MiR-1290 Induces EndMT And Promotes The Vascular Restenosis After Angioplasty By Targeting FGF2

ObjectiveRestenosis is a serious long-term adverse prognosis of endovascular treatment of lower extremity arterial occlusive disease(LEAOD),and the development of stent implantation is severely restricted by the terrible incidence rate of in-stent restenosis(ISR).Endothelial-tomesenchymal transition(End MT)is an important reason for restenosis but the specific mechanisms need to be further explored.Therefore,the purpose of this study is to screen significantly different micro RNAs(mi RNAs)through the whole transcriptome sequencing of the arterial segments of ISR patients and healthy volunteers,and explore its functions and downstream pathways both in vivo and in vitro.MethodFirstly,we collected 16 human artery samples from amputation surgery in clinic,including 8 ISR samples and 8 healthy blood vessel samples,then extracted tissue RNA,and screened out mi RNAs with significant differential expression in human tissues through whole transcriptome sequencing and quantitative real-time PCR(q RT-PCR),and identified mi R-1290 as the main research object of this topic.We explored the correlation between mi R-1290 and End MT in human umbilical vein endothelial cells(HUVECs)using techniques such as q RT-PCR,Western Blot(WB),and Pearson correlation analysis.Through further functional gain and loss experiments,we determined the regulatory effect of mi R-1290 on End MT at the cellular level.Subsequently,we screened and identified the direct downstream target fibroblast growth factor 2(FGF2)of mi R-1290 by bioinformatics analysis,RNA pull-down,double Luciferase reporter gene and other experiments,and verified its role in mi R-1290 regulating End MT pathway by further detecting the expression of End MT markers after co-transfection of mi R-1290 knockdown or overexpression and siFGF2.Finally,We constructed an animal model of carotid artery balloon injury in rats and injected antagomi R-1290 into the tail vein of the treatment group rats to verify whether mi R-1290 knockdown have an inhibitory effect on injury induced intimal hyperplasia.In addition,we further validated the cell function and downstream pathway results obtained in the HUVECs model at the animal level.ResultWe screened 129 differentially expressed mi RNAs(DE mi RNAs)in amputated and healthy arteries of ISR patients through RNA sequencing.Among them,mi R-1290 levels were significantly higher in arteries with ISR than in healthy arteries,and there was a correlation of expression between End MT markers and mi R-1290 in endothelial cells(ECs).As expected,further knockdown and overexpression experiments showed that the silencing of mi R-1290 prevented the transformation of growth factors-β 1(TGF-β 1)induced morphological changes of HUVECs towards the mesenchymal phenotype,also prevent the loss of endothelial marker CDH5 and the obtaining of interstitial markers(α-SMA and CDH2).On the contrary,overexpression of mi R-1290 can promote the expression of CDH5 while inhibite the expression changes of α-SMA and CDH2 and the transformation of cell morphology of HUVECs from oval to long spindle.In addition,we identified the direct downstream target FGF2 of mi R-1290 and demonstrated its involvement in the regulation of End MT by mi R-1290.Finally,we utilized a rat carotid artery injury model and found that mi R-1290 significantly increased after carotid artery injury,and the tail vein injection therapy of antagomi R-1290 demonstrated that knocking down mi R-1290 can alleviate End MT and alleviate the progression of restenosis in vivo.Conclusion and significanceOur data demonstrate the strong regulatory effect of mi R-1290 on End MT in vitro and in vivo,as well as the therapeutic effect in rats of mi R-1290 knock-down on endometrial hyperplasia after injury,which may serve as a biomarker source and therapeutic target for stent implantation in future LEAOD patients.