miR-15a/15b Cluster Modulates Survival of Mesenchymal Stem Cells to Improve Its Therapeutic Efficacy of Myocardial Infarction
miR-15a/15b Cluster Modulates Survival of Mesenchymal Stem Cells to Improve Its Therapeutic Efficacy of Myocardial Infarction
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miR-15a/15b簇调节间充质干细胞的存活以提高其对心肌梗死的治疗效果
DOI：
10.1161/jaha.118.010157
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发表时间：
2019
影响因子：
5.4
通讯作者：
Yu Bo
中科院分区：
文献类型：
--
作者：
Tu Yingfeng;Qiu Yan;Liu Li;Huang Tao;Tang Hao;Liu Youbin;Guo Wenguang;Jiang Hongchi;Fan Yuhua;Yu Bo
背景骨髓间充质干细胞（mesenchymal stem cells，MSCs）移植后存活率低，影响了其治疗缺血性心脏病的疗效. MicroRNA参与调节MSC的生存和功能。本研究旨在探讨miR-15 a/15 b对MSC存活的分子影响，重点关注血管内皮生长因子受体2的作用。方法和结果我们首先从FVB背景的荧光素酶转基因小鼠中分离供体luc（Luciferase）-MSC（5×105）。用miR-15 a/15 b模拟物或抑制剂转染Luc-MSC，并在氧葡萄糖剥夺条件下培养12小时，以模拟梗死心脏中的恶劣微环境;将它们进行MTT（3-（4，5-二甲基-2-噻唑基）-2，5-二苯基-2-H-溴化四氮唑？噻唑蓝溴化四唑）测定、生物发光成像、定量逆转录-聚合酶链反应、转移酶介导的脱氧尿苷三磷酸-地高辛缺口末端标记测定和流式细胞术。此外，通过蛋白质印迹法测定血管内皮生长因子受体2、蛋白激酶B、p（磷酸化）-蛋白激酶B、Bcl-2、Bax和caspase-3蛋白的水平。在体内，通过冠状动脉结扎在24只小鼠中诱导急性心肌梗死，随后接受Luc-MSC、Luc-MSC +miR-15 a/15 b抑制剂或PBS处理。采用生物发光成像和超声心动图测量，跟踪和评估治疗过程和治疗效果。接下来，进行离体成像和免疫组织化学以验证MSC的分布。我们证明miR-15 a/15 B靶向血管内皮生长因子受体2调节MSC存活，可能通过磷脂酰肌醇3-激酶/蛋白激酶B信号通路，这一点通过生物发光成像、免疫组织化学分析和超声心动图测量得到证实。miR-15 a/B的作用可以从luc-MSC信号的丢失推断。这一发现可能对miR-15 a/15 b修饰的MSC移植治疗心肌梗死具有实际的临床意义。
BackgroundThe poor viability of transplanted mesenchymal stem cells (MSCs) hampers their therapeutic efficacy for ischemic heart disease. MicroRNAs are involved in regulation of MSC survival and function. The present study was designed to investigate the molecular effects of miR‐15a/15b on MSC survival, focusing on the role of vascular endothelial growth factor receptor 2.Methods and ResultsWe first harvested donor luc(Luciferase)‐MSCs (5×105) isolated from the luciferase transgenic mice with FVB background. Luc‐MSCs were transfected with miR‐15a/15b mimics or inhibitors and cultured under oxygen glucose deprivation condition for 12 hours to mimics the harsh microenvironment in infarcted heart; they were subjected to MTT (3‐(4,5‐dimethyl‐2‐thiazolyl)‐2,5‐diphenyl‐2‐H‐tetrazolium bromide?Thiazolyl Blue Tetrazolium Bromide) assay, bioluminescence imaging, quantitative reverse transcription–polymerase chain reaction, transferase‐mediated deoxyuridine triphosphate–digoxigenin nick‐end labeling assay, and flow cytometry. Furthermore, the levels of vascular endothelial growth factor receptor 2, protein kinase B, p(Phosphorylate)‐protein kinase B, Bcl‐2, Bax, and caspase‐3 proteins were available by Western blotting assay. In vivo, acute myocardial infarction was induced in 24 mice by coronary ligation, with subsequent receipt of Luc‐MSCs, Luc‐MSCs+miR‐15a/15b inhibitors, or PBS treatment. The therapeutic procedure and treatment effects were tracked and assessed using bioluminescence imaging and echocardiographic measurement. Next, ex vivo imaging and immunohistochemistry were conducted to verify the distribution of MSCs. We demonstrated that miR‐15a/15b targeted vascular endothelial growth factor receptor 2 to modulate MSC survival, possibly via phosphatidylinositol 3‐kinase/protein kinase B signaling pathway, which was proved by bioluminescence imaging, immunohistochemistry analysis, and echocardiographic measurement.ConclusionsLuc‐MSCs could be followed dynamically in vitro and in vivo by bioluminescence imaging, and the role of miR‐15a/b could be inferred from the loss of signals from luc‐MSCs. This finding may have practical clinical implications in miR‐15a/15b–modified MSC transplantation in treating myocardial infarction.