微核醣核酸let-7b抗C型肝炎病毒感染的作用機制

Abstract

C型肝炎病毒為造成肝臟疾病的重要致病因子之一，病毒慢性感染引起肝臟纖維化，肝硬化，甚至導致肝癌，為公共衛生的重要課題。微核醣核酸（microRNA）是一non-coding小片段RNA，可透過降解mRNA或抑制蛋白質轉譯而達到調節標的基因表現的作用，進而參與了病毒感染的調控。Let-7b為一細胞內生性微核醣核酸，我們觀察到C型肝炎病毒感染會誘導let-7b表現。利用Ago2-IP實驗證實let-7b和C型肝炎病毒基因體具交互作用，接著，以標的預測分析及點突變實驗證實，let-7b會直接標的C型肝炎病毒基因體抑制C型肝炎病毒增殖。另一方面，我們證明let-7b也參與了調控宿主因子的表現。首先，利用microarray分析轉染let-7b細胞的轉錄圖譜，交集和C型肝炎病毒生活史相關的細胞因子，經由Gene Ontology 資料庫分析這些基因參與的生物途徑，發現let-7b調節表現增加的宿主基因，主要參與在第一型干擾素反應路徑。接著，搭配標的預測分析和報導載體標的驗證，我們發現let-7b標的JAK-STAT訊息傳遞路徑上的負調節因子SOCS1，進而促進第一型干擾素誘導的抗病毒基因表現。此外，let-7b也參與了第一型干擾素的產生。利用knockdown基因表現方式證實，let-7b透過RIG-I的訊息傳遞促進干擾素-β產量增加。因此，let-7b具有直接標的病毒基因與正調控抗病毒干擾素反應的雙重功能，具有發展成抗C型肝炎病毒藥物新標的的潛能。

Hepatitis C virus (HCV) is one of the most important etiologies for liver disease. Chronic HCV infection leads to liver fibrosis, cirrhosis, and hepatoma. It is an important issue for global public health. MicroRNA (miRNA) is a small non-coding RNA, which regulates gene expression through mRNA degradation or translational inhibition. Therefore, it also involved in regulation of virus infection. Let-7b is one of endogenous miRNAs, we found that let-7b is induced during HCV infection. Using Ago2-IP method, we found that let-7b interacts with HCV genome. The predicted target sites of let-7b on HCV genome are validated by both verified reporter analysis and site-directed mutagenesis analysis. These results demonstrated that let-7b directly targets the HCV genome to suppress HCV propagation. On the other hand, we further explore whether let-7b regulates expression of host genes to control HCV infection. Firstly, microarray analysis was performed to demonstrate the distinct expression profile of let-7b transfected cells. The let-7b regulated genes crossing with host factors which involved in HCV life cycle were selected. The engaged biological pathways were predicted by the Gene Ontology database. We found that most let-7b up-regulated genes are involving in type I interferon (IFN) signaling. To understand how let-7b regulates type I IFN signaling to interfere with HCV infection, target prediction combined with verified reporter analysis were performed. We demonstrated that let-7b targets SOCS1, a negative regulator of JAK/STAT signaling pathways, contributing to the expression of anti-viral genes induced by type I interferon. Further, let-7b also increases the production of type I IFN through RIG-I pathway by knockdown different genes which involved in IFN production. Therefore, let-7b has a dual function on directly targeting viral genome and regulation antiviral response of interferon. Let-7b thereby plays a potential therapeutic candidate for anti-HCV therapy.