Effect of mir-210-3p on The Function of c-kit+ Cardiac Progenitor Cells under Ischemia and Hypoxia

C-kit+ cardiac progenitor cells (CPCs) play an important role in improving the prognosis of ischemic heart disease. MicroRNAs have been shown to have a protective role in the cardiac repair process, but their specific functional mechanisms are still unclear. The aim of this study was to screen for differentially expressed miRNAs in c-kit+ CPCs under hypoxia and explore their effects on the function of c-kit+ CPCs. We found that miR-210-3p was significantly upregulated in hypoxia-treated c-kit+ CPCs through second-generation sequencing. Protein chip detection revealed that c-kit+ CPCs could express cytokines such as FGF basic, Angiogenin, and VEGF, and that hypoxia could enhance the secretion of some angiogenesis-related factors. Silencing miR-210-3p, on the other hand, resulted in a reduction in the secretion of these angiogenesis-related factors. In addition, we found that the conditioned medium of hypoxia-treated c-kit+ CPCs could promote the proliferation, migration, and tube formation ability of mouse cardiac microvascular endothelial cells (CMECs). Conversely, the conditioned medium of c-kit+ CPCs with silenced miR-210-3p after hypoxia inhibited the proliferation, migration, and tube formation ability of CMECs. These results suggest that c-kit+ CPCs may exert their pro-angiogenic effects through paracrine mechanisms, which are regulated by miR-210-3p. The upregulation of miR-210-3p in hypoxia-treated c-kit+ CPCs may enhance their paracrine function by regulating the secretion of angiogenic factors, thereby promoting angiogenesis in ischemic heart disease. Therefore, miR-210-3p plays a key role in regulating the paracrine function of c-kit+ CPCs and can affect the process of angiogenesis. The role of miR-210-3p has potential application value for the treatment of ischemic heart disease, and may become one of the important targets for future treatment of ischemic heart disease. Take healthy adult male C57BL/6 mice aged 4-6 weeks, digest their hearts using trypsin and collagenase, and use anti-c-kit-coupled magnetic beads for magnetic-activated cell sorting (MACS) to isolate c-kit cells. Culture the cells under hypoxia for 12 hours to establish the hypoxia model. Randomly divide the cultured cells into a normal control group and a hypoxia group, and extract cellular RNA for miRNA sequencing.

C-kit阳性心脏祖细胞（C-kit+ cardiac progenitor cells, CPCs）在改善缺血性心脏病预后方面发挥着重要作用。已有研究表明，微小RNA（microRNAs, miRNAs）在心脏修复过程中具有保护作用，但其具体功能机制尚未阐明。本研究旨在筛选缺氧条件下C-kit阳性心脏祖细胞中差异表达的miRNA，并探讨其对C-kit阳性心脏祖细胞功能的影响。通过二代测序发现，经缺氧处理的C-kit阳性心脏祖细胞中miR-210-3p显著上调。蛋白芯片检测结果显示，C-kit阳性心脏祖细胞可表达碱性成纤维细胞生长因子（FGF basic）、血管生成素及血管内皮生长因子（vascular endothelial growth factor, VEGF）等细胞因子，且缺氧可增强部分血管生成相关因子的分泌。反之，沉默miR-210-3p则会导致上述血管生成相关因子的分泌水平显著降低。此外，本研究发现经缺氧处理的C-kit阳性心脏祖细胞的条件培养基可促进小鼠心脏微血管内皮细胞（cardiac microvascular endothelial cells, CMECs）的增殖、迁移及管形成能力；而缺氧处理后沉默miR-210-3p的C-kit阳性心脏祖细胞的条件培养基则会抑制小鼠心脏微血管内皮细胞的上述生物学功能。上述结果表明，C-kit阳性心脏祖细胞可能通过旁分泌机制发挥促血管生成作用，而这一过程受miR-210-3p调控。缺氧处理的C-kit阳性心脏祖细胞中miR-210-3p的上调可通过调节血管生成因子的分泌增强其旁分泌功能，进而促进缺血性心脏病中的血管生成。因此，miR-210-3p在调控C-kit阳性心脏祖细胞的旁分泌功能及影响血管生成过程中发挥关键作用。miR-210-3p在缺血性心脏病治疗中具有潜在应用价值，有望成为未来缺血性心脏病治疗的重要靶点之一。选取4~6周龄健康成年雄性C57BL/6小鼠，采用胰蛋白酶与胶原酶消化其心脏组织，通过抗C-kit偶联磁珠进行磁激活细胞分选（magnetic-activated cell sorting, MACS）以分离C-kit阳性细胞。将分离得到的细胞置于缺氧环境中培养12小时以构建缺氧模型。将培养后的细胞随机分为正常对照组与缺氧组，提取细胞总RNA进行miRNA测序。