原发性胆汁性肝硬化中长链非编码RNA-GACAT1对肝内胆管细胞的调控机制研究

Primary biliary cirrhosis (PBC) is an autoimmune disease characterized by bile duct progressive non-suppurative inflammation. The intrahepatic bile duct cells (HiBEC) excessive apoptosis and abnormal activation has been proposed to account for the lesions be localizd on the bile duct. Our study found long-chain noncoding RNA-GACAT1 expression significantly increased and PTEN expression reduced in PBC patients' lesions liver tissue; GACAT1 binding with PTEN protein was found by RNA pulldown assay; stimulated by LPS, HiBECs which overexpression of PTEN to show excessive apoptosis and abnormal activation. This project intends to search for the following key issues: 1. the regulation mechanism of GACAT1 and PTEN in HiBEC apoptosis and abnormal activation; 2. the expression and regulation mechanism of DNA methylation and important transcription factors for GACAT1 in HiBEC; 3. using an mouse model to explore the possibility of regulating GACAT1 for the treatment and prevention of PBC. The above research, it will help us to clarify the mechanism of HiBEC excessive apoptosis and abnormal activation in PBC, and provide a theoretical basis to explore new ways of PBC immune intervention therapy.

原发性胆汁性肝硬化（PBC）以胆管内进行性非化脓性炎症反应为特征，患者肝内胆管细胞（HiBEC）异常活化及过度凋亡是导致特异性肝损伤的重要因素。本研究组前期发现，PBC患者病变肝组织中长链非编码RNA-GACAT1表达显著增高、PTEN表达显著降低；经RNA pulldown技术证实GACAT1与PTEN蛋白相结合；过表达PTEN的HiBEC呈现LPS刺激后过度凋亡和异常活化。本项目拟解决以下关键问题：1.GACAT1通过PTEN调节HiBEC过度凋亡及其异常活化的调控机制；2.DNA甲基化和重要转录因子对GACAT1在HiBEC中的表达调控机制；3.利用PBC小鼠模型探讨调节GACAT1以治疗和预防PBC的可能性。本研究将有助于阐明PBC中HiBEC过度凋亡和异常活化的调控机制，为探讨免疫干预治疗PBC的新途径提供理论依据。

本项目前期研究发现PBC患者病变肝组织中GACAT1表达显著降低，PTEN表达显著增高，进一步证实GACAT1与PTEN蛋白存在结合关系。PTEN过表达的HiBEC经LPS刺激后凋亡显著增加，TLR4/NF-kB信号通路过度活化，趋化因子分泌增多。我们首先研究GACAT1对HiBEC细胞功能的影响，在HiBEC中分别转染LV-GACAT1，通过流式细胞术发现转染后的细胞在LPS刺激24小时后，过表达lncRNA-GACAT1的HiBEC凋亡率显著降低，且TLR表达显著降低，相关的PTEN蛋白表达明显降低。.我们研究PBC患者GACAT1对肝内胆管细胞的调控机制，发现PBC患者GACAT1启动子区甲基化频率与对照组相比无差异，进一步我们对GACAT1的启动子区进行生物信息学预测，发现与自身免疫性疾病发展miR-106a-5p。我们构建miR-106a-5p的过表达载体以及抑制载体，发现与对照相比，过表达miR-106a-5p组，GACAT1表达增高约6倍，且PTEN蛋白水平明显降低。采用ELISA方法检测，发现上清液中转染组CXCL8,IL-10水平明显下降。说明GACAT1-miR-106a-5p-PTEN轴在PBC患者肝内胆管细胞调控机制起重要作用。.通过PBC模型小鼠体内实验发现，过表达GACAT1通过调控PTEN以及TLR4的蛋白水平来降低PBC小鼠胆管内淋巴细胞的浸润，进而缓解PBC小鼠的病程。.本研究初步阐明了GACAT1通过调控HiBEC细胞在PBC发病机制中发挥作用，为进一步深入理解PBC的发病机制提供了新的思路。