Faculty of Life Sciences, Northwest University, Xi'an 710069, China; State Key Laboratory of Cancer Biology, Department of Biochemistry and Molecular Biology, Fourth Military Medical University, Xi'an 710032, China.
College of Pulmonary and Critical Care Medicine, The 8th Medical Centre of Chinese PLA General Hospital, Beijing 100091, China.
Life Sci. 2023 Aug 1;326:121828. doi: 10.1016/j.lfs.2023.121828. Epub 2023 Jun 1.
Under various pathological conditions such as cancer, vascular smooth muscle cells (vSMCs) transit their contractile phenotype into phenotype(s) characterized by proliferation and secretion, a process called vSMC phenotypic transition (vSMC-PT). Notch signaling regulates vSMC development and vSMC-PT. This study aims to elucidate how the Notch signal is regulated.
Gene-modified mice with a SM22α-CreER transgene were generated to activate/block Notch signaling in vSMCs. Primary vSMCs and MOVAS cells were cultured in vitro. RNA-seq, qRT-PCR and Western blotting were used to evaluated gene expression level. EdU incorporation, Transwell and collagen gel contraction assays were conducted to determine the proliferation, migration and contraction, respectively.
Notch activation upregulated, while Notch blockade downregulated, miR-342-5p and its host gene Evl in vSMCs. However, miR-342-5p overexpression promoted vSMC-PT as shown by altered gene expression profile, increased migration and proliferation, and decreased contraction, while miR-342-5p blockade exhibited the opposite effects. Moreover, miR-342-5p overexpression significantly suppressed Notch signaling, and Notch activation partially abolished miR-342-5p-induced vSMC-PT. Mechanically, miR-342-5p directly targeted FOXO3, and FOXO3 overexpression rescued miR-342-5p-induced Notch repression and vSMC-PT. In a simulated tumor microenvironment, miR-342-5p was upregulated by tumor cell-derived conditional medium (TCM), and miR-342-5p blockade abrogated TCM-induced vSMC-PT. Meanwhile, conditional medium from miR-342-5p-overexpressing vSMCs significantly enhanced tumor cell proliferation, while miR-342-5p blockade had the opposite effects. Consistently, in a co-inoculation tumor model, miR-342-5p blockade in vSMCs significantly delayed tumor growth.
miR-342-5p promotes vSMC-PT through a negative-feedback regulation of Notch signaling via downregulating FOXO3, which could be a potential target for cancer therapy.
在癌症等各种病理条件下，血管平滑肌细胞（VSMC）将其收缩表型转变为增殖和分泌为特征的表型，这个过程称为 VSMC 表型转变（VSMC-PT）。Notch 信号通路调控 VSMC 的发育和 VSMC-PT。本研究旨在阐明 Notch 信号是如何被调控的。
利用 SM22α-CreER 转基因小鼠生成 VSMC 中激活/阻断 Notch 信号的基因修饰小鼠。体外培养原代 VSMC 和 MOVAS 细胞。采用 RNA 测序、qRT-PCR 和 Western blot 检测基因表达水平。EdU 掺入、Transwell 和胶原凝胶收缩实验分别用于测定增殖、迁移和收缩。
Notch 激活上调，而 Notch 阻断下调 VSMC 中的 miR-342-5p 和其宿主基因 Evl。然而，miR-342-5p 的过表达促进了 VSMC-PT，表现为基因表达谱的改变、迁移和增殖的增加以及收缩的减少，而 miR-342-5p 的阻断则表现出相反的效果。此外，miR-342-5p 的过表达显著抑制 Notch 信号通路，而 Notch 激活部分消除了 miR-342-5p 诱导的 VSMC-PT。机制上，miR-342-5p 直接靶向 FOXO3，而 FOXO3 的过表达挽救了 miR-342-5p 诱导的 Notch 抑制和 VSMC-PT。在模拟肿瘤微环境中，肿瘤细胞来源的条件培养基（TCM）上调 miR-342-5p，而 miR-342-5p 的阻断消除了 TCM 诱导的 VSMC-PT。同时，过表达 miR-342-5p 的 VSMC 的条件培养基显著增强了肿瘤细胞的增殖，而 miR-342-5p 的阻断则有相反的效果。一致地，在共接种肿瘤模型中，VSMC 中 miR-342-5p 的阻断显著延迟了肿瘤的生长。
miR-342-5p 通过下调 FOXO3 对 Notch 信号通路进行负反馈调节，从而促进 VSMC-PT，这可能成为癌症治疗的一个潜在靶点。
