, as cirrhosis and HCC take years to develop. Approximately 170 million people are infected worldwide with HCV. Most infected individuals do not clear the disease, but develop chronic infections that often lead to end-stage liver disease and HCC. Current treatment is limited to co-treatment with ribavirin and interferon , a therapy that is expensive and ineffective in 50% of infected individuals. Therefore, there is an urgent need to identify new and accessible viral and cellular targets for therapies against HCV and HCC. It has been shown recently that cellular microRNAs, especially those in the liver, can be readily inactivated by direct intravenous delivery of modified oligonucleotides. Thus, our finding that liver-specific microRNA miR-122 interacts directly with the viral 5’ noncoding region of HCV and that this interaction is essential to maintain intracellular abundance of the viral RNA, identifies a promising target for antiviral therapy. What are the host proteins required for microRNA expression in the liver? Recently, it has been found that host cell protein RCK/DDX6, a member of the DEAD box helicase family, is important for microRNA-mediated gene regulation in mammalian cells and that both miR-122 and RCK are greatly overexpressed in HCV-induced HCC in patient livers. Our preliminary results have shown that RCK is an essential positive regulator of HCV gene expression; depletion of RCK results in loss of HCV proteins. In this proposal, we propose to examine the mechanism by which RCK upregulates HCV gene expression at an apparently post-transcriptional step and by which RCK affects microRNA abundance and intracellular distribution in uninfected and HCV-infected liver cells. Using novel in situ hybridization technology, we will determine the effect of RCK expression on microRNA localization to specific cytoplasmic compartments known as P-bodies and stress granules. Finally, we propose experiments to identify targets for microRNAs whose abundance and distribution is affected by RCK, These studies will develop novel tools for HCV and HCC and are likely to point to new targets for antiviral therapy.