Department of Surgery, SUNY Upstate Medical University, Syracuse, NY; Department of Veterans Affairs Healthcare Network Upstate New York at Syracuse, Syracuse, NY.
Department of Surgery, SUNY Upstate Medical University, Syracuse, NY; Department of Veterans Affairs Healthcare Network Upstate New York at Syracuse, Syracuse, NY.
Surgery. 2014 Feb;155(2):228-33. doi: 10.1016/j.surg.2013.08.003. Epub 2013 Dec 5.
Thrombospondin-1 (TSP-1) is a matricellular glycoprotein released from platelets at sites of arterial injury and is important in neointima development after balloon angioplasty. MicroRNAs are small noncoding RNAs that function by binding target gene mRNA and inhibiting protein translation. MicroRNA-21 (miR-21) is up-regulated after angioplasty, and inhibition of miR-21 leads to decreased intimal hyperplasia. In this study, we examined the effects of miR-21 inhibition on vascular smooth muscle cell (VSMC) processes.
VSMCs were exposed to TSP-1 and miR-21 inhibitor for 20 minutes. TSP-1-induced migration was assessed with a modified Boyden microchemotaxis chamber and proliferation with calcein-AM fluorescence. Phosphorylated extracellular signaling kinase (ERK) 1/2 expression was determined by Western Blot and densitometry. Quantitative real-time polymerase chain reaction for TSP-1, hyaluronic acid synthase 2 (HAS2), and transforming growth factor beta 2 (TGFβ2) was performed. Statistical analysis was performed with analysis of variance (P < .05).
Inhibition of miR-21 blocked TSP-1-induced VSMC migration, proliferation, and ERK 1/2 phosphorylation (P < .05) and had no effect on TSP-1-stimulated expression of genes for TSP-1, HAS2, or TGFβ2 (P > .05).
Acute inhibition of miR-21 led to a decrease in VSMC migration and proliferation caused by TSP-1. The decrease in TSP-1's activation of ERK 1/2 after acute miR-21 inhibition indicates an active role for miR-21 in TSP-1's cell signaling cascade. No effect on TSP-1-induced expression of the pro-stenotic genes thbs1, tgfb2, or has2, occurred after acute miR-21 inhibition. These data indicate that miR-21 directly modulates cell function and signaling pathways in ways other than inhibition of protein translation.
血小板衍生的细胞外基质糖蛋白 1（TSP-1）在动脉损伤部位释放，并在球囊血管成形术后新生内膜形成中起重要作用。microRNAs 是一类小的非编码 RNA，通过与靶基因 mRNA 结合并抑制蛋白翻译来发挥作用。miR-21 在血管成形术后上调，抑制 miR-21 可减少内膜增生。在本研究中，我们研究了 miR-21 抑制对血管平滑肌细胞（VSMC）过程的影响。
用 TSP-1 和 miR-21 抑制剂处理 VSMC 20 分钟。用改良的 Boyden 微趋化实验检测 TSP-1 诱导的迁移，用 calcein-AM 荧光检测增殖。用 Western Blot 和密度计法检测磷酸化细胞外信号调节激酶（ERK）1/2 的表达。用实时定量聚合酶链反应（PCR）检测 TSP-1、透明质酸合酶 2（HAS2）和转化生长因子β 2（TGFβ2）的表达。采用方差分析（P <.05）进行统计学分析。
抑制 miR-21 阻断了 TSP-1 诱导的 VSMC 迁移、增殖和 ERK 1/2 磷酸化（P <.05），但对 TSP-1 刺激的 TSP-1、HAS2 或 TGFβ2 基因表达无影响（P >.05）。
急性抑制 miR-21 导致 TSP-1 诱导的 VSMC 迁移和增殖减少。急性 miR-21 抑制后 TSP-1 激活 ERK 1/2 的减少表明 miR-21 在 TSP-1 的细胞信号级联中发挥积极作用。急性 miR-21 抑制后，TSP-1 诱导的 thbs1、tgfb2 或 has2 等促狭窄基因的表达无变化。这些数据表明，miR-21 通过抑制蛋白翻译以外的方式直接调节细胞功能和信号通路。
