Maier Kristopher G, Ruhle Brian, Stein Jeffrey J, Gentile Karen L, Middleton Frank A, Gahtan Vivian
Division of Vascular Surgery and Endovascular Services, SUNY Upstate Medical University College of Medicine, 750 East Adams Street, Syracuse, NY, 13210, USA.
Department of Veterans Affairs VA Healthcare Network Upstate New York at Syracuse, Syracuse, USA.
Mol Cell Biochem. 2016 Jan;412(1-2):111-7. doi: 10.1007/s11010-015-2614-9. Epub 2016 Jan 4.
Thrombospondin-1 (TSP-1) is an important regulator of vascular smooth muscle cell (VSMC) physiology and gene expression. MicroRNAs (microRNA), small molecules that regulate protein translation, have emerged as potent regulators of cell function. MicroRNAs have been shown to be involved in intimal hyperplasia, atherosclerosis, and upregulated in the vasculature in diabetes. The purpose of this study was to identify microRNAs regulated by TSP-1 in vascular smooth muscle cells (VSMCs). Human VSMCs were treated for 6 h with basal media or TSP-1 both supplemented with 0.2% FBS. Cells were then snap frozen and RNA extracted. An Affymetrix GeneChip microRNA array analysis was performed in triplicate on three separate collections. Confirmatory qrtPCR was performed. Data were analyzed by ANOVA or t test, with significance set at p < 0.05. MicroRNAs identified were subjected to KEGG pathway analysis using the DIANA tools miRPath online tool. TSP-1 upregulated 22 microRNAs and downregulated 18 microRNAs in VSMCs (p < 0.05). The most upregulated microRNA was miR-512-3p (45.12 fold). The microRNA most downregulated by TSP-1 was miR-25-5p, which was decreased by 9.61. Of note, five members of the mir-17-92 cluster were downregulated. KEGG analysis revealed that thirty-three cellular signaling pathways were impacted by these microRNAs and that nine pathways were relevant to vascular disease. MicroRNAs regulate protein expression at the level of translation and may represent a significant mechanism by which TSP-1 regulates VSMC function. Several of the microRNAs identified have a role in vascular function. The miR-17-92 cluster family, which was found to exhibit reduced expression in this study, is known to be involved in angiogenesis and vascular function. TSP-1 regulates multiple microRNAs in VSMCs adding a new layer of complexity to TSP-1 regulation of VSMC function.
血小板反应蛋白-1（TSP-1）是血管平滑肌细胞（VSMC）生理功能和基因表达的重要调节因子。微小RNA（microRNA）是调节蛋白质翻译的小分子，已成为细胞功能的有力调节因子。微小RNA已被证明参与内膜增生、动脉粥样硬化，且在糖尿病患者的血管系统中表达上调。本研究的目的是鉴定TSP-1在血管平滑肌细胞（VSMC）中调节的微小RNA。将人VSMC用基础培养基或添加0.2%胎牛血清的TSP-1处理6小时。然后将细胞速冻并提取RNA。对三个独立样本进行三次重复的Affymetrix GeneChip微小RNA阵列分析。进行验证性qrtPCR。数据采用方差分析或t检验进行分析，显著性设定为p < 0.05。使用DIANA工具miRPath在线工具对鉴定出的微小RNA进行KEGG通路分析。TSP-1使VSMC中的22种微小RNA上调，18种微小RNA下调（p < 0.05）。上调幅度最大 的微小RNA是miR-512-3p（45.12倍）。TSP-1下调幅度最大的微小RNA是miR-25-5p，降低了9.61倍。值得注意的是，mir-17-92簇的五个成员下调。KEGG分析显示，这些微小RNA影响了33条细胞信号通路，其中9条通路与血管疾病相关。微小RNA在翻译水平调节蛋白质表达，可能是TSP-1调节VSMC功能的重要机制。鉴定出的几种微小RNA在血管功能中起作用。在本研究中发现表达降低的miR-17-92簇家族已知参与血管生成和血管功能。TSP-1调节VSMC中的多种微小RNA，为TSP-1对VSMC功能的调节增加了新的复杂性。
