Department of Pathology and Laboratory Medicine, University of Cincinnati Medical Center, Cincinnati, Ohio, United States of America.
PLoS One. 2013 Aug 28;8(8):e73304. doi: 10.1371/journal.pone.0073304. eCollection 2013.
microRNAs (miRs), a novel class of small non-coding RNAs, are involved in cell proliferation, differentiation, development, and death. In this study, we found that miR-221 translocation by microvesicles (MVs) plays an important role in cardioprotection mediated by GATA-4 overexpressed mesenchymal stem cells (MSC).
Adult rat bone marrow MSC and neonatal rat ventricle cardiomyocytes (CM) were harvested as primary cultures. MSC were transduced with GATA-4 (MSC(GATA-4)) using the murine stem cell virus (pMSCV) retroviral expression system. Empty vector transfection was used as a control (MSC(Null)). The expression of miRs was assessed by real-time PCR and localized using in situ hybridization (ISH). MVs collected from MSC cultures were characterized by expression of CD9, CD63, and HSP70, and photographed with electron microscopy. Cardioprotection during hypoxia afforded by conditioned medium (CdM) from MSC cultures was evaluated by lactate dehydrogenase (LDH) release, MTS uptake by CM, and caspase 3/7 activity. Expression of miR-221/222 was significantly higher in MSC than in CM and miR-221 was upregulated in MSC(GATA-4). MSC overexpression of miR-221 significantly enhanced cardioprotection by reducing the expression of p53 upregulated modulator of apoptosis (PUMA). Moreover, expression of PUMA was significantly decreased in CM co-cultured with MSC. MVs derived from MSC expressed high levels of miR-221, and were internalized quickly by CM as documented in images obtained from a Time-Lapse Imaging System.
Our results demonstrate that cardioprotection by MSC(GATA-4) may be regulated in part by a transfer of anti-apoptotic miRs contained within MVs.
微小 RNA（miRs）是一类新型的小非编码 RNA，参与细胞增殖、分化、发育和死亡。在本研究中，我们发现通过微泡（MVs）转移的 miR-221 在 GATA-4 过表达间充质干细胞（MSC）介导的心脏保护中发挥重要作用。
从成年大鼠骨髓 MSC 和新生大鼠心室心肌细胞（CM）中获得原代培养物。使用鼠干细胞病毒（pMSCV）逆转录病毒表达系统将 GATA-4 转导至 MSC（MSC（GATA-4））。空载体转染用作对照（MSC（Null））。通过实时 PCR 评估 miR 的表达，并通过原位杂交（ISH）定位。通过 CD9、CD63 和 HSP70 的表达来鉴定 MSC 培养物中收集的 MV，并通过电子显微镜拍摄照片。通过 CM 的乳酸脱氢酶（LDH）释放、MTS 摄取和 caspase 3/7 活性评估 MSC 培养物条件培养基（CdM）提供的缺氧期间的心脏保护作用。miR-221/222 在 MSC 中的表达明显高于 CM，并且 miR-221 在 MSC（GATA-4）中上调。MSC 过表达 miR-221 通过降低促凋亡 p53 上调调节剂（PUMA）的表达显著增强心脏保护作用。此外，与 MSC 共培养的 CM 中 PUMA 的表达明显降低。从 MSC 衍生的 MV 表达高水平的 miR-221，并在 Time-Lapse Imaging System 获得的图像中记录到 CM 快速内化。
我们的结果表明，MSC（GATA-4）的心脏保护作用可能部分受 MV 中包含的抗凋亡 miR 的转移调节。
