Department of Cardiology, Eastern Hospital, Sichuan Academy of Medical Sciences & Sichuan Provincial People's Hospital, Chengdu, China.
Eur Rev Med Pharmacol Sci. 2019 Jan;23(2):479-485. doi: 10.26355/eurrev_201901_16859.
The purpose of the study was to investigate the regulatory effect of microRNA-10-5p on TBX5 during myocardial differentiation of bone marrow mesenchymal stem cells (BMSCs) in rats.
Rat BMSCs were first isolated, cultured and identified by flow cytometry. Expression levels of BNP (Brain Natriuretic Peptide), α-actinin, and Islet-1 in BMSCs co-cultured with rat cardiomyocytes were detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Dual-luciferase reporter gene assay was conducted to verify the binding condition between microRNA-10-5p and TBX5. Subsequently, we detected TBX5 expression after overexpression or knockdown of microRNA-10-5p in BMSCs. Rescue experiments were conducted by overexpression of both microRNA-10-5p and TBX5 in BMSCs, and then, the expression levels of BNP, α-actinin, and Islet-1 were detected by qRT-PCR and Western blot.
Flow cytometry results showed positive-CD73 (99.3%), positive-CD90 (95.4%), and negative-CD34 (4.2%), which were consistent with immunophenotypic characteristics of BMSCs. TBX5 overexpression or microRNA-10-5p knockdown increased mRNA levels of BNP, α-actinin, and Islet-1 in BMSCs co-cultured with rat cardiomyocytes. Dual-luciferase reporter gene assay confirmed that microRNA-10-5p could bind to TBX5. Both mRNA and protein expressions of TBX5 were negatively regulated by microRNA-10-5p. The inhibited expression levels of BNP, α-actinin, and Islet-1 by microRNA-10-5p overexpression in BMSCs were partially reversed by TBX5 overexpression.
MicroRNA-10-5p regulates BMSCs differentiation into cardiomyocytes by binding to TBX5.
本研究旨在探讨 microRNA-10-5p 在大鼠骨髓间充质干细胞（BMSCs）向心肌细胞分化过程中对 TBX5 的调控作用。
首先通过流式细胞术分离、培养和鉴定大鼠 BMSCs。通过定量实时聚合酶链反应（qRT-PCR）检测与大鼠心肌细胞共培养的 BMSCs 中脑钠肽（BNP）、α-辅肌动蛋白和胰岛 1 的表达水平。通过双荧光素酶报告基因实验验证 microRNA-10-5p 与 TBX5 的结合情况。随后，检测 BMSCs 中转录 microRNA-10-5p 过表达或敲低后 TBX5 的表达情况。在 BMSCs 中转录 microRNA-10-5p 和 TBX5 过表达后进行挽救实验，然后通过 qRT-PCR 和 Western blot 检测 BNP、α-辅肌动蛋白和胰岛 1 的表达水平。
流式细胞术结果显示 CD73（99.3%）、CD90（95.4%）阳性，CD34（4.2%）阴性，与 BMSCs 的免疫表型特征一致。TBX5 过表达或 microRNA-10-5p 敲低均增加了与大鼠心肌细胞共培养的 BMSCs 中 BNP、α-辅肌动蛋白和胰岛 1 的 mRNA 水平。双荧光素酶报告基因实验证实 microRNA-10-5p 可与 TBX5 结合。microRNA-10-5p 负调控 TBX5 的 mRNA 和蛋白表达。microRNA-10-5p 过表达抑制 BMSCs 中 BNP、α-辅肌动蛋白和胰岛 1 的表达水平，通过 TBX5 过表达部分逆转。
microRNA-10-5p 通过与 TBX5 结合调节 BMSCs 向心肌细胞分化。
