miR21-HIF相互调控在肾脏缺血/缺氧耐受中作用机制的研究

Acute kidney injury is a common clinical critical disease, the main causes of which is renal ischemic / hypoxic injury。Ischemic / hypoxic tolerance or ischemic preconditioning is found to be the most powerful endogenous measure to prevent organ ischemic injury. MicroRNAs (miRNAs) as a kind of important endogenous small RNA molecules , play an important and ubiquitous role in regulating gene expression for various biological processes. But the role of miRNAs in renal ischemic / hypoxic tolerance hasn't been reported yet. miR-21 is one of the important hypoxia-related miRNAs. In our previous work, we found that delayed ischemic preconditioning induced high expression of miR-21 in renal tissue. In addition, miR-21 involved in the protective effect of the renal delayed ischemic preconditioning, the mechanism of which may be related with hypoxia-inducible factor (HIF) induced by ischemia pretreatment. This project want to further clarify the regulation of HIF on the miR-21 on the basis of the previous study, and verify that miR-21 indirectly regulate HIF by inhibiting its target gene proline hydroxylase subtypes -2. Therefore, we can know the role of miR-21 in renal ischemic / hypoxic tolerance around the interaction between HIF and miR-21. This research will supply a very important theoretical significance to elucidate the mechanism and prevention of renal ischemic / hypoxic injury, and set the base to find effective means of prevention.

急性肾损伤是一种常见的临床危重病症，缺血/缺氧肾损伤是急性肾损伤的主要发病原因，缺血/缺氧耐受或缺血预适应是迄今为止发现的最强有力预防缺血性器官损伤的内源性措施。微小RNA（microRNAs，miRNAs）作为一类具有重要调控作用的内源性小RNA分子，广泛参与机体多种生物学过程的调控，但miRNAs在肾脏缺血/缺氧耐受中的作用还未见报道。miR-21是重要的缺氧相关miRNAs之一，项目组前期工作发现晚期缺血预适应诱导肾脏miR-21高表达，并且miR-21参与肾脏晚期缺血预适应的保护作用，其作用机制可能与预缺血诱导的低氧诱导因子(HIF)有关。本研究拟在前期工作的基础上进一步明确HIF对miR-21的调控，并验证miR-21通过其靶基因脯氨酸羟化酶亚型-2对HIF的间接调控，围绕HIF与miR-21相互作用明确miR-21在肾脏缺血/缺氧耐受中的作用，对阐明肾脏缺血/缺氧损伤的机制和防

缺血/缺氧肾损伤是急性肾损伤的主要发病原因，缺血/缺氧耐受或缺血预适应是迄今为止发现的最强有力预防缺血性器官损伤的内源性措施。我们前期研究发现缺血预处理能通过诱导miR-21，保护肾脏减轻I/R损伤；同时我们在体外实验中发现，缺氧诱导肾小管上皮细胞miR-21高表达，抑制HIF-1α则miR-21的表达下调。本研究拟在前期工作的基础上明确HIF-1α与miR-21的相互调控关系，深入探讨HIF- miR21正反馈通路在肾脏缺血/缺氧耐受中的作用。我们依照研究计划，我们首先通过氙气预适应、晚期缺血预适应构建肾脏缺血缺氧耐受模型小鼠模型，发现预处理后肾脏HIF-1α和miR-21的表达趋势一致，同时原位杂交显示两者的表达部位也相似。在此基础上进行一系列的后续研究。我们分别通过体外细胞缺氧和体内注射氯化钴稳定肾脏HIF-1α的表达，体外发现肾小管上皮细胞HIF-1α可以增强miR-21的活性，再通过染色质免疫沉淀（CHIP）实验，证实HIF-1α可以与miR-21结合。由此提示HIF-1α在转录水平调控miR-21的表达。进一步在体内实验中证实HIF-1α对miR-21的调控。另一方面，通过生物信息学发现，我们寻找到HIF-1α诱导低氧功能的开关PHD2为miR-21的预测靶基因，并通过荧光素酶报告基因检测，证实miR-21与PHD2 3’UTR结合，并在体外低氧或低氧/复氧处理的肾小管上皮细胞中证实miR-21对PHD2的抑制作用，进一步在晚期缺血预适应小鼠模型中证实miR-21表达与PHD2蛋白水平有一定的负相关性。利用LNA anti miR-21抑制体外缺氧/复氧肾小管上皮细胞和体内预适应诱导的肾脏miR-21的表达，证实miR-21通过抑制PHD2，诱导HIF-1α及下游靶基因VEGF高表达，减轻肾脏损伤。最后，我们构建首个肾小管上皮细胞特异性转基因小鼠，并证实miR-21在肾脏特异性高表达，同时定位在肾小管上皮细胞。进一步发现miR-21高表达小鼠肾脏缺血/再灌注损伤轻于同窝miR-21阴性小鼠。本项目的研究成果为研究急性肾损伤的修复机制及治疗策略提供新的理论依据，并且为今后miRNAs在肾脏疾病中作用的研究提供新思路，并且对细胞或机体缺氧反应调节的研究有着深远的意义。肾小管上皮细胞特异性miR-21转基因小鼠的构建为今后肾脏miR-21的研究提供有力工具。