| BackgroundAtherosclerotic cardiovascular disease is the first cause of death in all regions of the world.In most countries with low medical levels,the dangers of atherosclerosis have surpassed infectious diseases.Atherosclerosis can lead to a variety of clinical manifestations,included transient ischemic attack(TIA),aortoiliac occlusion and coronary heart disease(CHD),etc.Although its clinical symptoms are diverse,the disease itself has general commonality.They all represent the deposition of a large number of cholesteryl esters in the arterial wall and the formation of complex advanced plaques,and it finally leads to ischemic changes in the corresponding blood supply area of the arteries.Ninety percent of carotid stenosis is caused by atherosclerosis.It can lead to severe symptoms of cerebral ischemia,such as amaurosis,blurred vision,hemiplegia,etc.which makes patients’ life seriously restricted and causes serious social and economic problem.The rate of disability and mortality are very high.Carotid stenosis is often insidious,it was found after cerebral ischemia appear.It is generally believed that the symptoms of cerebral ischemia is caused by two main reason.One is plaque or thrombus fall off easily form embolus leading to cerebral ischemia,the other one is arterial stenosis causes the hypoperfusion of the distal brain tissue.In recent years,studies have shown that the main reason of ischemic stroke is the plaque falling off,while hypoperfusion caused less stroke.Therefore,it is of great significance to study the stability of carotid artery plaque and to explore the early prediction of biomarkers of unstable plaque and the mechanism of action.It can effectively reduce the occurrence of adverse events such as stroke.lncRNAs are defined as transcripts that are longer than 200 nt and do not code for proteins.More and more evidences show that lncRNA play an important role in vascular diseases.It regulates the function of vascular wall,activates macrophages,regulates lipid metabolism and immune inflammation in the development and progression of atherosclerosis.However,there is no clear report on the role and mechanism of lncRNA in the stability of carotid artery plaque.ObjectiveIn the early stage,the lncRNA and mRNA expression profiles between stable plaque group and unstable plaque group were screened by gene chip in our centre.The difference of lncASAP2-7:1 expression is high.Next we will verify the differential expression level of lncASAP2-7:1 between stable and unstable plaque,and detect the correlation between lncASAP2-7:1 and the thickness of fibrous cap and other plaque composition.Using bioinformatics analysis to predict the biological function of lncASAP2-7:1.We further verified the role of lncASAP2-7:1 in regulating the function of vascular smooth muscle cells and the possible mechanism through cell experiments,and then elaborated its role in regulating plaque stability.Methods(1)Clinical sample studies: Carotid atherosclerotic plaque specimens from patients undergoing carotid endarterectomy in Changhai Hospital Affiliated to the Naval Military Medical University from August 2015 to December 2016 were collected.The plaque of the carotid artery were cut into a small segment.One part of the plaque is stored in liquid nitrogen for extracting RNA and validating lncASAP2-7:1 differential expression level in gene chip by real time PCR.The other part of the plaque were stored in the formalin solution and used for paraffin sections,HE staining,scanning electron microscopy to determine the plaque stability and measure the fibrous cap thickness and lipid core area etc.Finally,the correlation of lncASAP2-7:1 expression level and plaque composition was statistically analyzed.Combined with the results of gene chip,the biological functions of lncASAP2-7:1 were analyzed by bioinformatics.The hospital ethics review committee reviewed and approved the study,and all the patients informed consent.(2)Cell experimental study: lncASAP2-7:1 overexpressed and interfered lentivirus were packaged,and then transfected human aortic smooth muscle cells,which were divided into lncASAP2-7:1 up regulation group,up regulated control group,down regulation group,down regulated control group and blank control group.Cell scratch test was used to detect the effect of lncASAP2-7:1 on the migration of vascular smooth muscle cells.The content of type I collagen were detected by enzyme-linked immunosorbent assay,which reflected the function of lncASAP2-7:1 on protein secretion of vascular smooth muscle cells.Flow cytometry was used to detect the effect of lncASAP2-7:1 on the apoptosis of smooth muscle cells.The effect of lncASAP2-7:1 on the proliferation of smooth muscle cells was detected by cck8 and flow cytometry.Results(1)Clinical sample studies: From August 2015 to December 2016,22 carotid atherosclerotic plaques in Vascular surgery of Changhai Hospital were collected,including 11 unstable plaques and 11 stable plaques.The relative expression level of lncASAP2-7:1 was 6.679±5.883 in patients with unstable plaque,while it was 1.192±0.583 in the stable plaque patients.Real time PCR results confirmed that expression level of lncASAP2-7:1 in unstable plaque group was significantly higher than the stable plaque group(p <0.0001).The fibrous cap thickness of unstable plaque is 120.80±31.29 μm,and the thickness of stable plaque is 163.51±34.34μm.The lipid core area of unstable plaque is 33.45%±9.76%,and the area of stable plaque is 27.84%±7.38%.The thickness of fibrous cap in stable plaque was significantly higher than that of unstable plaque(p<0.01),and there was no significant statistical significance in the lipid core area of two groups.The relative expression of lncASAP2-7:1 in carotid plaque is negative correlated to the thickness of fibrous cap(r=-0.667,p<0.01),and has no significant correlation with the area of lipid core.(r=0.340,p>0.05).Bioinformatics analysis suggests that lncASAP2-7:1 may be involved in biological processes,such as cell migration,secretion and protein transport.(2)Cell experimental study: We interfered human aortic smooth muscle cells with the lncASAP2-7:1 overexpressed and interfered lentivirus,and verified the migration,secretion,proliferation and apoptosis of vascular smooth muscle cells,and found that lncASAP2-7:1 can inhibit the migration of vascular smooth muscle cells and the synthesis of type I collagen.No obvious effect on cell proliferation and apoptosis.ConclusionThe expression level of lncASAP2-7:1 in unstable plaque group was significantly higher than the stable plaque group and negative correlated to the thickness of fibrous cap.Cell experiments confirm that lncASAP2-7:1 overexpression inhibits migration of vascular smooth muscle cells and secretion of type I collagen,and further affects the thickness of fibrous cap,resulting in plaque instability,leading to stroke and other adverse events. |
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