过表达GATA-4骨髓间充质干细胞以外泌体修复心肌损伤的相关microRNA

A molecular study on myocardial repair via exosomes secreted from GATA-4-overexpressed bone marrow mesenchymal stem cells

背景:GATA-4是与心脏发育特异相关的锌指样转录因子,前期实验发现过表达GATA-4的骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)分泌的外泌体(exosome)与BMSCs共培养时可以促使BMSCs表达出更多的心肌特异性抗原。当其与心肌细胞在低氧环境下培养可以抑制心肌细胞的凋亡。目的:探讨过表达GATA-4的BMSCs分泌exosome修复心肌损伤的相关microRNA,从分子水平对其生物学效应进行探讨。方法:(1)通过慢病毒载体GV308携带GATA-4转染小鼠BMSCs,构建过表达GATA-4小鼠BMSCs,并加入基因开启剂强力霉素,然后采用ExoQuick-TC法提取分泌的exosome;(2)实验设5组:GATA-4-BMSCs-exosome+BMSCs共培养组、空载体-BMSCs-exosome+BMSCs共培养组、BMSCs-exosome+BMSCs共培养组、BMSCs单独培养组、心肌细胞单独培养组。培养24 h采用Q-PCR定量检测心肌特异性抗原cTnT、α-actin、connexin 43、Desmin的表达;(3)实验设5组:GATA-4-BMSCs-exosome+心肌细胞共培养组、空载体-BMSCs-exosome+心肌细胞共培养组、BMSCs-exosome+心肌细胞共培养组、心肌细胞单独培养组,在低氧(体积分数为1%)无血清培养条件下培养24 h诱导细胞凋亡。以正常条件下单独培养的心肌细胞作为阴性对照组,采用流式细胞技术检测各组细胞的凋亡率;(4)采用Agilent microRNA芯片检测过表达GATA-4小鼠BMSCs分泌exosome内有关细胞分化及抗凋亡的microRNA。结果与结论:(1)Q-PCR和流式细胞技术检测结果显示:过表达GATA-4-BMSCs分泌的exosome可以有效促进BMSCs向心肌细胞转化且具有极强的抗凋亡能力;(2)Agilent microRNA芯片检测结果显示:涉及细胞分化的关键microRNA:mmu-miR-199a-3p(microRNA为上调);mmu-miR-1894-5p(microRNA为下调)。涉及细胞抗凋亡的关键microRNA:mmu-miR-199a-3p、mmu-miR-20a-5p、mmu-miR-330-3p,这3个基因均为上调。其中mmu-miR-199a-3p即涉及细胞分化又涉及细胞抗凋亡,为重点首先需要验证的microRNA。

BACKGROUND： GATA-4 is a zinc finger transcription factor that is specifically associated with cardiac development. Previous experiments have found that exosomes secreted from GATA-4-overexpressed bone marrow mesenchymal stem cells （BMSCs） co-cultured with BMSCs can make BMSCs express more myocardial-specific antigens. When co-cultured with myocardial cells in a hypoxic environment, these exosomes can inhibit apoptosis in myocardial cells.OBJECTIVE： To identify from a molecular level the basic and essential exosomes secreted from GATA-4-overexpressed mouse BMSCs that has a significant role in cardiac repair after myocardial infarction. METHODS： （1） GATA-4-overexpressed mouse BMSCs were constructed by transfecting mouse BMSCs with GV308 （a lentiviral vector）-carrying GATA-4 before adding doxycycline for gene induction. ExoQuick-TC （SBI Inc.） was then used to extract the secreted exosomes. （2） The BMSCs were co-cultured with GATA-4-BMSCs-Exosome, free-vector-BMSCs-Exosomes, and BMSCs-Exosome. Another BMSCs and mouse myocardial cells were cultured alone. The expression levels of myocardial specific antigens, cTnT, α-actin, connexin 43, and Desmin, were assessed via qPCR quantification at 24 hours of culture. （3） The mouse myocardial cells were then co-cultured with GATA-4-BMSCs-Exosome, free-vector-BMSCs-Exosomes, and BMSCs-Exosome, and were subsequently mono-cultured in hypoxia and in serum-free cultures to construct the apoptosis-positive control group. Normally cultured myocardial cells were as negative controls. Cell apoptosis rates in different groups were determined by flow cytometry. （4） The microRNAs, which were associated with cellular differentiation or anti-apoptosis, in the exosomes secreted from GATA-4-overexpressed mouse BMSCs were detected by Agilent microRNA microarray. RESULTS AND CONCLUSION： Results from Q-PCR and flow cytometry suggested that the exosomes secreted from GATA-4-overexpressed mouse MBSCs effectively facilitated the differentiation of BMSCs to form myocardial cells and reduce apoptosis. The Agilent microRNA microarray test results showed that the key microRNAs associated with differentiation included mmu-miR-199a-3p （up-regulated） and mmu-miR-1894-5p （down-regulated）, while the key microRNAs associated with the anti-apoptotic function included mmu-miR-199a-3p, mmu-miR-20a-5p, and mmu-miR-330-3p, all of which were up-regulated. Among these microRNAs, mmu-miR-199a-3p was the only one associated with both cellular differentiation and anti-apoptosis, and it was therefore considered as the most primary microRNA to be validated.