缺氧环境中外泌体miR-20a-5p通过CCND1/Hexokinase-2保护急性肾损伤小管上皮细胞线粒体的机制研究

线粒体异常导致肾小管上皮细胞坏死脱落，是急性肾损伤的重要机制。我们前期发现：缺氧时，小管上皮细胞分泌富含miR-20a-5p的外泌体能较好保护线粒体，促进小管上皮细胞增殖，减轻肾损害；蛋白组学结果发现miR-20a-5p干预后，肾组织Hexokinase-2（己糖激酶-2，定位于线粒体外膜的糖酵解关键蛋白）表达显著升高。通过双荧光素酶报告试验，我们证实定位在线粒体外膜、抑制线粒体活性的CCND1是miR-20a-5p下游靶基因。近期有研究报道，CCND1可竞争Hexokinase-2的线粒体外膜结合位点，损害线粒体，抑制细胞增殖。由此我们提出科学假设：缺氧时，外泌体miR-20a-5p下调CCND1进而促进Hexokinase-2富集于小管上皮细胞线粒体外膜，保护线粒体、促进ATP产生，保护肾脏。我们拟采用CCND1基因敲入小鼠及细胞实验阐明缺氧时外泌体miR-20a-5p对肾保护机制。

Mitochondria abnormality leads to necrosis and shedding of renal tubular epithelial cells, which is an important pathogenesis for acute kidney injury. We previously found that exosomes rich in miR-20a-5p secreted by hypoxic tubular epithelial cells protected mitochondria and promoted the proliferation of tubular epithelial cells to reduce damage. Proteomics analysis showed that after the intervention of miR-20a-5p, the expression of renal tissue Hexokinase-2 was significantly increased ,which is a key protein located at the mitochondrial outer membrane and closely related to glycolysis. Through the dual-luciferase reporting assay, we found that CCND1, which located in the mitochondrial outer membrane and inhibits mitochondrial activity, is the downstream target gene of miR-20a-5p. Current reports showed that CCND1 and Hexokinase-2 compete for the mitochondrial outer membrane binding site, causing mitochondrial damage and inhibiting cell proliferation. Therefore, we proposed the scientific hypothesis that : during hypoxia, exosomal miR-20a-5p down-regulates CCND1 and promotes Hexokinase-2 enrichment in the outer mitochondrial membrane of tubule epithelial cells, protecting mitochondria, promoting ATP production, and protecting the kidneys. We intend to use CCND1 gene knock-in mice and cell experiments to elucidate the protective mechanism of exosomal miR-20a-5p on kidney during hypoxia.