Molecular Mechanism Of Linc01278 Regulates ACTG2 To Control Vascular Smooth Muscle Cell Proliferation By Sponging MiR-500b-5p In Aortic Dissection

BackgroundAortic dissection(AD)is one of the most dangerous diseases in cardiovascular disease.It develops rapidly and once diagnosis is confirmation,necessary treatment should be arrangement,or it will be caused disastrous consequences.The main etiology of AD mainly relevant with the blood enters the middle layer of ascending aortic artery through the intima tear,and flows forward in the vascular dissection driven by blood pressure,thus tearing the blood vessels to cause symptoms.The common clinical symptoms are sudden chest and back pain.The common pathogenic factors of AD includes hereditary factors such as Marfan syndrome,Ehlers-Danlos syndrome,and non hereditary factors such as hypertension,atherosclerosis,syphilitic arteritis,traumatic aneurysm,etc.,in which hypertension is considered to be the most important inducing factor.At present,the specific pathogenesis of AD has not been determined,which is mainly related to the imbalance of proliferation and apoptosis of vascular smooth muscle cells.Previous studies have shown that the proliferation of vascular smooth muscle in AD is significantly higher than that in the normal ascending aorta.A series of experiments in vivo and in vitro have been carried out to verify the effect of the increase or decrease of the marker protein encoding the contraction of smooth muscle cells on the proliferation of aortic smooth muscle cells.At present,there is a lack of early and effective diagnostic measures for this catastrophic disease,especially the serological diagnostic markers.At present,there is no serum diagnostic and therapeutic marker of AD that integrates specificity and sensitivity.Most of the patients with AD were diagnosed by computed tomography angiography.However,there are many diseases with chest and back pain,among which myocardial infarction is the primary suspect.Most of the patients suspected AD only after no obvious abnormality was found in the coronary angiography.Some of the patients died of rupture during frequent transport and examination.Therefore,more rapid,effective and simple diagnosis and treatment methods need to solve the current situation of AD diagnosis and treatment.Long none coding RNA(lnc RNA)is a kind of linear non coding RNA.The existing research shows that lnc RNA can combine with micro RNA and affect the expression of coding genes,thus affecting the development of diseases.In cardiovascular diseases,there have been many reports that lnc RNA is related to the pathogenesis of heart failure,ischemic heart disease and heart valve disease.However,there are few studies on the pathogenesis of AD,especially the effect of lnc RNA on the contraction of vascular smooth muscle in the middle layer of AD.ObjectiveIn this study,the gene expression profiles of lnc RNA,mi RNA and m RNA were detected by high-throughput microarray second-generation sequencing technology of the tissues from ascending aorta of AD patients and normal ascending aorta tissues of the control group.Through modern high-throughput bio-informatics analysis of the second-generation sequencing results,the differential expression of lnc RNA,mi RNA and m RNA was found,and gene ontology analysis and signal pathway were carried out for the differentially expressed genes analysis.In order to screen out the key lnc RNA,mi RNA and m RNA signal pathway of vascular smooth muscle contraction in the pathogenesis of AD.Finally,identify the key mechanism of AD,diagnosis markers and potential therapeutic targets for early diagnosis in the future are proposed.Method1.Collection of ascending aorta tissue.The tissues of ascending aorta(experimental group)which were resected around the intimal tear and the tissues of ascending aorta(control group)which were removed by the perforating device of ascending aorta during the operation of coronary artery bypass grafting.All the tissues were cleaned and stored in-80 ? liquid nitrogen.2.Detection of lnc RNA,mi RNA and m RNA gene expression profile in AD disease.The high-throughput second-generation microarray was used to sequence the ascending aorta tissues of 6 AD patients and 6 ischemic heart disease patients. According to the selection criteria of P < 0.05 and |Fold Chang| ? 2.0,significant differential expression of lnc RNA,mi RNA and m RNA were preliminarily screened.At the same time,10 AD ascending aorta tissues and 10 ischemic heart disease ascending aorta tissues were randomly selected for real-time quantitative PCR to verify the accuracy of the gene chip.3.Bioinformatics analysis of differential expression of lnc RNA,mi RNA and m RNA in ascending aorta of AD.After integration of the differentially expressed m RNA in AD tissue and the gene sequencing results of aortic dissection m RNA in GEO database(gse52093),the target m RNA was screened by gene ontology analysis,KEGG signal pathway analysis,and the lnc RNA and mi RNA were predicted by targetscan and Diana tools.The final lnc RNA and mi RNA were determined by venn analysis.The target lnc RNA,mi RNA and m RNA and vascular smooth muscle were discussed about proliferation and the pathogenesis of AD.4.The mechanism of linc01278 on the proliferation of aortic smooth muscle cells in AD.The specific location of linc01278 in vascular smooth muscle cells was detected by in situ hybridization.After plasmid overexpression of linc01278 and RNA interference reduced linc01278,the expression levels of linc01278,mir-500b-5p and ACTG2 were measured.The expression levels of SMA,SM22?,MYH11 and calponin.4)Detection of MMP2,MMP9 and scratch test to verify the migration of VSMC after transfection.5.Double luciferase reporter gene experiment.The binding sites of linc01278 with mir-500b-5p and mir-500b-5p with ACTG2 were predicted by targetscan database.The binding sites of mir-500b-5p with linc01278 and ACTG2 were further verified by double luciferase experiments.Results1.The ascending aorta of human aortic dissection and ischemic heart disease patients were successfully obtained,and RNA was extracted.The concentration and purity of RNA were detected,which met the further experimental standard.2.The differential expression of lnc RNA and m RNA in the ascending aorta around the aortic dissection was detected by high throughput second generation microarray.There are 2084 differentially expressed lnc RNAs,730 differentially up-regulated genes and 1354 differentially down-regulated genes in lnc RNA gene expression chip.2834 differentially expressed m RNA,1928 differentially up-regulated genes and 906 differentially down-regulated genes.Five up-regulated and five down-regulated differentially expressed lnc RNA and five up-regulated and five down-regulated differentially expressed m RNA were randomly selected to verify the expression by q RT-PCR between 10 cases of AD and 10 cases of ischemic heart disease ascending aorta.The results of q RT-PCR and microarray showed the same trend,which proved that the microarray results were accurate and reliable.3.The regulatory network of ce RNA was constructed,and the m RAN chip and the human AD gene chip GSE52093 from GEO database were integrated.There are 144 co-expression up-regulated genes and 96 co-expression down-regulated genes.All the differentially expressed genes were analyzed by signal pathway analysis.The down regulated genes were enriched in vascular smooth muscle contraction pathway,and the up regulated genes were enriched in cell cycle pathway.ACTG2,BDNF,DMD,cnn1 and myocd were the hub genes of aortic dissection.The mi RNAs with binding sites were predicted by targetscan.The predicted 214 mi RNAs and our mi RNA microarray were analyzed by venn analysis,and 13 coexpressed mi RNAs were selected.Using Diana tools to predict the lnc RNA with 13 mi RNA binding sites.All predicted lnc RNA and our lnc RNA gene chip were integrated,and finally got six lnc RNAs with common differential expression.Then we construct the regulatory network of ce RNA.According to the modern high-throughput bio-informatics calculation method,we finally predicted that linc01278(down-regulated expression)-mir-500b-5p-actg2(down-regulated expression)regulatory pathway plays an important role in the proliferation of ascending aortic smooth muscle in patients with aortic dissection.4.Linc01278 regulates the proliferation and phenotype transformation of aortic smooth muscle cells.Using RNA interference technology to knock down linc01278 in vascular smooth muscle cells,we found that the expression of mir-500b-5p increased with the decrease of linc01278 expression,which increased the ACTG2.ACTG2 is an important factor in vascular smooth muscle contraction pathway,through the proliferation of vascular smooth muscle cells and phenotype transformation.The final results showed that gene marker of SMA,SM22?,MYH11 and calponin decreased at the same time,which indicated that the contraction ability of vascular smooth muscle increased while the secretion ability decreased.The above phenomena can be reversed by mir-500b-5p inhibitor,which can weaken the contractile ability and enhance the secretory ability of vascular smooth muscle.However,in the over expression cell experiment of linc01278,the result was opposite to that of RNA interference.When the expression of linc01278 increased,the contraction ability of vascular smooth muscle decreased and the secretion ability increased.These phenomena can be reversed by mir-500b-5p mimics.5.The binding of linc01278,mir-500b-5p and ACTG2 was confirmed by double Luciferase Report.Pgl6-mir-linc01278(WT),pgl6-mir-actg2-3'UTR(WT)and mir-500b-5p mimics were constructed respectively.After transfection of 293 T cells and fluorescence detection,it was found that the binding sites of mir-500b-5p to linc01278 and ACTG2 could be inhibited.Conclusion1.Gene chip showed 2084 differentially expressed lnc RNAs and 2834 differentially expressed m RNAs in ascending aorta of patients with aortic dissection compared with those in non aortic dissection patients.2.The high-throughput bio-informatics analysis of lnc RNA,mi RNA and m RNA gene expression profiles suggested that linc01278 may regulate the expression of ACTG2 by competitively binding mir-500b-5p,thereby affecting the abnormal proliferation and phenotype transformation of vascular smooth muscle cells caused by the smooth muscle contraction pathway of vascular smooth muscle cells.3.After knocking down linc01278,the proliferation and migration ability of VSMCs increased,but after over expression linc01278,the proliferation and migration ability of VSMCs decreased.4.Dual luciferase reporter analysis verified the binding site between mi R-500b-5p and linc01278 or ACTG2.In conclusion,the low expression of linc01278 in aortic dissection ascending aortic smooth muscle tissue inhibits the expression of ACTG2 by reducing the competitive adsorption of mir-500b-5p,thus promoting the contraction of vascular smooth muscle cells,and enhancing the proliferation and migration ability of vascular smooth muscle cells.These results provide a new theoretical basis for the mechanism and molecular pathway of non coding RNA in the pathogenesis of aortic dissection.At the same time,it is the potential marker and drug treatment target for early diagnosis in the future.