prep基因DNA去甲基化促进胆管细胞增殖进而调控肝再生的分子机制研究

There is important basic and clinical significance in revealing the molecular mechanisms of important regulatory factor in liver regeneration. Previous work found that after the knock out of the prep gene, liver regeneration takes on serious defects. Besides, further study found that the proliferation of the biliary cells was obviously inhibited. This indicates that prep gene may affect liver regeneration by regulating the proliferaion of bile duct cells. In addition, BSP analysis found demethylation change in the prep promoter region during liver regeneration, which implies the possibility that in this process DNA demethylation participates in regulating the prep transcriptional expression. Therefore, we put forward the assumption that DNA demethylation mediates prep to promote the proliferation of biliary cells, and then regulates liver regeneration. In order to verify this assumption and discuss the detail molecular mechanisms of prep regulating liver regeneration, this project is divided into four parts: 1) the function analyses of Prep in the regulation of liver regeneration; 2) prep regulates the proliferation of biliary cell and furtherly promotes liver regeneration; 3) the study of the prep transcription activity regulated by DNA demethylation; 4) molecular mechanisms of prep regulating liver regeneration .

揭示肝再生重要调控因子发挥功能的分子机制具有重要的基础和临床意义。前期工作已经发现prep敲除后肝受损后再生出现严重缺陷，同时胆管细胞的增殖明显受到抑制，提示prep通过调控胆管细胞的增殖从而影响肝再生的可能性。此外，BSP(Bisulfite sequencing PCR)分析发现prep启动子区域其在再生过程中出现去甲基化变化，暗示在此过程中DNA去甲基化控制prep的自身的表达调控。因此，我们提出DNA去甲基化介导prep促进胆管细胞增殖，进而调控肝受损后再生这一假说，为证实该假说并阐明prep调控肝脏受损后再生的详细分子机理，本项目将开展以下四项工作: 1）Prep缺失致使肝受损后再生缺陷的功能研究；2）DNA去甲基化调控prep转录活性的研究；3）Prep调控胆管细胞增殖进而促进肝脏受损再生的功能研究；4）prep调控肝脏受损后再生的具体分子机制。

Prolyl endopeptidase (prep)是一种丝氨酸肽酶，本项目研究了prep在斑马鱼极限肝脏再生过程中发挥着重要的功能。我们发现在肝脏被完全杀除后胆管上皮细胞的prep被重新启动表达，并且在再生过程中持续高表达。当prep突变或者被抑制剂抑制之后，肝脏再生过程受到了抑制，prep主要调控胆管上皮细胞的增殖，而不影响胆管上皮细胞的去分化与再分化，prep被热激过表达后加速肝脏再生的进程。Prep在肝脏再生过程中被重新启动表达是受到DNA去甲基化酶tet2的调控，tet2减少prep启动子中CpG位点的甲基化水平，从而使prep重新高表达。随后，prep通过PI3K-AKT-mTOR信号通路促进胆管上皮细胞增殖从而促进肝脏再生。本项目为深入理解肝脏再生的关键调控因子prep的功能及其作用机制做出贡献。