Mesenchymal stem cell-derived exosomes ameliorate cardiomyocyte apoptosis in hypoxic conditions through microRNA144 by targeting the PTEN/AKT pathway
Mesenchymal stem cell-derived exosomes ameliorate cardiomyocyte apoptosis in hypoxic conditions through microRNA144 by targeting the PTEN/AKT pathway
复制标题
间充质干细胞来源的外泌体通过靶向 PTEN/AKT 通路的 microRNA144 改善缺氧条件下的心肌细胞凋亡
DOI：
10.1186/s13287-020-1563-8
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发表时间：
2020-01-23
影响因子：
7.5
通讯作者：
Wang, Jingfeng
中科院分区：
文献类型：
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作者：
Wen, Zhuzhi;Mai, Zun;Wang, Jingfeng
背景越来越多的证据表明，干细胞来源的外泌体microRNAs（miRNAs）可能是一种有前途的缺氧条件下的心脏保护疗法。本研究旨在探讨骨髓间充质干细胞（MSC）来源的外泌体中的miRNA-144（miR-144）在缺氧条件下对心肌细胞凋亡的保护作用及其机制。使用总外泌体分离试剂分离MSC衍生的外泌体，并通过纳米颗粒运输分析以及使用TSG 101和CD 63作为标记物的蛋白质印迹法确认。使用AnaeroPack方法建立H9 C2细胞的缺氧生长条件。测试的处理条件包括用外来体预孵育、用miR-144模拟物或抑制剂转染或用PTEN抑制剂SF 1670处理的H9 C2细胞，所有这些都在缺氧生长条件下。流式细胞仪检测7-ADD和Annexin V联合应用对细胞凋亡的影响。采用real-time PCR和western blotting方法检测H9 C2细胞中AKT/p-AKT、Bcl-2、caspase-3、HIF-1α、PTEN和Rac-1的表达水平。外泌体介导的H9 C2细胞凋亡保护伴随着p-AKT水平的增加。发现miR-144在MSC衍生的外泌体中高度富集。用miR-144抑制剂转染细胞削弱了外泌体介导的细胞凋亡保护。此外，用miR-144模拟物处理在缺氧条件下生长的细胞导致PTEN表达降低，p-AKT表达增加，并阻止H9 C2细胞凋亡，而用miR-144抑制剂处理导致缺氧条件下PTEN表达增加，p-AKT表达降低，并增强H9 C2细胞凋亡。我们还通过荧光素酶报告基因检测验证了PTEN是miR-144的靶点。此外，细胞与SF 1670，一个PTEN特异性抑制剂，导致增加p-AKT表达和减少H9 C2细胞apoptosis.ConclusionsThese研究结果表明，MSC衍生的外泌体抑制细胞凋亡损伤在缺氧条件下通过提供miR-144的细胞，在那里它的目标是PTEN/AKT通路。MSC衍生的外泌体可能是一种有前途的治疗载体，以促进miRNA疗法的递送，从而改善缺血性疾病。
BackgroundA growing body of evidence suggests that stem cell-derived exosomal microRNAs (miRNAs) could be a promising cardioprotective therapy in the context of hypoxic conditions. The present study aims to explore how miRNA-144 (miR-144), a miRNA contained in bone marrow mesenchymal stem cell (MSC)-derived exosomes, exerts a cardioprotective effect on cardiomyocyte apoptosis in the context of hypoxic conditions and identify the underlying mechanisms.MethodsMSCs were cultured using the whole bone marrow adherent method. MSC-derived exosomes were isolated using the total exosome isolation reagent and confirmed by nanoparticle trafficking analysis as well as western blotting using TSG101 and CD63 as markers. The hypoxic growth conditions for the H9C2 cells were established using the AnaeroPack method. Treatment conditions tested included H9C2 cells pre-incubated with exosomes, transfected with miR-144 mimics or inhibitor, or treated with the PTEN inhibitor SF1670, all under hypoxic growth conditions. Cell apoptosis was determined by flow cytometry using 7-ADD and Annexin V together. The expression levels of the miRNAs were detected by real-time PCR, and the expression levels of AKT/p-AKT, Bcl-2, caspase-3, HIF-1α, PTEN, and Rac-1 were measured by both real-time PCR and western blotting.ResultsExosomes were readily internalized by H9C2 cells after co-incubation for 12 h. Exosome-mediated protection of H9C2 cells from apoptosis was accompanied by increasing levels of p-AKT. MiR-144 was found to be highly enriched in MSC-derived exosomes. Transfection of cells with a miR-144 inhibitor weakened exosome-mediated protection from apoptosis. Furthermore, treatment of cells grown in hypoxic conditions with miR-144 mimics resulted in decreased PTEN expression, increased p-AKT expression, and prevented H9C2 cell apoptosis, whereas treatment with a miR-144 inhibitor resulted in increased PTEN expression, decreased p-AKT expression, and enhanced H9C2 cell apoptosis in hypoxic conditions. We also validated that PTEN was a target of miR-144 by using luciferase reporter assay. Additionally, cells treated with SF1670, a PTEN-specific inhibitor, resulted in increased p-AKT expression and decreased H9C2 cell apoptosis.ConclusionsThese findings demonstrate that MSC-derived exosomes inhibit cell apoptotic injury in hypoxic conditions by delivering miR-144 to cells, where it targets the PTEN/AKT pathway. MSC-derived exosomes could be a promising therapeutic vehicle to facilitate delivery of miRNA therapies to ameliorate ischemic conditions.