Research Center of Guangdong General Hospital, Guangdong Provincial Cardiovascular Institute, Guangdong Academy of Medical Sciences, Guangzhou 510080, China.
Life Sci. 2012 Jun 27;90(25-26):1020-6. doi: 10.1016/j.lfs.2012.05.011. Epub 2012 Jun 4.
Upregulation of microRNA 16 (miR-16) contributed to the differentiation of human bone marrow mesenchymal stem cells (hMSCs) toward myogenic phenotypes in a cardiac niche, the present study aimed to determine the role of miR-16 in this process.
hMSCs and neonatal rat ventricular myocytes were co-cultured indirectly in two chambers to set up a cardiac microenvironment (niche). miRNA expression profile in cardiac-niche-induced hMSCs was detected by miRNA microarray. Cardiac marker expression and cell cycle analysis were determined in different treatment hMSCs. Quantitative real-time PCR and Western blot were used to identify the expression of mRNA, mature miRNA and protein of interest.
miRNA dysregulation was shown in hMSCs after cardiac niche induction. miR-16 was upregulated in cardiac-niche-induced hMSCs. Overexpression of miR-16 significantly increased G1-phase arrest of the cell cycle in hMSCs and enhanced the expression of cardiac marker genes, including GATA4, NK2-5, MEF2C and TNNI3. Differentiation-inducing factor 3 (DIF-3), a G0/G1 cell cycle arrest compound, was used to induce G1 phase arrest in cardiac-niche-induced hMSCs, and the expression of cardiac marker genes was up-regulated in DIF-3-treated hMSCs. The expression of CCND1, CCND2 and CDK6 was suppressed by miR-16 in hMSCs. CDK6, CCND1 or CCND2 knockdown resulted in G1 phase arrest in hMSCs and upregulation of cardiac marker gene expression in hMSCs in a cardiac niche.
miR-16 enhances G1 phase arrest in hMSCs, contributing to the differentiation of hMSCs toward myogenic phenotypes when in a cardiac niche. This mechanism provides a novel strategy for pre-modification of hMSCs before hMSC-based transplantation therapy for severe heart diseases.
微小 RNA16（miR-16）的上调促进了人心肌基质细胞（hMSC）在心脏龛位中向肌性表型的分化，本研究旨在确定 miR-16 在这一过程中的作用。
通过 miRNA 微阵列检测心脏龛位诱导的 hMSC 中的 miRNA 表达谱。将 hMSC 和新生大鼠心室肌细胞在两个室中间接共培养，建立心脏微环境（龛位）。在不同处理的 hMSC 中测定心脏标志物表达和细胞周期分析。采用定量实时 PCR 和 Western blot 鉴定感兴趣的 mRNA、成熟 miRNA 和蛋白的表达。
心脏龛位诱导后 hMSC 中出现 miRNA 失调。miR-16 在心脏龛位诱导的 hMSC 中上调。miR-16 的过表达显著增加了 hMSC 细胞周期的 G1 期停滞，并增强了心脏标志物基因的表达，包括 GATA4、NK2-5、MEF2C 和 TNNI3。分化诱导因子 3（DIF-3）是一种 G0/G1 细胞周期阻滞化合物，用于诱导心脏龛位诱导的 hMSC 中的 G1 期停滞，并且 DIF-3 处理的 hMSC 中心脏标志物基因的表达上调。CCND1、CCND2 和 CDK6 在 hMSC 中被 miR-16 抑制。在心脏龛位中，CCDK6、CCND1 或 CCND2 的敲低导致 hMSC 的 G1 期停滞和心脏标志物基因表达的上调。
miR-16 增强了 hMSC 中的 G1 期停滞，有助于 hMSC 在心脏龛位中向肌性表型分化。该机制为严重心脏病患者基于 hMSC 的移植治疗前 hMSC 的预修饰提供了一种新策略。
