| [Background and objective]Ischemic stroke(IS)is a kind of common cerebrovascular disease that seriously threatens human life.However,its etiology and pathogenesis have not been fully elucidated.Epidemiological data showed that IS was a very complex disease caused by various factors including heredity and environment.This complexity brought great difficulties for its effective prevention and treatment.Therefore,it has been an important task for the contemporary medical community to investigate the etiology of IS.Recently,with the progress of science and technology,genome-wide association studies(GWAS)was widely used in the etiology research of human diseases,so the role of genetical genes in the pathogenesis of IS,especially the long non-coding RNA(Lnc RNA)and micro RNA(mi RNA),became the focus of our research.Lnc RNA GAS5 was a member of the Lnc RNA family and attracted more and more attention in recent years.Its gene was located in the q25 region of human chromosome 1,had a total length of 4,087 nt and consisted of 12 exons.It played an important role in inflammation,vascular endothelial cell damage and the formation of atherosclerotic plaque.The formation of atherosclerotic plaque was the pathological basis of IS,so it was speculated that Lnc RNA GAS5 might play an important role in the pathogenesis of IS.In the pilot study,our research members searched the db SNP database and found that there were several gene polymorphisms in Lnc RNA GAS5 gene.However,it was not clear whether these gene polymorphisms were related to IS.In this study,the effect of Lnc RNA GAS5 gene polymorphisms on IS susceptibility and gene expression was revealed by case-control method,and its mechanism was further explored.In order to elucidate the molecular mechanism of how Lnc RNA GAS5 was involved in atherosclerosis which was the basis of IS,human umbilical vein endothelial cells(HUVECs)treated by ox LDL were used as the cell model.The effect of Lnc RNA GAS5 expression on the apoptosis and viability of HUVECs was detected,and its mechanism was further explored.Eventually,the role and molecular mechanism of Lnc RNA GAS5 in the genesis of IS were revealed,and a new target for IS diagnosis and therapy was provided.[Methods]1.The Lnc RNA GAS5 gene polymorphisms were detected by SNPscan technology in 416 IS patients and 416 controls.Their distribution frequencies were computed and the relationship between their distribution frequencies and IS susceptibility was analyzed.2.The expression level of Lnc RNA GAS5 in the plasma of 60 IS patients and 60 controls was detected by q RT-PCR.The expression level of Lnc RNA GAS5 in the plasma of IS patients and controls with different rs145204276 genotypes was analyzed and the effect of Lnc RNA GAS5 gene polymorphism on its own expression was explored.The plasmid containing Lnc RNA GAS5 promoter with rs145204276 wild/mutant(ins/del)allele was constructed.The effect of rs145204276 ins/del allele on the activity of Lnc RNA GAS5 promoter was measured by dual-luciferase reporter assay.3.The Lnc RNA GAS5 up or down-regulation lentivirus was constructed and infected HUVECs.CCK-8 assay and flow cytometry were used to analyze the effect of up or down-regulation of Lnc RNA GAS5 on the apoptosis and activity of HUVECs and atherosclerotic HUVECs model.4.The plasmid containing wild/mutant Lnc RNA GAS5,HIF3 A or FBN1 gene DNA fragment was constructed and transfected into HEK-293 T cells.The targeted relationships between Lnc RNA GAS5 and mi R-29b-3p,mi R-29b-3p and HIF3A/FBN1 were verified by dual-luciferase reporter assay.5.After infected by the Lnc RNA GAS5 up or down-regulation lentivirus,HUVECs were cultured and collected.The expression levels of IS-related molecules and proteins were detected by q RT-PCR or Western blot.At the cellular level,the role and molecular mechanism of Lnc RNA GAS5 in the regulation of HIF3 A and FBN1 by competitively binding to mi R-29b-3p in the genesis of IS were revealed.[Results]1.The six loci in Lnc RNA GAS5 gene(rs55829688,rs145204276,rs2067079,rs75315904,rs1951625 and rs2235095)were polymorphic in IS patients and controls.There were three genotypes at rs145204276 locus including ins/ins,ins/del and del/del.Compared with genotype ins/ins or ins/ins+ins/del,genotype del/del significantly increased the risk of IS(del/del vs.ins/ins:OR=2.07,95% CI=1.30-3.29,P=0.002;ins/ins+ins/del vs.del/del: OR=2.08,95% CI=1.35-3.22,P=0.001).In addition,compared with allele ins,allele del significantly increased the risk of IS(del vs.ins: OR=1.27,95%CI=1.03-1.574,P=0.024).2.The expression level of Lnc RNA GAS5 in the plasma of IS patients was1.43±0.39 times that of controls(P<0.001).The expression level of Lnc RNA GAS5 in the plasma of IS patients with rs145204276 genotype del/del was1.31±0.30 times that of IS patients with rs145204276 genotype ins/ins(P=0.001),and the expression level of Lnc RNA GAS5 in the plasma of controls with rs145204276 genotype del/del was 1.26±0.42 times that of controls with rs145204276 genotype ins/ins(P=0.025).The result of dual-luciferase reporter assay showed that the relative luciferase activity of rs145204276 del Lnc RNA GAS5 promoter was 1.21±0.02 times that of rs145204276 ins Lnc RNA GAS5 promoter(P=0.002).3.After infected by Lnc RNA GAS5 up-regulation lentivirus,HUVECs viability decreased with the increase of culture time,the expression level of Lnc RNA GAS5 increased by 2.45±0.26 times(P<0.001),the apoptosis rate of HUVECs increased from 11.84±1.26% to 30.52±0.85%,and the apoptosis rate of the atherosclerotic HUVECs model increased from 27.83±1.56% to34.66±0.92%.After infected by Lnc RNA GAS5 down-regulation lentivirus,HUVECs viability increased with the increase of culture time,the expression level of Lnc RNA GAS5 decreased by 0.27±0.07 times(P<0.001),the apoptosis rate of HUVECs decreased from 11.84±1.26% to 6.15±2.40%,and the apoptosis rate of the atherosclerotic HUVECs model decreased from27.83±1.56% to 21.75±0.37%.4.The result of dual-luciferase reporter assay showed that the relative luciferase activity of the plasmid containing wild Lnc RNA GAS5 gene DNA fragment was 0.42±0.11 times that of the plasmid containing mutant Lnc RNA GAS5 gene DNA fragment(P<0.001);the relative luciferase activity of the plasmid containing wild HIF3 A gene DNA fragment was 0.58±0.09 times that of the plasmid containing mutant HIF3 A gene DNA fragment(P<0.001);the relative luciferase activity of the plasmid containing wild FBN1 gene DNA fragment was 0.71±0.05 times that of the plasmid containing mutant FBN1 gene DNA fragment(P<0.001).5.After HUVECs were infected by Lnc RNA GAS5 up-regulation lentivirus,the expression level of mi R-29b-3p decreased by 0.77±0.05 times(P<0.001);the expression levels of HIF3 A and FBN1 increased by 1.68±0.10 times(P<0.001)and 1.58±0.06 times(P<0.001),respectively;the ratio of Cleaved-caspase3/Caspase3 increased by 2.97±0.10 times(P<0.001).After HUVECs were infected by Lnc RNA GAS5 down-regulation lentivirus,the expression level of mi R-29b-3p increased by 1.33±0.06 times(P<0.001);the expression levels of HIF3 A and FBN1 decreased by 0.83±0.05 times(P=0.040)and 0.67±0.04 times(P<0.001),respectively;the ratio of Cleaved-caspase3/Caspase3 decreased by 0.41±0.03 times(P<0.001).[Conclusion]1.The rs145204276 polymorphism of Lnc RNA GAS5 gene was significantly related to IS susceptibility.Compared with genotype ins/ins and allele ins,genotype del/del and allele del significantly increased the risk of IS,respectively.2.The expression level of Lnc RNA GAS5 in the plasma of IS patients was significantly higher than that of controls.The expression level of Lnc RNA GAS5 in the plasma of individuals carrying rs145204276 genotype del/del was significantly higher than that of individuals carrying genotype ins/ins.The relative luciferase activity of rs145204276 del Lnc RNA GAS5 promoter was significantly higher than that of rs145204276 ins Lnc RNA GAS5 promoter,and rs145204276 polymorphism regulated the expression level of Lnc RNA GAS5 by influencing the activity of Lnc RNA GAS5 promoter.3.After the expression level of Lnc RNA GAS5 was up-regulated,the activity of HUVECs decreased and the apoptosis of HUVECs increased;after the expression level of Lnc RNA GAS5 was down-regulated,the activity of HUVECs increased and the apoptosis of HUVECs decreased.Lnc RNA GAS5 was the upstream regulatory gene of mi R-29b-3p,while HIF3 A and FBN1 were the downstream target genes of mi R-29b-3p.After the expression level of Lnc RNA GAS5 was up-regulated,the expression level of mi R-29b-3p decreased,the expression levels of HIF3 A and FBN1 increased,and the ratio of Cleaved-caspase3/Caspase3 increased.After the expression level of Lnc RNA GAS5 was down-regulated,the expression level of mi R-29b-3p increased,the expression levels of HIF3 A and FBN1 decreased,and the ratio of Cleaved-caspase3/Caspase3 decreased.Taken together,our study showed that Lnc RNA GAS5 regulated the expression of HIF3 A and FBN1 by competitively binding to mi R-29b-3p,and finally promoted the occurrence of IS. |
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