Numerous miRNAs have been detected in mitochondria, which play important roles in many physiological and pathophysiological processes. However, the dynamic changes of miRNA distribution in mitochondria and their mechanisms in reactive oxygen species (ROS)-induced endothelial injury remain unclear. Therefore, miRNA levels in whole cells and mitochondria of H₂O₂-treated endothelial cells were analyzed by small RNA sequencing in the present study. The results showed that H₂O₂ significantly reduced the relative mitochondrial distribution of dozens of miRNAs in human umbilical vein endothelial cells (HUVECs). Among the high-abundance miRNAs, miR-301a-3p has the most significant changes in the redistribution between cytosol and mitochondria confirmed by absolute quantitative polymerase chain reaction (qPCR). To unravel the mechanism of miR-301a-3p distribution in mitochondria, RNA pull-down followed by label-free quantitative proteomic analysis was performed, and RNA-binding protein Musashi RNA binding protein 2 (MSI2) was found to specifically bind to miR-301a-3p. Western blotting and immunofluorescence colocalization assay showed that MSI2 was located in mitochondria of various cell types. H₂O₂ significantly downregulated MSI2 expression in whole endothelial cells, promoted the distribution of MSI2 in cytosol and decreased its distribution in the mitochondria. Moreover, overexpression of MSI2 increased the mitochondrial distribution of miR-301a-3p, whereas inhibition of MSI2 decreased its distribution in mitochondria. Thus, MSI2 might be responsible for the distribution of miR-301a-3p between cytosol and mitochondria in endothelial cells. Our findings revealed for the first time that MSI2 was involved in the regulation of miRNA distribution in mitochondria and provided valuable insight into the mechanism of mitochondrial distribution of miRNAs.

线粒体中已检测到许多miRNA，它们在许多生理和病理生理过程中都起着重要作用。然而，miRNA在线粒体中分布的动态变化及其在活性氧（ROS）诱导的内皮损伤中的机制尚不清楚。因此，在本研究中，通过小RNA测序分析了H ₂ O ₂处理的内皮细胞的全细胞和线粒体中的miRNA水平。结果表明，H ₂ O ₂显着降低了人脐静脉内皮细胞（HUVEC）中数十种miRNA的相对线粒体分布。在高丰度的miRNA中，通过绝对定量聚合酶链反应（qPCR）证实，miR-301a-3p在细胞质和线粒体之间的重新分布具有最显着的变化。为了揭示线粒体中miR-301a-3p分布的机制，进行了RNA下拉，然后进行无标记的定量蛋白质组分析，发现RNA结合蛋白Musashi RNA结合蛋白2（MSI2）与miR- 301a-3p。免疫印迹和免疫荧光共定位分析表明，MSI2位于各种细胞类型的线粒体中。H ₂ O ₂显着下调了整个内皮细胞中MSI2的表达，促进了MSI2在细胞质中的分布，并降低了其在线粒体中的分布。此外，MSI2的过表达增加了miR-301a-3p的线粒体分布，而对MSI2的抑制则降低了其在线粒体中的分布。因此，MSI2可能负责内皮细胞中溶质和线粒体之间miR-301a-3p的分布。我们的发现首次揭示了MSI2参与线粒体中miRNA分布的调控，并为miRNA的线粒体分布机理提供了有价值的见解。