微小 RNA-146a 通过调节 Kruppel 样因子 4 促进胚胎干细胞向血管平滑肌细胞分化。
MicroRNA-146a Promotes Embryonic Stem Cell Differentiation towards Vascular Smooth Muscle Cells through Regulation of Kruppel-like Factor 4.
机构信息
Department of Cardiology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China.
Hubei Key Laboratory of Biological Targeted Therapy, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China.
出版信息
Curr Med Sci. 2023 Apr;43(2):223-231. doi: 10.1007/s11596-023-2736-3. Epub 2023 Apr 19.
OBJECTIVE
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.
METHODS
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.
RESULTS
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).
CONCLUSION
Our data support that miR-146a promotes ESC-VSMC differentiation through regulating KLF4 and modulating the transcription factor activity of VSMCs.
目的
血管平滑肌细胞（VSMC）分化自干细胞，是与高血压、动脉粥样硬化和再狭窄等血管重构相关疾病有关的 VSMC 数量增加的一个来源。微小 RNA-146a（miR-146a）已被证明参与细胞增殖、迁移和肿瘤代谢。然而，miR-146a 在胚胎干细胞（ESC）向 VSMC 分化中的功能作用知之甚少。本研究旨在确定 miR-146a 在 ESC 向 VSMC 分化中的作用。
方法
将小鼠 ESC 分化为 VSMC，并通过 Western blot 和 RT-qPCR 分析细胞提取物。此外，使用转染了 miR-146a/ mimic 和质粒的 ESC 进行荧光素酶报告基因检测。最后，向 C57BL/6J 雌性小鼠注射 mimic 或过表达 miR-146a 的 ESC，并对这些小鼠的组织样本进行免疫组织化学、Western blot 和 RT-qPCR 检测。
结果
miR-146a 在 VSMC 分化过程中显著上调，同时伴随着 VSMC 特异性标记基因平滑肌肌动蛋白-α（SMαA）、平滑肌 22（SM22）、平滑肌肌球蛋白重链（SMMHC）和 h1-钙调蛋白。此外，miR-146a 的过表达增强了体外和体内的分化过程。同时，作为 miR-146a 的预测靶标之一的 Kruppel 样因子 4（KLF4）在过表达 miR-146a 的 ESC 中的表达明显降低。重要的是，抑制 KLF4 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 上调了与 VSMC 分化相关的转录因子，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。
结论
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。
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