Long Noncoding RNA MIAT Regulated Cell Migration Via Sponging Of MiR-326 In VSMCs

BackgroundCoronary heart disease（CHD）,the leading cause for cardiovascular related death,is inseparable with the progression of atherosclerosis（AS）.The pathological features of AS is primarily due to the deposition of fibrin and lipids in arteries.Vessel smooth muscle cells（VSMCs）from media abnormally migration to intima,which result in thickness of intima,is the initial reason for AS.Long noncoding RNAs（lnc RNA）,transcribed by RNA polymerase II,belong to a transcript with length of less than 200 nt without capacity of coding protein.Lnc RNA are closely connected to aberrant expression of atherosclerosis and several disorders and development of atherosclerosis.Myocardial infarction associated transcript（MIAT）are located on hμMan chromosome 22q12.1,containing five exon.MIAT are aberrantly expressed in amount of diseases including atherosclerosis and shared extensive participatory among muscle hypertrophy,heart failure,acute myocardial infarction and diabetic cardiomyopathy.There remain no reports about that whether MIAT could participate migration of vascular smooth muscle,and relevant mechanisms remain to be elusive.With length of about 22-25 nt,micro RNA（mi RNA）was identified to possess no protein-coding capacity.mi RNA share broad participatory in pathophysiological processes in inflammation,tu Mor,vascular disease,neurodegenerative disease and so on.mi R-326 are extensively expressed in various tμMor diseases,and are involved in tμMor cell proliferation,migration,invasion and similar biology activities.Studies with respect to mi R-326 participating VSMCs migration and functions on atherosclerosis are still poor.Based on above study background,we are interested in discovering the role of MIAT/mi R-326 axis in atherosclerosis and figuring out its regulatory mechanisms.Our research aims at providing a new therapeutic target and experiment basis for studying coronary atherosclerotic heart disease in molecularr level.ObjectiveThe current research aimed to explore the expression of MIAT in atherosclerosis tissue and cell models.Meanwhile,the present research aimed to uncover the effect of MIAT on VSMCs cells migration.And the current study aimed to confirm whether the facilitative effect of MIAT working on VSMCs cell migration was achieved through mi R-326 sponging.Materials and methods46 paired plasma samples in patients with atherosclerosis and in patients without atherosclerosis were clinically collected.The expression of MIAT and mi R-326 in the plasma samples was detected by a RT-q PCR assay.And the correlation between MIAT and mi R-326 was analyzed by Spearman correlation analysis.An online software GEO2 R was applied to measure the differential expression of MIAT and mi R-326 in atherosclerosis-related datasets GSE28829 and GSE24258.Diverse concentration（10μg/m L for low-As group and 50 μg/m L for high-As group）of oxidized low density lipoprotein（ox-LDL）was used to simulate the different stage of atherosclerosis cells models,with PBS as a control.The expression of monocyte chemotactic protein 1（MCP-1）were confirmed by a western blot assay.Specific MIAT small interfering RNAs were transfected into the constructed atherosclerosis cell models.And the expression of MIAT was qualified by a RT-q PCR assay,whilst the cell migration ability changes of VSMCs was determined by a transwell assay.Mi R-326 mimic was transfected into the constructed atherosclerosis cell models,and the mi R-326 level was confirmed by a RT-q PCR assay,the cell migration ability changes of VSMCs was checked by a transwell assay.The targeted binding sites between MIAT and mi R-326 were theoretical predicted.The targeted binding effect between MIAT and mi R-326 were verified by a dual luciferase assay.MIAT overexpression plasmids and mi R-326 mimic were co-transfected into constructed atherosclerosis cell models.A transwell assay was performed to determine the effect of mi R-326 work on MIAT-mediated facilitation of VSMCs migration.ResultsMIAT was upregulated and mi R-326 was downregulated in 46 plasma samples of patients with atherosclerosis as comparing with that in plasma samples of patients with non-atherosclerosis.There was a negative correlation between MIAT and mi R-326,r =-0.6591,P = 0.0002.The expression of MIAT in plaque samples of advanced atherosclerosis was remarkably higher than that in in plaque samples of early atherosclerosis according to GEO dataset GSE28829,P < 0.0001.The expression of mi R-326 in platelet samples of FAMI was significantly lower than that in healthy control,P = 0.0034.MCP-1 was upregulated in low-AS and high-AS group as comparing with that in control group,indicated that the construction of atherosclerosis cell models was successful.Specific MIAT si RNAs were transfected into low-AS and high-AS cells.And the expression of MIAT was downreuglated in the aforementioned cell models.Also,the cell migration ability of these cell models were weakened.Mi R-326 mimic was transfected into low-AS and high-AS cells,and mi R-326 level was prominently upreuglated in these cell models.And,the cell migration ability of these cell models were suppressed.MIAT supplied certain mi R-326 binding sites as illsutrated by bioinformatics prediction.The targeted binding effect between MIAT and mi R-326 was verified by a dual luciferase assay.Transfection of MIAT overexpression plasmids strengthened the migration ability of low-AS and high-AS cells.While,the facilitative effect was attenuated by a co-transfection of MIAT overexpression plasmids and mi R-326 mimic.ConclusionUpregulated MIAT and downregulated mi R-326 were expressed in atherosclerosis tissue and cell models.A negative correlation between MIAT and mi R-326 were found in atherosclerosis.MIAT promoted migration in ox-LDL induced VSMCs.On the contrary,mi R-326 suppressed migration in ox-LDL induced VSMCs.MIAT could bind to mi R-326 via the theoretical mi RNA response elements.MIAT promoted migration via sponging of mi R-326 in ox-LDL induced VSMCs.