利用類轉錄活化因子核酸酶基因剃除小鼠模型探討微型核糖核酸130a與B型肝炎病毒之間的交互作用

Abstract

B型肝炎病毒(HBV)感染為全球主要的健康問題，B型肝炎病毒感染可能會造成急性或慢性肝炎，長期的慢性肝炎可能會演變成肝硬化甚至是肝癌。雖然目前已經有疫苗能有效地預防B型肝炎病毒的感染，但仍然有許多人在施打疫苗後無法成功地產生對抗B型肝炎病毒的抗體以及疫苗對於慢性B型肝炎病人並無治療的效果。現今的第一線抗B型肝炎病毒藥物為第一型干擾素及核苷酸類似物，但兩者的作用效果皆有限且副作用強，容易產生抗藥性，目前的治療方法像是抑制病毒的進入或是抑制病毒顆粒的組裝等藥物皆無法根除病毒的共價閉合環狀去氧核醣核酸(cccDNA)，病毒仍然潛藏在肝細胞中。慢性B型肝炎病毒感染仍影響全球3.5億人口且每年約有78萬6千人口死於慢性B型肝炎病毒導致的肝硬化及肝癌，目前仍急須找尋有效的治療方法來對抗B型肝炎病毒感染。 微型核醣核酸(micro-RNA)為小片段非編碼的核糖核酸，能透過影響轉錄後的標的信使核糖核酸(messenger RNA)的轉譯及降解來調控基因的表現，已經有許多研究指出微型核醣核酸影響病毒與宿主之間的關係以及病毒的生命週期，近年來有些微型核醣核酸被發現能夠調控細胞蛋白或B型肝炎病毒轉錄本(transcript)，進而直接或間接地影響B型肝炎病毒的複製。 我們實驗室先前發現微型核醣核酸130a能夠調控兩個B型肝炎病毒轉錄因子/共同活化子: PGC1a與PPARg，進而抑制B型肝炎病毒的信使核糖核酸與蛋白質的表現及病毒的複製。 本次實驗，我們利用類轉錄活化因子核酸酶(TALEN)方法製作微型核醣核酸130a剃除鼠，研究B型肝炎病毒與微型核醣核酸130a在活體內的關係，我們利用高壓流體注射法(hydrodynamic injection)從小鼠尾靜脈打入B型肝炎病毒質體，發現到帶有PGC1a與PPARg高度表現的微型核醣核酸130a剃除鼠會有較高的病毒複製，另外我們從剃除鼠的尾靜脈打入微型核醣核酸130a表現的腺病毒來回補微型核醣核酸130a，發現能夠回過頭來抑制B型肝炎病毒的複製。 總和來說，我們在小鼠模型中驗證了微型核醣核酸130a能夠抑制B型肝炎病毒的複製，微型核醣核酸130a或許能夠作為有效的治療對策來抑制B型肝炎病毒。

Hepatitis B virus (HBV) infection is a major public health problem throughout the world. HBV infection causes acute or chronic hepatitis which may cause liver cirrhosis or hepatocellular carcinoma (HCC). Although effective HBV vaccine are available to prevent HBV infection, some individual can’t produce antibody against HBV and existing vaccine has no therapeutic effect on the chronically infected populations. The first-line antiviral HBV therapy reagents include type I interferon and nucleotide analogs but both have limited efficacy; severe side effects and drug resistance were found in many cases. Furthermore, current therapeutic methods such as viral entry inhibitors and capsid assembly inhibitors cannot eradicate HBV because viral cccDNAs still exist in hepatocytes. Chronic HBV infection still influences 350 million people worldwide and estimated 786,000 people die every year due to complications of HBV-related liver cirrhosis and HCC. Exploration of more effective therapeutic strategies for HBV infection is extremely urgent. micro-RNAs (miRNAs) are small, non-coding RNAs that can regulate gene expression at post-transcriptional level through translational repression or degradation of targeted mRNAs. So far, several studies have implicated that miRNAs influence the virus and host interaction and affect the virus life cycle. Recently, some miRNAs have been found to directly or indirectly regulate HBV replication by targeting cellular protein or HBV transcript. Previously, our laboratory found that miR-130a inhibited HBV replication by targeting two HBV transcriptional factor/coactivator: PGC1a and PPARg, thereby inhibits viral mRNA transcription and thus influence viral protein expression and virus replication. In this study, we create the miR-130a knockout (KO) mice by TALEN approach to dissect the relationship between HBV and miR-130a in vivo. After HBV plasmids tail-vein hydrodynamic injection, these miR-130a KO mice can promote the HBV replication due to higher expression levels of PGC1a and PPARg. In addition, the I.V. injection of miR-130a-expressing adenovirus in miR-130a KO mice, which replenishes the miR-130a expression, would inhibit the HBV replication reversely. Taken together, we validate that miR-130a have an inhibitory effect on HBV replication in vivo and increase miR-130a might be an effective strategy to limit HBV replication.