Objective

The pathogenesis of coronary artery calcification (CAC) in coronary heart disease (CHD) is mediated by exosomes derived from vascular smooth muscle cells (VSMCs). However, little is known about their underlying mechanism. In this study, we aimed to investigate the differentially expressed miRNAs in VSMCs undergoing induced calcification.

Methods

A cellular calcification model was established using the mouse VSMC line MOVAS-1. Calcium deposition was evaluated by Alizarin Red staining. Exosome sizes were determined by Nanoparticle Tracking Analysis (NTA), and exosome morphology was examined by transmission electron microscopy (TEM). The expression of exosome and calcification biomarkers was analyzed by quantitative real-time PCR (qPCR) and western blotting. Differential miRNA profiles were determined by deep sequencing and bioinformatics. Protein levels in VSMCs experiencing interference by a miR-324-3p inhibitor were detected by western blotting.

Results

The MOVAS-1 calcification model was confirmed by Alizarin Red staining and expressional alteration of α-SMA, BMP-2, OPN, and MGP. Exosomes from the calcification model showed expression of exosomal biomarkers and regular exosome diameters, which caused significant calcification in MOVAS-1 cells. In total, 987 and 92 miRNAs were significantly upregulated and downregulated in exosomes from the cellular calcification model as compared with those from MOVAS-1 cells, respectively. Target genes of differential miRNAs were involved in various biological processes such as development, metabolism, and cellular component organization and biogenesis as well as multiple signaling pathways such as protein kinase B (AKT) signaling. The most differentially expressed miRNAs were validated by qPCR, which showed that mmu-let-7e-5p was downregulated and mmu-miR-324-3p was upregulated in exosomes from the MOVAS-1 cellular calcification model. The expression of IGF1R was increased, and the expressions of PIK3CA and MAP2K1 were reduced in MOVAS-1 transfected with a miR-324-3p inhibitor.

Conclusion

microRNA profiles were significantly altered in exosomes from VSMCs undergoing calcification.

中文翻译：

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与冠状动脉钙化相关的血管平滑肌细胞的外泌体中差异表达的microRNA图谱。

目的

冠心病（CHD）中冠状动脉钙化（CAC）的发病机理是由来自血管平滑肌细胞（VSMC）的囊泡介导的。但是，对其基本机制知之甚少。在这项研究中，我们旨在研究经历诱导钙化的VSMC中差异表达的miRNA。

方法

使用小鼠VSMC系MOVAS-1建立了细胞钙化模型。通过茜素红染色评价钙沉积。通过纳米粒子跟踪分析（NTA）确定外泌体的大小，并通过透射电子显微镜（TEM）检查外泌体的形态。通过定量实时PCR（qPCR）和western印迹分析外泌体和钙化生物标志物的表达。通过深度测序和生物信息学确定了差异miRNA谱。通过蛋白质印迹法检测受到miR-324-3p抑制剂干扰的VSMC中的蛋白水平。

结果

茜素红染色和α-SMA，BMP-2，OPN和MGP的表达改变证实了MOVAS-1钙化模型。来自钙化模型的外泌体显示出外泌体生物标志物的表达和规则的外泌体直径，这在MOVAS-1细胞中引起了显着的钙化。与来自MOVAS-1细胞的外显子相比，来自细胞钙化模型的外泌体中总共有987和92个miRNA显着上调和下调。差异miRNA的靶基因参与了各种生物学过程，例如发育，代谢，细胞组分的组织和生物发生，以及多种信号通路，例如蛋白激酶B（AKT）信号传导。表达最差的miRNA已通过qPCR验证，这表明在MOVAS-1细胞钙化模型的外泌体中，mmu-let-7e-5p被下调，mmu-miR-324-3p被上调。在用miR-324-3p抑制剂转染的MOVAS-1中，IGF1R的表达增加，而PIK3CA和MAP2K1的表达减少。

结论

在经历钙化作用的VSMC的外来体中，microRNA谱显着改变。