OBJECTIVE: This research aims to explore the possible function and the underlying mechanisms of miR-199a-5p in the context of RS.
METHODS: Primary ASMCs were extracted from the femoral arteries of both healthy individuals and patients with PAD or RS. The expression levels of miR-199a-5p were assessed using both qRT-PCR and in situ hybridization techniques. To examine the impacts of miR-199a-5p, a series of experiments were performed, including flow cytometry, TUNEL assay, EdU assay, CCK8 assay, Transwell assay, and wound closure assay. A rat carotid balloon injury model was employed to elucidate the mechanism through which miR-199a-5p mitigated neointimal hyperplasia.
RESULTS: MiR-199a-5p exhibited downregulation in RS patients and was predominantly expressed within ASMCs. Elevated the expression of miR-199a-5p resulted in an inhibitory effect of proliferation and migration in ASMCs. Immunohistochemistry and a dual-luciferase reporter assay uncovered that RS exhibited elevated expression levels of both HIF-1α and E2F3, and they were identified as target genes regulated by miR-199a-5p. The co-transfection of lentiviruses carrying HIF-1α and E2F3 alongside miR-199a-5p further elucidated their role in the cellular responses mediated by miR-199a-5p. In vivo, the delivery of miR-199a-5p via lentivirus led to the mitigation of neointimal formation following angioplasty, achieved by targeting HIF-1α and E2F3.
CONCLUSIONS: MiR-199a-5p exhibits promise as a prospective therapeutic target for RS since it alleviates the condition by inhibiting the proliferation and migration of ASMCs via its regulation of HIF-1α and E2F3.
目的:本研究旨在探索miR-199a-5p在RS背景下的可能功能和潜在机制。
方法:从健康个体和PAD或RS患者的股动脉中提取原发性ASMC。使用qRT-PCR和原位杂交技术评估miR-199a-5p的表达水平。为了检查miR-199a-5p的影响,进行了一系列实验,包括流式细胞术,TUNEL检测,EdU分析,CCK8测定,Transwell分析,和伤口闭合试验。采用大鼠颈动脉球囊损伤模型来阐明miR-199a-5p减轻新生内膜增生的机制。
结果:MiR-199a-5p在RS患者中表现出下调,主要在ASMC中表达。miR-199a-5p的表达升高导致ASMC中增殖和迁移的抑制作用。免疫组织化学和双荧光素酶报告分析发现,RS表现出HIF-1α和E2F3的表达水平升高,并且它们被鉴定为miR-199a-5p调节的靶基因。携带HIF-1α和E2F3的慢病毒与miR-199a-5p的共转染进一步阐明了它们在miR-199a-5p介导的细胞应答中的作用。在体内,通过慢病毒递送miR-199a-5p导致血管成形术后新内膜形成的缓解,通过靶向HIF-1α和E2F3实现。
结论:MiR-199a-5p有望作为RS的前瞻性治疗靶标,因为它通过调节HIF-1α和E2F3抑制ASMC的增殖和迁移来缓解病情。