| Objective:Vascular endothelial cells(VECs)damage plays an important role in the pathogenesis of atherosclerosis(AS),while appropriate autophagy levels in VECs is atheroprotective.To date,the underlying mechanisms have not been elucidated.MicroRNAs are newly discovered epigenetic factor involved in the regulation of cell proliferation,differentiation and apoptosis by specifically down-regulating the post-transcriptional expression of their target genes.The present study aimed to define the role of microRNA-214-3p(miR-214-3p)in the regulation of endothelial autophagy and elucidate the underlying mechanism.Methods:ApoE knockout(ApoE-/-)mice fed with a high-fat diet for 12 weeks were used to establish an AS model.After sorting CD31+VECs from aortic segment using the flow cytometry,we examined the levels of miR-214-3p and a few autophagy-related genes(ATGs).We analyzed the relationship between miR-214-3p and ATG5 and ATG12 protein levels.Bioinformatics analysis and dual luciferase reports gene assay were performed to confirm the target genes of miR-214-3p.In vitro experiments,human umbilical vein endothelial cells(HUVECs)were transfected with miR-214-3p mimic/inhibitor following the stimulation with 100 μg/mL oxidized low-density lipoprotein(ox-LDL)for 12h.Western blotting was used to detect the expression levels of autophagy-related proteins.Transmission electron microscopy(TEM)was used to determine changes of cytoplasmic autophagosomes.Immunofluorescence assay was used to examine the expression and distribution of cytoplasmic protein microtubule-associated protein 1 light chain 3B-II(LC3B-II).Oil red O staining was used to detect lipid accumulation in the cytoplasm.Adhesion of THP-1 monocyte to HUVECs was performed.CCK-8 assay was used to determine the cell viability.TUNNEL assay was used to measure cellular apoptosis.Caspase-3/cleaved caspase-3 protein levels were also measured.Results:In the AS mouse model,the expression level of miR-214-3p was significantly up-regulated,while the levels of autophagy-related proteins were markedly down-regulated in VECs.Furthermore,the expression levels of miR-214-3p were negatively correlated with ATG5 and ATG12 protein levels(R=0.461,p=0.003;R=0.412,p=0.013).Vitro experiments showed that ox-LDL stimulation induced the autophagy of population doublings(PDL)5 young HUVECs,with the decreased miR-214-3p level.Overexpression of miR-214-3p inhibited ox-LDL-induced autophagy,as indicated by the down-regulation of ATG5,ATG12-ATG5,ATG12 and LC3B-Ⅱprotein levels,and up-regulation of SQSTM1/p62 protein levels.Consistently,the fluorescence intensity of LC3B-Ⅱ protein in the cytoplasm was weakened,with the number of autophagosomes in the cytoplasm markedly decreased.As expected,overexpression of miR-214-3p reduced the cell viability of PDL5 HUVEC and increased apoptosis.In addition,miR-214-3p promoted lipid accumulation in the cytoplasm of VECs and the adhesion of THP-1 cells to VECs.Conversely,in PDL36 senescence HUVECs,ox-LDL stimulation suppressed the autophagy,with the increased miR-214-3p level.Reduced expression of miR-214-3p restored oxLDL-suppressd autophagy,increased cell viability,and decreased apoptosis,reduced lipid accumulation and THP-1 cell adhesion.Conclusion:In a mouse AS model,miR-214-3p expression was significantly increased in atherosclerotic vessles and its levels were negatively correlated with the protein levels of ATG5 and ATG12.In cultured HUVECs,miR-214-3p promotes endothelial cell injury by blocking oxLDL-induced autophagy possibly through an ATG5-dependent mechanism.Our findings may provide a potential new target for treating endothelial injury in AS. |
PDF Full Text Request