血管平滑肌细胞（VSMC）来源于干细胞的分化是参与血管重构相关疾病（如高血压、动脉粥样硬化和再狭窄）中不断增加的VSMC数量的一种来源。 microRNA-146a（miR-146a）已被证明参与细胞增殖、迁移和肿瘤代谢。然而，对miR-146a在胚胎干细胞（ESCs）中VSMC分化的功能作用知之甚少。本研究旨在确定miR-146a在ESCs中VSMC分化中的作用。将小鼠ESCs分化为VSMCs，并通过Western blotting和RT-qPCR分析细胞提取物。此外，使用转染miR-146a / mimic和质粒的ESCs进行荧光素酶报告基因试验。最后，将C57BL / 6J雌鼠注射miR-146a / overexpressing ESCs，对来自这些小鼠的组织样本进行免疫组化、Western blotting和RT-qPCR实验。在VSMC分化期间，miR-146a显著上调，伴随着VSMC特异性标记基因平滑肌α-肌动蛋白（SMαA）、平滑肌22（SM22）、平滑肌肌重链（SMMHC）和h1-卡波林。此外，miR-146a的过表达增强了细胞内和体内的分化过程。与此同时，miR-146a的过表达使Kruppel-like因子4（KLF4）的表达急剧降低。重要的是，抑制KLF4的表达增强了miR-146a过表达诱导的分化ESC中VSMC特异性基因表达。此外，miR-146a上调了VSMC分化相关转录因子（包括血清反应因子（SRF）和肌细胞增强因子2c（MEF-2c））的mRNA表达水平和转录活性。我们的数据支持miR-146a通过调节KLF4和调节VSMC的转录因子活性来促进ESC-VSMC分化。 ©2023。作者（们）。

Vascular smooth muscle cell (VSMC) differentiation from stem cells is one source of the increasing number of VSMCs that are involved in vascular remodeling-related diseases such as hypertension, atherosclerosis, and restenosis. MicroRNA-146a (miR-146a) has been proven to be involved in cell proliferation, migration, and tumor metabolism. However, little is known about the functional role of miR-146a in VSMC differentiation from embryonic stem cells (ESCs). This study aimed to determine the role of miR-146a in VSMC differentiation from ESCs.Mouse ESCs were differentiated into VSMCs, and the cell extracts were analyzed by Western blotting and RT-qPCR. In addition, luciferase reporter assays using ESCs transfected with miR-146a/mimic and plasmids were performed. Finally, C57BL/6J female mice were injected with mimic or miR-146a-overexpressing ESCs, and immunohistochemistry, Western blotting, and RT-qPCR assays were carried out on tissue samples from these mice.miR-146a was significantly upregulated during VSMC differentiation, accompanied with the VSMC-specific marker genes smooth muscle-alpha-actin (SMαA), smooth muscle 22 (SM22), smooth muscle myosin heavy chain (SMMHC), and h1-calponin. Furthermore, overexpression of miR-146a enhanced the differentiation process in vitro and in vivo. Concurrently, the expression of Kruppel-like factor 4 (KLF4), predicted as one of the top targets of miR-146a, was sharply decreased in miR-146a-overexpressing ESCs. Importantly, inhibiting KLF4 expression enhanced the VSMC-specific gene expression induced by miR-146a overexpression in differentiating ESCs. In addition, miR-146a upregulated the mRNA expression levels and transcriptional activity of VSMC differentiation-related transcription factors, including serum response factor (SRF) and myocyte enhancer factor 2c (MEF-2c).Our data support that miR-146a promotes ESC-VSMC differentiation through regulating KLF4 and modulating the transcription factor activity of VSMCs.© 2023. The Author(s).