基于生物学网络研究多顺反子miR-23b/27b/24簇靶向JNK信号通路调控肝再生

Mammalian liver shows enormous regeneration capacity, which is beneficial to the prevention of liver disease. In the previous study we found that liver-specific miR-122 controls liver developmental timing by targeting key transcription factors. Recently, we further found that the express profiles of miRNA are changed during liver regeneration, including polycistronic miR-23b/27b/24 cluster. The translational impacts of miRNA research remain limited by a paucity of empirical miRNA targeting data, particularly in primary tissues. Therefore, to elucidate the roleof miRNAs in liverregeneration, two third partial hepatectomy in mice was used as liver regeneration model, and high-throughput profile of miR:target interactionsin human liver tissues was applied. We decipher Ago2:RNA interactions using photoactivatable-ribonucleotide-enhanced crosslinking and immunoprecipitation (PAR-CLIP) to generate the first transcriptome-wide map of miRNA targeting events in human liver cells, detecting liver Ago2 binding sites. Our initial exploration of this interaction would reveal an abundance of miRNA target sites in gene coding regions, including new miRNA functions in regulating hepatocyte proliferationand metabolism. Also, we might uncover several clinically-relevant interactions involving common genetic variants that the miR-23b/27b/24 cluster targeting events in hepatopathy-associated genes, and demonstrate the miR-23b/27b/24 cluster regulate liver regeneration by targeting the JNK signaling pathway. Overall, these data could provide a critical resource for bolstering translational miRNA research in liver regeneration.

肝脏再生是受损肝脏修复的重要方式之一，课题组前期研究发现肝脏通过特异性高表达的miR-122直接靶向发育关键转录因子来控制肝脏的发育时序。进而发现肝再生过程中，以多顺反子miR-23b/27b/24簇为代表的miRNA表达谱发生显著改变，但因缺乏正常生理条件下尤其是肝脏再生过程中miRNA的作用靶向数据，暂无法完全阐明miR-23b/27b/24簇对肝再生的影响，故亟待对miRNA在正常肝脏再生过程中的功能进行分类。本项目拟以小鼠2/3肝切除为研究模型，利用PAR-CLIP技术，全面解析肝脏再生过程中全转录组范围内miRNA的结合位点，通过挖掘miR-23b/27b/24簇和靶标的相互作用关系及调控网络，进一步阐明miR-23b/27b/24靶向作用JNK信号通路在肝再生过程中的调控机制，以期为肝再生提供miRNA调控机制的数据资源。

本研究首先构建了CLIP-seq 实验平台，以小鼠2/3肝切除后再生为研究模型，利用AGO2 CLIP-seq研究体系，从正常生理角度，阐明了肝脏再生过程中全转录组范围内miRNA的结合位点。进一步系统解析丰度变化显著的miRNA及其靶标，特别是肝再生过程中关键的多顺反子miR-23b/27b/24簇的靶标和相关信号通路，构建出miR-23b/27b/24-靶标相互作用的基因调控网络。进一步，对小鼠肝发育过程中的肝组织样品进行了AGO2 CLIP-seq, 对比肝脏发育过程和肝再生过程中的miRNA 和mRNA靶标数据，从肝脏发育角度揭示再生环境下，miRNA的表达变化和靶标互作共同调控肝再生的分子机制。而且，在本项目实施过程之中，以CLIP-seq实验平台为基础，我们解析了肌分化过程中的miRNA和mRNA靶标互作网络，阐明了mamiRs （myogenesis associated miRNAs） 通过调控多级磷酸化因子靶向JNK/MAPK信号通路控制骨骼肌分化（Xie S et al. cell death diff, 2018）,以及miR-101同时作用于MEF2A和lncRNA MALAT1对肌分化起到“刹车”作用（Liu S, Xie S et al. Sci China Life Sci,2020）。这些成果扩展了CLIP-seq 的实验技术的应用。通过以CLIP-seq 实验平台为基础对肝再生、肝发育以及肌分化的研究，我们更加清楚的理解了RNA和RNA结合蛋白之间互作调控模式，为更好的研究分子间作用方式和功能奠定了基础。