Molecular Mechanism And Clinical Value Of Secreted MiR-139 In Atherogenesis Induced By Lipoprotein

Background:Atherosclerotic cardiovascular disease(CVD)has become one of the main causes of threat to human health.Until now,the pathogenesis mechanism of atherosclerosis has not been fully elucidated,and there are also lack of high sensitivity and specific molecular markers.Atherosclerosis(As)is the main pathological basis of CVD.The abnormality of lipid metabolism including the upregulating of oxidized lipoprotein(ox-LDL)acts as important pathogenic factors in As.MicroRNAs(miRNAs)are endogenous noncoding single chain small RNA of approximately 19-22 nucleotides in length,which regulate gene expression at the post transcriptional level.MiRNAs play an important role in As progression,which is induced by lipoprotein.What's more,miRNAs are involved in the occurrence and development of CVD.Recent studies have shown that there are abundant and stably expressed miRNAs in human peripheral blood circulation.The circulatory miRNAs with physiological function can be encapsulated by the exosome secreted by different types of cells and transported to the receptor cells for intercellular communication,and involved in the pathological process of a variety of diseases,including CVD.Therefore,exploring the key secreted miRNAs and its mechanism will provide new and more sensitive and specific biomarkers for early diagnosis of disease,and new therapies can be developed in AsObjectives:(1)To screen the differently expressed miRNAs between macrophages and foam cell models,and to analyze its expression in cells and their culture.(2)To investigate whether the level of miRNA has changed in the serum and exosome of patients with As,in order to provide new ideas and new methods for As diagnosis and treatment of As patients.(3)To explore the mechanism of specific changes in the secreted miRNA in the process of As.Methods:(1)Phorbol-12-myristate-13-acetate(PMA)converted THP-1 cell into macrophages,and then the macrophages was transformed into foam cells through the addition of 100 ?g/ml ox-LDL for 48h.Whether the foam cell model was successfully constructed was identified by oil red O staining.The foam cells and macrophages model were detected by Affymetrix miRNA chip for seeking the differently expressed miRNAs.Real time fluorescent quantitative polynucleotide chain reaction(qRT-PCR)were used to verify the level of miRNA in macrophages and foam cells.Exosomes in the cell culture fluid of both cells were extracted to further detect the expression of the specific miRNA.(2)30 AMI patients and 30 healthy controls were collected respectively,and qRT-PCR technology was used to detect the altered miRNA in serum,exosome and exosome free serum.ROC curve was used to analyze the sensitivity and specificity of the miRNAs.(3)Bioinformatics software was used to predict the target gene,which was also related to As.The targeting effect was verified by luciferase reporter gene experiment and Western Blot.Dil dye labelled exosome was co-cultured with HUVEC and further confirmed to enter cell by laser scanning confocal microscopy.We detected miRNA level in HUVEC after co-culture with exosome Apoptosis,Western Blot and qRT-PCR technology were used to investigate the function of miRNA to HUVEC.The targeting effect relationship was verified by luciferase reporter gene experiment,and Western Blot were used to validate the relation overexpression or suppression of specific expression.Results:(1)As compared with macrophages,the levels of miR-99,miR-139,miR-1973 and miR-6757 were upregulated in foam cells.qRT-PCR results showed that compared with macrophages,the levels of miR-99(P=0.0177)and miR-139(P=0.0240)in foam cells are significantly higher than that in macrophages.There were no significant differences of miR-1973 and miR-6757 between foam cells and macrophages(P>0.05).When compared with macrophages,miR-139 level in exosome secreted by foam cells increased significantly(P=0.0216).(2)Compared with healthy controls,miR-99 in AMI patients serum(P=0.0406),exosome(P=0.0049)and exosome free serum(P=0.047)were increased significantly.miR-139 in serum of AMI patients increased significantly(P=0.0051),while miR-139 in exosome and exosome free serum can not be steadily detected due to low content.The ROC curve results showed that the AUC under the ROC curve of the patients with miR-99 and miR-139 were 0.631(95%CI 0.489-0.773)and 0.650(95%CI 0.511?0.789),respectively,and the sensitivity of them both were 67%.(3)The bioinformatics software predicted that the insulin-like growth factor 1 receptor(IGF-1R)may be the target gene of miR-139.Laser scanning confocal microscopy confirmed that exosome could enter HUVEC successfully.What' more,exosome which was secreted by foam cells could significantly increase the level of miR-139 in HUVEC(P=0.0153).In foam cells,secreted miR-139 could twice the apoptosis of HUVEC,when compared with macrophage group.The results of luciferase reporter assays showed that the activity of firefly luciferase can as low as 1/3(P<0.001),or increased two times(P<0.05)by overexpressing or silencing miR-139 and when the seed sequence was mutated,the activity of firefly luciferase showed no difference between experimental group and control group.We experimentally validated that miR-139 can be combined with the 3 '-UTR of IGF-1R mRNA.Conclusions:ox-LDL induced increased levels of miR-99 and miR-139 in foam cells.At the same time,secreted miR-139 can be used as an intercellular communication molecule to negatively regulate the target protein IGF-1R in HUVEC,reduce its expression and promote the apoptosis of HUVEC.This experiment suggested that miR-139 may be an important molecule to promote the occurrence and development of As.It also provided a new idea for the diagnosis and treatment of As.