Molecular Mechanism Of MiR-375 Promoting Endothelial Cell Senescence

Vascular ageing is an independent risk factor for cardiovascular diseases,such as hypertension,coronary artery disease,and stroke,which are common but life threatening.Vascular endothelial cell senescence is the cellular basis of vascular ageing,and studies have shown that epigenetics,especially micro RNA,plays an important role in vascular endothelial cell senescence.In previous studies,miR-375 was found to be highly expressed in various cellular senescence models.This suggest miR-375 may be a new potential target for regulating endothelial cell senescence.To study the role and mechanism of miR-375 in endothelial cell senescence.First,Human Umbilical Vein Endothelial Cells(HUVECs)were used to construct a cell model.We found that overexpression of miR-375 increased positive SA-?-gal staining by 60%,and significantly decreased the proliferation rate of endothelial cell,indicating that miR-375 promotes endothelial cell senescence.Next,to find the target gene of miR-375,we combined bioinformatics prediction tools and RNAseq database of cellular senescence model to initially identify 12 candidate target genes.Then cloned a 300-500 bp fragment that contained the potential binding site of miR-375.8 genes were obtained by dual-luciferase reporter assay.To further identify whether miR-375 regulate the target gene via the binding site.We mutate the binding sites and 4 genes,YAP1,STX6,PAFAH1B1,RIDA,are found with restored luciferase activity,indicating that miR-375 specifically binding with these four genes,of which STX6,PAFAH1B1 and RIDA were discovered for the first time.Finally,si RNA was used to knockdown STX6,PAFAH1B1 and RIDA expression and we found that only STX6 and RIDA significantly increased SA-?-gal staining and decreased the proliferation rate of endothelial cells.At the same time,western-blot results showed that overexpression of miR-375 significantly inhibited the expression of STX6 and RIDA proteins.These results indicate that STX6 and RIDA are target genes of miR-375 associated with cell senescence.In summary,our study found a novel micro RNA,miR-375,that promotes endothelial cell senescence,and identified YAP1,STX6 and RIDA are the target genes of miR-375 associated with cell senescence,indicating that miR-375 may promote endothelial cell senescence by negatively regulating multiple target genes.Our study provides a new perspective for in-depth understanding of vascular ageing and the development of ageing-related diseases.