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. 2023 Apr 19;43(2):223–231. doi: 10.1007/s11596-023-2736-3

MicroRNA-146a Promotes Embryonic Stem Cell Differentiation towards Vascular Smooth Muscle Cells through Regulation of Kruppel-like Factor 4

Qing Zhang 1,2,3,#, Rong-rong Pan 4,#, Yu-tao Wu 5,✉, Yu-miao Wei 1,2,3,✉
PMCID: PMC10112997  PMID: 37072613

Abstract

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.

Electronic supplementary material

The online version of this article (10.1007/s11596-023-2736-3) contains supplementary material, which is available to authorized users.

Key words: microRNA-146a, embryonic stem cells, differentiation, vascular smooth muscle cells, Kruppel-like factor 4

Supplementary data

Appendix (10MB, pdf)

Footnotes

This work was funded by the National Natural Science Foundation of China (No. 82070376 and No. 81873491), the Natural Science Foundation of Zhejiang Province (No. LY21H020005), the Zhejiang Medical Science and Technology Project (No. 2019KY376 and No. 2018KY071), and a Ningbo Science and Technology Project (No. 202002N3173).

Conflict of Interest Statement

The authors declare that they have no conflicts of interest.

These authors contributed equally to this work.

Contributor Information

Qing Zhang, Email: 21718081@zju.edu.cn.

Rong-rong Pan, Email: panrong_0131@126.com.

Yu-tao Wu, Email: banshuirentaotao@126.com.

Yu-miao Wei, Email: ymwei12@163.com.

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