piR-823在肝纤维化中通过翻译起始因子EIF3B启动TGF-β1翻译促进HSC活化

There are rich piRNAs in the liver which is one of the small non-coding RNA. Studies show that the expression of piR-823 is increased in liver cirrhosis and hepatocellular carcinoma compared to normal liver. TGF-β1 is the most important profibrotic factor for liver fibrosis which actives hepatic stellate cells (HSC). EIF3B is the translation initiation factor to start the translation of TGF-β1. In the previous study we found that the exprssion of piR-823 increased in activated HSC and it promoted the activation of HSC when the exprssion of piR-823 increased. Meanwhile piR-823 combined with EIF3B in HSC by using RNA pull down method and LC-MSMS. We speculate that piR-823 actives hepatic stellate cells through promoting TGF -β1 translation via conjuncting with EIF3B. We aim to clarify the changes of expression of piR-823 in HSC in the dynamic process of hepatic fibrosis and change the expression of piR-823 in HSC to investigate the activation of HSC. Finally explore the mechanisms of piR-823 activating HSC. It provides a new mechanism of hepatic fibrosis.

piRNA 是一类小分子非编码RNA，在肝脏表达丰富。其中一种piR-823在肝硬化、肝癌前病变、肝癌中表达异常。TGF-β1可活化肝星状细胞（HSC），是最重要的促肝纤维化细胞因子，EIF3B蛋白可调控TGF-β1的翻译。我们前期研究发现：活化的HSC中piR-823表达升高，过表达piR-823可促进HSC活化；RNA pull down及质谱分析发现，piR-823可与EIF3B结合。据此推测，piR-823可能通过与EIF3B结合促进TGF-β1翻译，进而活化HSC，促进肝纤维化。本项目拟在动物模型和HSC中观察肝纤维化中，HSC的 piR-823表达；过表达/敲低piR-823表达，观察其对HSC活化的调控作用；在整体水平过表达/敲低piR-823，证明其功能；并进一步验证piR-823与EIF3B结合促进TGF-β1翻译，明确其分子机制。本项目为阐明肝纤维化机制提供了新思路。

背景与目的：piRNA 是一类小分子非编码RNA，在肝脏表达丰富。其中一种piR-823在肝硬化、肝癌前病变、肝癌中表达异常。TGF-β1可活化肝星状细胞（HSC），是最重要的促肝纤维化细胞因子。本课题旨在观察 HSC 活化过程中 piR-823 的表达变化情况，阐明piR-823对HSC活化的调控作用及其可能的分子机制。.研究方法：应用四氯化碳腹腔注射法及胆总管结扎建立小鼠肝纤维化模型，并提取小鼠原代HSCs。将人工合成的piR-823正义序列及反义序列转染入LX-2和小鼠原代HSCs中，应用 CCK-8 及 BrdU 方法检测LX-2及小鼠原代HSCs细胞的活性及增殖能力。用带有GFAP特异启动序列的AAV病毒，携带piR-823 antisense转染小鼠，以敲低其肝内HSCs中piR-823的表达，运用多种方法综合评价小鼠肝纤维化情况。应用RNA沉淀技术及液质联质谱分析寻找 piR-823在HSCs中特异结合的蛋白。应用脂质体将 siRNA转染入细胞改变目标蛋白的表达，观察其对HSCs的影响。.结果：由肝纤维化小鼠肝脏提取的HSCs中piR-823表达较对照组明显升高；而体外培养过程中自发活化的HSCs中，piR-823表达随HSCs活化程度增强而增多。上调 piR-823 表达可促进 HSCs 活化；敲低piR-823表达可抑制HSCs活化；敲低小鼠体内原代HSCs中piR-823表达可减弱其肝纤维化程度。piR-823可特异结合蛋白EIF3B。敲低EIF3B后，TGF-β1蛋白水平表达减少。上调piR-823表达后，TGF-β1蛋白表达增多。敲低piR-823表达，TGF-β1蛋白表达减少。敲低EIF3B可抑制 piR-823上调TGF-β1蛋白表达的作用。.结论：肝星状细胞活化过程中，piR-823表达增加。升高 piR-823 表达可促进 HSCs 活化，抑制 piR-823表达可抑制HSCs活化，减轻肝纤维化程度。在HSCs中，piR-823可通过与EIF3B特异结合，上调TGF-β1蛋白表达，进而影响HSCs的活化。