Study Of Circulating MiR-26a-5p On Severe Coronary Artery Disease Diagnosis And Its Regulatory Mechanisms

Coronary artery disease(CAD)is responsible for worldwide deaths,accounting for about 16%deaths per year followed by strokes(about 11%).In China,about 15%deaths are caused by CAD,leading to the second largest cause of death,followed by stroke(21%).In 2017,approximately 7%patients with CAD received percutaneous coronary intervention(PCI).Although the risk factors of CAD has been studied quite thoroughly,the available detections are not enough to identify patients with CAD who are in the pre-myocardial infarction(MI)event.For example,some patients with normal or non-specific ECG need PCI when they are diagnosed with CAD.In a large number of patients with suspected CAD,it is clearly impractical to perform coronary angiography in all of them.The formation of atherosclerosis(AS)is an underlying pathophysiological mechanism in the development of coronary heart disease,peripheral arterial disease(PAD),cerebrovascular(CVD)and carotid artery disease.MicroRNAs(miRNAs)are non-coding RNA molecules of approximately 22 nucleotides in length that play key roles in a variety of biological processes by negatively regulating relevant target genes at the post-transcriptional level,and thus play a key role in a variety of biological processes.In fact,miRNA dysregulation is associated with the development and progression of many human diseases such as cancer and cardiovascular diseases.miRNAs can be stably present in extracellular spaces such as urine,blood,while those are stable in various body fluids,such as serum and plasma,are called circulating miRNAs.Recently,many studies have shown that circulating miRNAs are potential diagnostic and predictive biomarkers.At present,there are many studies on circulating miRNAs in myocardial infarction(MI),but there is no report on those in non-MI patients with severe coronary heart disease who need to be treated with PCI.Objective:This study aims to identify circulating miRNAs in non-MI patients with severe CAD who need to be treated with PCI,evaluate their diagnostic values,and investigate futher molecular mechanisms of screened circulating miRNAs.Methods:All patients with severe CAD were those in whom at least one main coronary artery was>70%stenosed confirmed by coronary angiography,and received primary PCI.Patients had no other heart disease such as MI and other major malignant diseases.Controls were subjects with normal coronary angiography and without atherosclerotic vascular disease.A series of molecular cell experiments were performed to clarify its related molecular mechanism.1.Analysis of circulating miRNA expression profile in patients with severe CAD.The miRNA microarray was used to identify circulating miRNA expression profiles of severe CAD.Bioinformatics analyses were performed to screen the differentially expressed miRNAs between controls and severe CAD.Target genes of differentially expressed miRNAs were predicted,which were subjected to GO and KEGG analysis.2.Assessing the diagnostic performance of circulating miRNAs in severe CAD.The differentially expressed miRNAs identified by miRNA microarray were further validated by RT-qPCR.And the differentially expressed miRNAs were further identified in 2 independent groups of samples.The plasma expression levels of differentially expressed miRNAs in patients with severe CAD were analyzed.The potential of the screened miRNAs as novel potential markers for CAD was evaluated by the receiver operating characteristic(ROC)curve.3.Exploring the molecular mechanism of hsa-miR-26a-5p in regulating atherosclerotic endothelial dysfunctionWe selected hsa-miR-26a-5p with the highest AUC to further explore its molecular mechanism in atherosclerosis.OxLDL was used to induce oxidative stress in human umbilical vein endothelial cells HUVECs,and RT-qPCR was used to identify the effect of oxLDL stimulation of endothelial cells on hsa-miR-26a-5p levels,which was used to construct an in vitro atherosclerosis cell model.A series of functional assays were performed on the model,including flow cytometry to detect apoptosis,CCK-8 assay and Matrigeltubing assay to detect cell viability and angiogenesis,respectively,to analyze the effect of inhibiting hsa-miR-26a-5p expression on endothelial dysfunction.The functional role of hsa-miR-26a-5p on endothelial inflammation was determined by stimulating endothelial cells using the pro-inflammatory factors TNF-α or IL-1β,and measuring the protein expression levels of VCAM-1 and ELAM-1 in endothelial cells using western blot.The target genes of hsa-miR-26a-5p were predicted by TargetScan,combined with dual luciferase reporter gene experiments and western blot to determine whether this targetregulatory relationship holds.A rescue experimental subgroup was set up to test the role of the hsa-miR-26a-5p/ITPR1 axis on endothelial dysfunction by flow cytometry,CCK-8,and adult tube experiments.Further,the collected CdM binding was used to detect whether ITPR1 partially acts on paracrine mechanisms by tube formation experiments.The downstream signaling pathway of ITPR1,Apelin/APJ,was obtained using GeneCards,and the regulatory relationship between ITPR1 and Apelin/APJ signaling pathway was examined using western blot,while CCK-8,scratch healing assay,and flow cytometry were used to explore the role of ITPR1/Apelin/APJ in endothelial dysfunction.4.Exploring the molecular mechanism of has-miR-26a-5p targeting ITPR1 to regulate atherosclerotic foam cell formationNext,the role of hsa-miR-26a-5p in the foam cells formation of atherosclerotic was further analyzed.The foam cells model was established by treating human monocyte THP1 cell line with oxLDL in vitro.The hsa-miR-26a-5p levels in oxLDL-stimulated THP-1 was detected by RT-qPCR.The foam cell formation and intracellular lipid accumulation were determined by oil red O staining and TC/FC kit,and the levels of miR-26a-5p target gene ITPR1 and Apelin/APJ pathway protein expression in cells by western blot.The molecular mechanism of hsa-miR-26a-5p in atherosclerotic foam cell formation by targeting ITPR1 through Apelin/APJ signaling pathway was clarified.5.Exploring the effect of miR-26a-5p on atherosclerosis in ApoE-/-miceAnimal experiments were performed to validate the role of miR-26a-5p in atherosclerosis.In this study,ApoE-/-mice were induced by high-fat diet as atherosclerosis model,and miR-26a-5p agomir and miR-26a-5p antagomir were injected by tail vein.After mice were sacrificed,eye blood and aorta were collected.The aortic plaque,lipid and lipoprotein content in serum were observed by macroscopic oil red O staining,ELISA,and miR-26a-5p target gene ITPR1 expression was detected by Western blot.The aim of this study was to investigate the expression of miR-26a-5p in mice and the effect on the development of atherosclerosis mice.Results:1.MiRNA microarray results showed that there were 9 differentially expressed circulating miRNAs(i.e.ebv-miR-BART12,ebv-miR-BART16,let-7i-5p,miR-130a-3p,miR-26a-5p,miR-3149,miR-3152-3p,miR-32-3p,and miR-149-3p)between severe CAD and controls.Combined with the RT-qPCR results,4 miRNAs(hsa-let-7i-5p,hsa-miR-323p,hsa-miR-3149,and hsa-miR-26a-5p)were found to be consistent with the trend of miRNA microarray results.Therefore,4 miRNAs were further analyzed in 2 independent cohorts.The circulating expression levels of hsa-miR-32-3p,hsa-miR-3149,and hsa-miR26a-5p of patients with severe CAD were significantly higher than those of controls in experimental cohort,validation cohort,and combined cohort(P<0.05).The circulating expression levels of hsa-let-7i-5p of patients with severe CAD in experimental cohort and combined cohort were significantly upregulated(P<0.05).Although the circulating expression levels of hsa-let-7i-5p of patients with severe CAD in validation cohort were higher than those of controls,but the difference was not significant(P=0.053).2.The ROC curve analysis revealed that the AUC values of hsa-let-7i-5p,hsa-miR-323p,hsa-miR-3149 and hsa-miR-26a-5p in experimental cohort were 0.634,0.745,0.795,and 0.818,respectively,while those of hsa-let-7i-5p,hsa-miR-32-3p,hsa-miR-3149 and hsamiR-26a-5p in validation cohort were 0.611,0.638,0.827,and 0.849,respectively.The diagnostic performance of these 4 miRNA panels was further evaluated,and the results of 3-miRNA(hsa-miR-32-3p,hsa-miR-3149,and hsa-miR-26a-5p)and 4-miRNA(hsa-let-7i5p,hsa-miR-32-3p,hsa-miR-3149 and hsa-miR-26a-5p)all had better diagnostic performancehad than any individual miRNA(P<0.05),while the difference with hsa-miR26a-5p,which had the highest individual miRNA AUC,did not reach statistical significance(P>0.05).The AUC of the 2-miRNA(hsa-miR-32-3p and hsa-miR-3149)was lower than that of hsa-miR-26a-5p.The results of the validation group showed that hsa-let-7i-5p was not analyzed for miRNA panels for its non-significant difference in plasma expression levels between the two groups.The AUC of both 2-miRNA(hsa-miR-32-3p and hsa-miR-3149)and 3-miRNA(hsa-miR-32-3p,hsa-miR-3149 and hsa-miR-26a-5p)was 0.940,with significantly higher diagnostic performance than individual miRNAs(P<0.01).Based on the findings,we selected hsa-miR-26a-5p with the highest AUC to further explore its molecular mechanism in atherosclerosis.3.RT-qPCR assay showed upregulated expression of hsa-miR-26a-5p in oxLDL-induced endothelial HUVECs.The in-vitro functional experiments showed that the inhibition of hsamiR-26a-5p could significantly reverse the effect of oxLDL on cell apoptosis,migration and the tube formation.Furthermore,western blot was carried out to detect the expression of endothelial cell adhesion molecules VCAM-1 and E-selection and found that the knockout of hsa-miR-26a-5p could significantly inhibit endothelial cell inflammation.These results indicated that endothelial dysfunction can be alleviated by inhibiting the expression of hsamiR-26a-5p.ITPR1 was found and validated as the potential target gene of hsa-miR-26a-5p by target prediction.The results of the dual-luciferase reporter gene assay showed that hsamiR-26a-5p could target to inhibit the expression of ITPR1.To explore whether hsa-miR-26a5p affects endothelial cell dysfunction by targeted regulation of ITPR1 expression,a rescue experiment was designed in this study,and overexpression of ITPR1 was found to rescue endothelial dysfunction caused by high expression levels of hsa-miR-26a-5p.A series of study results indicated that hsa-miR-26a-5p can affect endothelial cell dysfunction by targeting ITPR1,thereby playing a critical role in the formation of atherosclerosis.In addition,this study further investigated the role of ITPR1 in promoting endothelial cell network formation,attempting to explore the mechanism of ITPR1-mediated endothelial cell angiogenesis.We found that after the treatment of the CdM generated by transfected oe-ITPR1 cells,the number of endothelial tubules and branch points in HUVECs increased significantly.Combined with Western blot results,we found that the angiogenesis effect of ITPR1 is at least partially mediated by paracrine,and VEGF is not a potential mediator of this mechanism.In addition,Apelin/APJ signal pathway is an important pathway of ITPR1.Up-regulation of ITPR1 significantly increased the expression of pathway-related molecules Apelin-13,while CCK-8,scratch healing and tube formation experiments showed that silent ITPR1 significantly inhibited endothelial cell viability,migration and angiogenesis,while induced Apelin/APJ signal pathway significantly reduced this inhibitory effect.Accordingly,silencing ITPR1 aggravates the apoptotic injury,and on this basis,the apoptotic injury of endothelial cells is significantly reduced after the induction of Apelin/APJ signal pathway.4.The expression of hsa-miR-26a-5p was upregulated in THP-1 treated with oxLDL and PMA.Combined with oil red O staining results,hsa-miR-26a-5p promoted foam cell formation and enhanced lipid accumulation during foam cell formation.In addition,overexpression of hsa-miR-26a-5p target gene ITPR1 inhibited the formation of foam cells and lipid accumulation.In addition,the hsa-miR-26a-5p targeting ITPR1 was found to regulate foam cell formation by rescue experiments,and ITPR1 inhibited foam cell formation through Apelin/APJ.5.Significantly high levels of miR-26a-5p were found in the aortic tissue of AS mice.Hyperlipidemia was worsened in mice injected with miR-26a-5p agomir,and miR-26a-5p promoted hyperlipidemia in AS mice.And oil red O staining also showed that miR-26a-5p promoted plaque formation in AS mice.In addition,western blot also verified that miR-26a5p negatively correlated with the level of ITPR1 in AS mice.Conclusion:1.The findings showed that circulating expression levels of hsa-let-7i-5p,hsa-miR-323p,hsa-miR-3149,and hsa-miR-26a-5p were significantly upregulated in severe CAD patients.The elevated circulating expression levels of hsa-miR-32-3p,hsa-miR-3149,and hsa-miR-26a-5p were significant compared with controls.2.In severe CAD,the highest AUC value for a single miRNA was hsa-miR-26a-5p,suggesting that hsa-miR-26a-5p is potentially a novel non-invasive and important biomarker for screening patients with severe CAD.3-miRNA(hsa-miR-32-3p,hsa-miR-3149,and hsamiR-26a-5p)may have better diagnostic performance than individual miRNAs,and could be analyzed and clarified by continuing studies with large samples for the further research,which is expected to improve the diagnostic efficiency of severe CAD.3.In this study,an in vitro atherosclerotic cell model was successfully established by oxLDL-induced oxidative stress.Down-regulation of hsa-miR-26a-5p expression promoted endothelial cell activity,angiogenic capacity,and reduced apoptotic damage and endothelial inflammation.ITPR1,a potent target gene of hsa-miR-26a-5p,promotes endothelial angiogenesis,at least in part through a paracrine mechanism,and also reverses hsa-miR26a-5p-induced endothelial dysfunction by inducing the Apelin/APJ signaling pathway.4.In this study,oxLDL was applied to establish foam cell model on human monocyte THP-1 cell line in vitro.MiR-26a-5p promoted foam cell formation,intracellular lipid accumulation.And miR-26a-5p was involved in atherosclerotic foam cell formation by targeting ITPR1 through the Apelin/APJ signaling pathway,thus participating in the molecular mechanism of atherosclerotic foam cell formation.5.ApoE-/-mice were induced by a high-fat diet as an atherosclerosis model,and miR26a-5p agomir and miR-26a-5p antagomir were injected via tail vein.MiR-26a-5p promoted hyperlipidemia,atherosclerotic plaque formation in AS mice.