miR-146a靶向ALDH2调控氧化应激的机制及其在肾脏缺血再灌注损伤中的作用

Renal ischemia-reperfusion (IR) injury significantly increased mortality in renal transplant recipients. To date, no effective clinical treatment is available. Oxidative stress due to excessive production of reactive oxygen species (ROS) and subsequent lipid peroxidation plays a critical role in renal IR injury. Recent study found that miRNA may be involved in the regulation of oxidative stress, but the mechanism remains unclear. We found that several miRNA, including miR-146a, significantly upregulated within 24h after H2O2 treatment in mouse tubular epithelial cell by high throughput miRNA array, and further validated the expression by qRT-PCR. As our previous studies showed that the aldehyde dehydrogenase 2 (ALDH2) is a key molecule involved in the regulation of oxidative stress after kidney IR, we focused on miRNAs that have potential in targeting ALDH2. We found that miR-146a might target ALDH2 via computational miRNA target-gene prediction and Firefly Luciferase Reporter Gene Assay, and verified by cell transfection test. On this basis, this project intends to study the mechanism of miR-146a targeting ALDH2 in the modulation of oxidative stress during kidney IR. Syngeneic kidney transplantation cold ischemia model and bilateral kidney warm ischemia model will be used to better mimic the clinical condition. The crosstalk between pathways modulated by miR-146a targeting ALDH2 and TLR signaling will also be investigated.The current project should be useful for better understanding of the role and mechanism of ALDH2 in kidney IR and future clinical treatment of IR injury.

氧化应激在肾脏缺血再灌注(IR)损伤中发挥关键作用，近期研究发现miRNA可能参与调控氧化应激，但机制仍不明确。我们采用miRNA芯片发现肾小管上皮细胞在H2O2刺激后miR-146a等表达上调，并经qRT-PCR法证实。结合我们所发现的乙醛脱氢酶2（ALDH2）在调控肾脏IR后氧化应激中的关键作用，经信息学预测和双荧光报告基因系统筛选发现miR-146a可能靶向ALDH2，并经预实验证实。由此提出假说：miR-146a可能靶向ALDH2参与调控肾脏IR后氧化应激。本项目拟研究IR对肾脏miR-146a表达的调控及其机制；联用小鼠同系肾移植冷/热缺血损伤模型，结合miR-sponge技术，探讨miR-146a靶向ALDH2的机制及在肾脏IR损伤中的作用，采用TLR通路相关缺陷小鼠，分析miR-146a调控ALDH2与其调控TLR通路的相互作用。本研究可为肾脏IR的临床治疗提供新的理论依据。

氧化应激在肾脏缺血再灌注(IR)损伤中发挥关键作用，我们在前期研究中发现乙醛脱氢酶2（ALDH2）参与调控肾脏IR后氧化应激反应，经过预实验，我们推测miR-146a可能靶向ALDH2参与调控肾脏IR损伤。因此，本项目旨在研究IR对肾脏miR-146a和ALDH2表达的影响，探讨miR-146a靶向ALDH2的机制及在肾脏IR损伤中的作用。本项目通过原代培养肾小管上皮细胞（TEC）和腹腔巨噬细胞（MΦ）进行体外实验，通过小鼠肾蒂钳夹构建热IR损伤模型，通过小鼠肾移植构建冷IR损伤模型进行研究。结果显示：原代TEC和MΦ在H2O2 刺激后miR-146a表达显著升高，小鼠热/冷IR损伤后肾组织miR-146a表达亦显著升高；体内外实验均发现IR刺激后ALDH-2 mRNA表达升高，但在小鼠肾脏热IR模型中发现ALDH-2蛋白表达在IR后早期迅速降低，随后升高。采用IL-17C缺陷小鼠，我们进一步验证了IR所致氧化应激对miR-146a以及ALDH-2表达的调控作用。我们通过信息学预测和报告基因系统验证发现miR-146a可靶向ALDH2基因，经小鼠MΦ转染miR-146a mimics（或inhibitor）可显著抑制（或增加）ALDH-2 mRNA表达。体外转染mimics上调miR-146a表达后再行模拟IR，发现TNF-α表达以及LDH释放显著高于对照组，提示miR-146a靶向ALDH2可促进IR后炎症因子表达；而转染inhibitor下调miR-146a表达后再行模拟IR，TNF-α表达以及LDH释放仍显著高于对照组，推测可能与miR-146a其余通路的抗炎作用有关。本课题发现了IR所致氧化应激对miR-146a表达的调控作用，明确了miR-146a靶向ALDH2在肾脏IR损伤中的调控机制，研究结果为肾脏IR的临床治疗提供新的理论依据。