Inflammatory smooth muscle cells (iSMCs) generated from adventitial stem/progenitor cells (AdSPCs) have been recognised as a new player in cardiovascular disease, and microRNA-214-3p (miR-214-3p) has been implicated in mature vascular SMC functions and neointimal hyperplasia. Here, we attempted to elucidate the functional involvements of miR-214-3p in iSMC differentiation from AdSPCs and unravel the therapeutic potential of miR-214-3p signalling in AdSPCs for injury-induced neointimal hyperplasia. The role of miR-214-3p in iSMC differentiation from AdSPCs was evaluated by multiple biochemistry assays. The target of miR-214-3p was identified through binding site mutation and reporter activity analysis. A murine model of injury-induced arterial remodelling and stem cell transplantation was conducted to study the therapeutic potential of miR-214-3p. RT-qPCR analysis was performed to examine the gene expression in healthy and diseased human arteries. miR-214-3p prevented iSMC differentiation/generation from AdSPCs by restoring sonic hedgehog-glioma-associated oncogene 1 (Shh-GLI1) signalling. Suppressor of fused (Sufu) was identified as a functional target of miR-214-3p during iSMC generation from AdSPCs. Mechanistic studies revealed that miR-214-3p over-expression or Sufu inhibition can promote nuclear accumulation of GLI1 protein in AdSPCs, and the consensus sequence (GACCACCCA) for GLI1 binding within smooth muscle alpha-actin (SMαA) and serum response factor (SRF) gene promoters is required for their respective regulation by miR-214-3p and Sufu. Additionally, Sufu upregulates multiple inflammatory gene expression (IFNγ, IL-6, MCP-1 and S100A4) in iSMCs. In vivo, transfection of miR-214-3p into the injured vessels resulted in the decreased expression level of Sufu, reduced iSMC generation and inhibited neointimal hyperplasia. Importantly, perivascular transplantation of AdSPCs increased neointimal hyperplasia, whereas transplantation of AdSPCs over-expressing miR-214-3p prevented this. Finally, decreased expression of miR-214-3p but increased expression of Sufu was observed in diseased human arteries. We present a previously unexplored role for miR-214-3p in iSMC differentiation and neointima iSMC hyperplasia and provide new insights into the therapeutic effects of miR-214-3p in vascular disease.

中文翻译：

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miR-214-3p-Sufu-GLI1是控制干细胞和新内膜增生性炎症平滑肌细胞分化的新型调节轴

由外膜干/祖细胞（AdSPC）产生的炎性平滑肌细胞（iSMC）被公认为是心血管疾病的新参与者，microRNA-214-3p（miR-214-3p）与成熟的血管SMC功能有关。新内膜增生。在这里，我们试图阐明miR-214-3p在从AdSPCs分化为iSMC的功能中的作用，并阐明在adSPCs中miR-214-3p信号转导对损伤引起的内膜增生的治疗潜力。通过多种生化分析评估了miR-214-3p在iSMC从AdSPC分化中的作用。通过结合位点突变和报道分子活性分析，确定了miR-214-3p的靶标。小鼠损伤诱导的动脉重塑和干细胞移植的模型进行了研究，以研究miR-214-3p的治疗潜力。进行RT-qPCR分析以检查在健康和患病的人动脉中的基因表达。miR-214-3p通过恢复与声音刺猬神经胶质瘤相关的癌基因1（Shh-GLI1）信号传导，阻止了iSMC从AdSPC分化/生成。在从AdSPC生成iSMC的过程中，融合抑制基因（Sufu）被确定为miR-214-3p的功能靶标。机理研究表明，miR-214-3p的过表达或Sufu抑制可促进AdSPC中GLI1蛋白的核积累，以及平滑肌α-肌动蛋白（SMαA）和血清反应因子（SRF）中GLI1结合的共有序列（GACCACCCA） ）基因启动子是miR-214-3p和Sufu各自调节所必需的。此外，Sufu上调了iSMC中的多种炎症基因表达（IFNγ，IL-6，MCP-1和S100A4）。体内，将miR-214-3p转染到受伤的血管中会导致Sufu表达水平降低，iSMC生成减少并抑制新内膜增生。重要的是，AdSPC的血管周围移植可增加新内膜增生，而过度表达miR-214-3p的AdSPC的移植可防止这种情况。最后，在患病的人动脉中观察到miR-214-3p的表达降低但Sufu的表达升高。我们提出了miR-214-3p在iSMC分化和新内膜iSMC增生中未曾探索的作用，并为miR-214-3p在血管疾病中的治疗作用提供了新见解。AdSPC的血管周移植增加了新内膜增生，而过度表达miR-214-3p的AdSPC移植阻止了这种情况。最后，在患病的人动脉中观察到miR-214-3p的表达降低但Sufu的表达升高。我们提出了miR-214-3p在iSMC分化和新内膜iSMC增生中未曾探索的作用，并为miR-214-3p在血管疾病中的治疗作用提供了新见解。AdSPC的血管周移植增加了新内膜增生，而过度表达miR-214-3p的AdSPC移植阻止了这种情况。最后，在患病的人动脉中观察到miR-214-3p的表达降低但Sufu的表达升高。我们提出了miR-214-3p在iSMC分化和新内膜iSMC增生中未曾探索的作用，并为miR-214-3p在血管疾病中的治疗作用提供了新见解。