Overexpression of GATA4 enhances the antiapoptotic effect of exosomes secreted from cardiac colony-forming unit fibroblasts via miRNA221-mediated targeting of the PTEN/PI3K/AKT signaling pathway
Overexpression of GATA4 enhances the antiapoptotic effect of exosomes secreted from cardiac colony-forming unit fibroblasts via miRNA221-mediated targeting of the PTEN/PI3K/AKT signaling pathway
复制标题
GATA4 的过表达通过 miRNA221 介导的 PTEN/PI3K/AKT 信号通路靶向增强心脏集落形成单位成纤维细胞分泌的外泌体的抗凋亡作用
DOI：
10.1186/s13287-020-01759-8
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发表时间：
2020-06-26
影响因子：
7.5
通讯作者：
Chen, Lijuan
中科院分区：
文献类型：
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作者：
Hao, Chunshu;Lu, Zhengri;Chen, Lijuan
背景GATA4是一种早期心脏特异性转录因子，内源性GATA4阳性细胞在心肌损伤后的心脏保护中发挥着关键作用。作为治疗细胞的功能性旁分泌单位，外泌体可以部分复制其亲本细胞的修复特性。在这里，我们研究了源自过表达 GATA4 的心脏集落形成单位成纤维细胞 (cCFU-Fs) 的外泌体 (cCFU-Fs(GATA4)) 的心脏保护能力，以及这些外泌体使用 microRNA (miRNA) 递送来调节心肌梗死 (MI) 中靶蛋白的潜在机制。方法 通过超速离心从 cCFU-F 中收获外泌体。进行 miRNA 阵列以确定源自 cCFU-Fs(GATA4)(GATA4-Exo) 和对照 cCFU-Fs (NC-Exo) 的外泌体之间的差异 miRNA 表达。双荧光素酶报告基因测定证实，miR221 直接靶向十号染色体上的磷酸酶和张力蛋白同源物 (PTEN) 基因的 3' 非翻译区 (UTR)。分别通过超声心动图和Masson三色染色评估心脏功能和心肌梗塞面积。结果与NC-Exo相比，GATA4-Exo提高了H9c2细胞的存活率，减少了细胞凋亡。结扎左冠状动脉前降支（LAD）后，在缺血区域边缘直接进行 GATA4-Exo 心肌内移植，可显着恢复心脏收缩功能并减少梗塞面积。微阵列分析显示 GATA4-Exo 中 miR221 表达显着增加。 qPCR 证实，用 GATA4-Exo 处理的 H9c2 细胞中的 miR221 水平高于用 NC-Exo 处理的细胞。与转染 miR221 抑制剂的细胞相比，转染 miR221 模拟物的 H9c2 细胞在暴露于缺氧条件后表现出显着更高的存活率。双荧光素酶报告基因测定证实 PTEN 基因是 miR221 的靶标。 Western blot分析显示，用GATA4-Exo处理的H9c2细胞表现出较低的PTEN蛋白表达和较高的p-Akt表达。结论 GATA4过表达增强了cCFU-F来源的外泌体对心肌缺血损伤的保护作用。从机制上看，至少部分是由于GATA4-Exo转移的miR221抑制PTEN表达，激活磷脂酰肌醇3激酶（PI3K）/AKT信号通路，从而减轻心肌细胞（CM）的凋亡。
Background GATA4 is an early cardiac-specific transcription factor, and endogenous GATA4-positive cells play a critical role in cardioprotection after myocardial injury. As functional paracrine units of therapeutic cells, exosomes can partially reproduce the reparative properties of their parental cells. Here, we investigated the cardioprotective capabilities of exosomes derived from cardiac colony-forming unit fibroblasts (cCFU-Fs) overexpressing GATA4 (cCFU-Fs(GATA4)) and the underlying mechanism through which these exosomes use microRNA (miRNA) delivery to regulate target proteins in myocardial infarction (MI). Methods Exosomes were harvested from cCFU-Fs by ultracentrifugation. miRNA arrays were performed to determine differential miRNA expression between exosomes derived from cCFU-Fs(GATA4)(GATA4-Exo) and control cCFU-Fs (NC-Exo). A dual-luciferase reporter assay confirmed that miR221 directly targets the 3 ' untranslated region (UTR) of the phosphatase and tensin homolog on chromosome ten (PTEN) gene. Cardiac function and myocardial infarct size were evaluated by echocardiography and Masson trichrome staining, respectively. Results Compared with NC-Exo, GATA4-Exo increased the survival and reduced the apoptosis of H9c2 cells. Direct intramyocardial transplantation of GATA4-Exo at the border of the ischemic region following ligation of the left anterior descending (LAD) coronary artery significantly restored cardiac contractile function and reduced infarct size. Microarray analysis revealed significantly increased miR221 expression in GATA4-Exo. qPCR confirmed higher miR221 levels in H9c2 cells treated with GATA4-Exo than in those treated with NC-Exo. miR221 mimic-transfected H9c2 cells demonstrated a significantly higher survival rate following exposure to hypoxic conditions than those transfected with miR221 inhibitor. A dual-luciferase reporter gene assay confirmed the PTEN gene as a target of miR221. Western blot analysis showed that H9c2 cells treated with GATA4-Exo exhibited lower PTEN protein expression and higher p-Akt expression. Conclusion GATA4 overexpression enhances the protective effect of cCFU-F-derived exosomes on myocardial ischemic injury. In terms of the mechanism, it is at least partly due to the miR221 transferred by GATA4-Exo, which inhibits PTEN expression, activates the phosphatidylinositol 3 kinase (PI3K)/AKT signaling pathway, and subsequently alleviates apoptosis of myocardial cells (CMs).