目的:内皮-间质转化(Enditional-to-间质转化)是再狭窄的重要原因,但其机制有待进一步探讨。因此,本研究的目的是筛选显著不同的microRNAs(miRNAs)并评估其功能和下游通路。
方法:本研究使用全转录组重测序和实时定量逆转录PCR(qRT-PCR)筛选了在来自再狭窄患者和健康志愿者的人动脉段之间具有显著差异的几种miRNA。我们使用Westernblot探索miR-1290和EndMT之间的相关性,qRT-PCR,皮尔逊相关分析和进一步的功能得失实验。随后,我们通过生物信息学分析确定了miR-1290的直接下游靶标,RNA下拉,双荧光素酶报告基因等功能实验。最后,大鼠颈动脉球囊损伤模型证明了miR-1290调节因子的治疗潜力。
结果:我们筛选了129个差异表达的miRNA。其中,miR-1290水平在再狭窄动脉中显著高于健康动脉,正如预期的那样,EndMT在miR-1290过表达时功能增强,当miR-1290敲低时相对减弱。此外,成纤维细胞生长因子-2(FGF2)被确立为miR-1290的下游靶标。最后,我们利用动物模型,发现低miR-1290水平可以缓解EndMT和再狭窄的进展.
结论:我们的研究证明了miR-1290对EndMT的强调节作用,子宫内膜增生和再狭窄,可作为下肢动脉闭塞性疾病患者支架植入术的生物标志物和治疗靶点。
OBJECTIVE: Endothelial-to-mesenchymal transition (EndMT) is an important reason for restenosis but the underlying mechanisms need to be further explored. Therefore, the purpose of this study is to screen significantly different microRNAs (miRNAs) and assess their functions and downstream pathways.
METHODS: This study screened several miRNAs with significant differences between human arterial segments from restenosis patients and healthy volunteers using whole transcriptome resequencing and real-time quantitative reverse transcription PCR (qRT-PCR). We explored the correlation between miR-1290 and EndMT using Western blot, qRT-PCR, Pearson correlation analysis and further functional gain and loss experiments. Subsequently, we identified the direct downstream target of miR-1290 by bioinformatics analysis, RNA pull-down, double Luciferase reporter gene and other functional experiments. Finally, rat carotid artery balloon injury model demonstrated the therapeutic potential of miR-1290 regulator.
RESULTS: We screened 129 differentially expressed miRNAs. Among them, miR-1290 levels were significantly higher in restenosis arteries than in healthy arteries, and as expected, EndMT was functionally enhanced with miR-1290 overexpression and comparatively weakened when miR-1290 was knocked down. In addition, fibroblast growth factor-2 (FGF2) was established as the downstream target of miR-1290. Finally, we utilized an animal model and found that low miR-1290 levels could alleviate EndMT and the progression of restenosis.
CONCLUSIONS: Our study demonstrated the strong regulatory effects of miR-1290 on EndMT, endometrial hyperplasia and restenosis, which could be useful as biomarker and therapeutic target for stent implantation in patients with arterial occlusive disease of the lower extremities.