The Role Of MicroRNA In Macrophage Foam Cell-derived Exosomes On Proliferation Of Vascular Smooth Muscle Cells

Atherosclerosis(As)is the pathogenesis of cardiovascular diseases,seriously endangers human health.In the process of As,the formation of macrophage foam cells plays a key role and the abnormal proliferation and migration of vascular smooth muscle cells(VSMCs)are the important pathological basisExosomes are a class of membranous vesicles from 30 to 100 nm that released from living cells,it carries a variety of substances,including protein,mRNA and miRNA,through the functional molecules,regulating the target cell gene regulatory network,extensive participation in intercellular communication.MicroRNA(miRNA)is a kind of non-coding RNA with a regulated function in eukaryotes.Its size is about 23 nucleotides and is highly conserved.Typically it binds to the 3'untranslated region(3'UTR)of the target gene,degrading or inhibiting the expression of the target mRNA.In the formation process of As,macrophage foam cell formation and the abnormal proliferation and migration of VSMCs play an important role.We explore whether macrophage foam cells and VSMCs can communicate with exosomes,so as to explore the new mechanism of proliferation and migration of VSMCs.We first isolated and identified exosomes by differential centrifugation,and tracked whether the VSMCs could uptake the macrophage foam cell exosomes by PKH67 tracer.MiRNA microarray was used to detect miRNAs differentially expressed in exosomes of macrophage foam cells.Overexpression or inhibition miRNA stable VSMCs lines were built by lentivirus vectors,and then explored the effects of miRNA on the proliferation and migration of VSMCs.The target gene of miRNA was predicted and validated by bioinformatics and dual luciferase reporter gene methods.The results are as follows:1.The morphology of the exosomes was detected by transmission electron microscopy(TEM),and the diameter of the samples was between 30~100 nm,showing the round or cup-like shape.Detection of exosomes labeled protein CD9,CD63,CD81,was positive expression.MiRNA microarray screened a total of 188 miRNAs associated with macrophage foam cells(up or down more than 2 times).Compared with the macrophage exosomes,macrophage foam cell exosomes can significantly inhibit the proliferation of VSMCs.2.The negative control(NC),LV-miR-483 and Anti-miR-483-3p stable VSMCs lines were built by lentivirus infection and screened by 2 ?g/ml puromycin.Compared with NC group VSMCs,the expression of miR-483-3p in LV-miR-483 was increased by about 48 times,indicating that we successfully constructed stable VSMCs lines.MTT assay and EdU method were used to analyze the proliferation of VSMCs,compared with the NC group,the proliferative ability of VSMCs in LV-miR-483 group was inhibited,and the proliferation ability of VSMCs in Anti-mi R-483-3p group was significantly promoted.Proliferating Cell Nuclear Antigen(PCNA)protein levels showed the same trend.Scratch assay showed that overexpression of miR-483-3p significantly inhibited the migration of VSMCs,whereas the migration ability of VSMCs in Anti-miR-483-3p group was significantly inhibited.It indicates that miR-483-3p can inhibits the proliferation and migration of VSMCs.3.The miR-483-3p potential target genes OGT and XPO1 which related to cell proliferation and migration predicted by TargetScanHuman and miRDB bioinformatics software.Dual luciferase reporter assay showed a significant decrease in luciferase activity in the co-transfected group of miR-483-3p mimics with luciferase-OGT 3'-UTR expression vectors.The level of OGT protein in miR-483-3p overexpression group was significantly down-regulated.These results suggest that miR-483-3p in macrophage foam cell derived exosomes inhibits the proliferation and migration of VSMCs by targeting OGT,which provides an important theoretical basis for the pathogenesis of cardiovascular disease.