The Molecular Mechanism By Which MiR-33a-5p Promotes Endothelial Cell Senescence

Vessels run through various tissues and organs across the body and have the function of transportation and homeostasis.The basic structure of blood vessels consists of intima,media,and adventitia,which are composed of endothelial cells,smooth muscle cells,fibroblasts,and connective tissues.Vascular endothelial cells not only regulate active blood substances,but also play an important role in regulating vascular smooth muscle cells.Therefore,the dysfunction of vascular endothelial cells play an important role in the regulation and maintenance of vascular function.There is a lot of evidence that endothelial cell senescence is closely related to various cardiovascular and cerebrovascular diseases(hypertension,coronary heart disease,stroke,etc.),however the mechanism of endothelial cell senescence is not clear.micro RNA is a kind of epigenetic regulation that upon binding to the 3'-UTR region of m RNA can reduce the efficiency of protein translation.Some studies have shown that micro RNA is closely related to the senescence of vascular endothelial cells.We found that micro RNA-33a(hsa-mi R-33a-5p)increased with age in the blood vessels.Later,it was reported that hsa-mi R-33a-5p could promote the pathogenesis of vascular atherosclerosis.However,whether hsa-mi R-33a-5p contributes to atherosclerosis by promoting the senescence of vascular endothelial cells has not been reported.In this study,we used human umbilical vein endothelial cells as a model to elucidate the effects of micro RNA-33 a and its mechanism in endothelial cell senescence.The research contents include the following aspects:1.Overexpression of hsa-mi R-33a-5p resulted in an increase in the percentage of SA-beta-gal positive cells(4-folds)and a decrease in cell proliferation detected through colony formation assay(the percentage of proliferation cells was decreased 0.62-folds).Flow cytometry analysis showed that cells were arrested in G2/M phase.These results suggest that hsa-mi R-33a-5p can promote endothelial cell senescence.2.In order to elucidate the mechanism of endothelial cell senescence induced by hsa-mi R-33a-5p,we screened three RNAseq datasets of human umbilical vein endothelial cell senescence(replicative senescence,DOX-,and MMC-induced senescence)through bioinformatics predictive tools such as mi RWalk(predicted target module;minimum seed sequence length is 7,P < 0.05),combined with the characteristics of micro RNA "no change or decreased expression of target gene m RNA level" in RNAseq datasets.Nine potential target genes of hsa-mi R-33a-5p were identified.3.In order to confirm that these predicted target genes are regulated by hsa-mi R-33a-5p,we constructed the binding sequences of these nine target genes in the plasmids of double luciferase reporter gene.It was found that the expression of ABCA1,BTBD2,and ARID5 B was significantly reduced by hsa-mi R-33a-5p.If the binding sites of hsa-mi R-33a-5p on these sequences were mutated,the luciferase activities of ABCA1,BTBD2,and ARID5 B were no longer affected by hsa-mi R-33a-5p.These results suggest that hsa-mi R-33a-5p inhibits the expression of ABCA1,BTBD2 and ARID5 B through specific binding with target genes.4.To confirm which of the hsa-mi R-33a-5p target gene promotes endothelial cell senescence,we knocked down ABCA1,BTBD2,and ARID5 B in endothelial cells,and found that knocking down BTBD2 gene can significantly inhibit cell proliferation and promote endothelial cell senescence,while there was no significant difference between ABCA1 and ARID5 B knocked down groups and control group.Based on the above research,for the first time,we found that hsa-mi R-33a-5p can induce endothelial cell senescence,and identified ABCA1,BTBD2,and ARID5 B as its downstream target genes.We demonstrated for the first time that BTBD2 can promote endothelial cell senescence,confirming that hsa-mi R-33a-5p promotes endothelial cell senescence by regulating the expression of its target gene BTBD2.This study provides a basis for understanding the epigenetic regulation in endothelial cell senescence,and provides a new intervention target for preventing the pathogenesis and development of cardiovascular and cerebrovascular diseases in the elderly through endothelial cell senescence.