下调Let-7促进糖酵解保护供肝缺血再灌注损伤的机制研究

Strategies aiming at microRNA and its targeting genes have the potential to alleviate ischemia reperfusion injury (IRI). Let-7 family is the first discovered microRNA in human, but its role on IRI remains largely unknown. Our previous research showed that let-7 can modulate liver regeneration after surgical injury. The preliminary data shows the evidence that down-regulation of Let-7 can help to alleviate liver IRI by enhancing glycolysis. Based on the previous studies and our preliminary data, we hypothesize that Let-7 levels might affect IRI, down-regulation of Let-7 might help to alleviate IRI through enhancing glycolysis, the process might be due to activated PI3K-AKT-mTOR pathway and HIF-1α pathway. To test the above hypothesis, we simulate IRI process in cell culture, establish liver IRI model in let-7 overexpressing and liver specific knock out transgenic mice to test the relationship between let-7 and IRI. We also test the glycolysis levels, the important proteins expression in PI3K-AKT-mTOR and HIF-1α pathway, and then we use the inhibitors of above pathways to verify our hypothesis. The study will investigate the effect of let-7 on liver IRI and its mechanism, the results will provide a new strategy on how to attenuate IRI and improve the patients’ outcome after liver transplantation.

针对微小RNA及其调控的基因有望成为改善肝脏缺血再灌注损伤（ischemia reperfusion injury, IRI）的新方法。Let-7是最早被发现的微小RNA之一，但在肝脏IRI中的作用未见明确报道。我们前期研究证实Let-7精细调控肝再生，预实验提示下调Let-7可通过促进糖酵解改善肝脏IRI，结合文献分析该过程与激活PI3K-AKT-mTOR及HIF-1α通路相关。本课题拟结合体外和体内实验，在肝脏差异表达Let-7的小鼠中建立肝脏IRI模型，检测肝脏损伤、凋亡、炎症因子、糖酵解、PI3K-AKT-mTOR及HIF-1α通路相关分子的表达，并使用以上通路抑制剂，证实下调Let-7通过促进糖酵解改善肝脏IRI，该过程与激活PI3K-AKT-mTOR及HIF-1α通路相关。阐明Let-7对肝脏IRI的影响及其具体机制，将为临床肝移植改善IRI提供新的思路及理论基础。

缺血/再灌注损伤（IRI）是常规器官移植过程中不可避免的事件，影响移植后的预后。越来越多的证据表明microRNAs在IRI的调控中起着重要的作用。我们先前的研究表明let-7b的下调可以促进多种损伤后的肝再生。然而，let-7b在肝脏IRI中的作用尚不清楚。我们在体外建立LO2和AML12缺氧-复氧（H/R）细胞系，模拟肝脏IRI过程。我们检测了10对IRI后供肝组织中let-7b的表达水平。然后，我们构建了LV-let-7b-LO2细胞系。采用qRT-PCR和Western-blot检测p-STAT3和SOCS4的表达水平。采用双荧光素酶报告分析法来确定let-7b调节SOCS4的机制。LO2缺氧复氧损伤模型和临床组织结果显示，let-7b在IRI中表达明显下调。let-7b上调显著提高活性氧水平和降低细胞活力。p-STAT3的表达与let-7b的表达一致。同时，SOCS4的表达降低。双荧光素酶报告分析表明let-7b能与SOCS4的3'UTR结合。此外，SOCS4的过度表达可以逆转肝I/R损伤期间let-7b慢病毒引起的STAT3激活。let-7的下调可能通过增加SOCS4的表达，抑制STAT3通路的激活，从而保护肝组织免受IRI的损伤。