The Effect And Related Mechanisms Of Long Noncoding RNA MALAT1 Gene Polymorphism And Expression Level For Acute Coronary Syndrome

Objective:（1）To explore the association between single nucleotide polymorphisms（SNPs）of MALAT1 gene（rs3200401,rs4102217,rs600231）and acute coronary syndrome（ACS）from the perspective of population genetic epidemiology,and the relationship with myocardial ischemia injury biomarkers.（2）To investigate the value of lnc RNA MALAT1 as a new marker for ACS in early diagnosis and predictive prognosis.（3）To clarify the molecular genetic mechanisms of lnc RNA MALAT1 in the vascular endothelial cell injury.Methods:（1）The three single nucleotide polymorphisms of MALAT1 gene were genotyped by the technique of multiple SNP typing(SNPscan^TM).The case-control study consecutively recruited 929 ACS patients and 1053 healthy controls,and using Logistic regression to analyze its correlation with ACS.（2）The case-control and prospective cohort study recruited 159 ACS patients and 148 healthy controls.To measure the relative expression of lnc RNA MALAT1 in the peripheral blood mononuclear cells（PBMCs）at admission by quantitative real-time PCR（q RT-PCR）.To evaluate the ability for diagnosis of ACS by using the receiver operating characteristics（ROC）,through area under the curve（AUC）of the relative expression of lnc RNA MALAT1.During the follow-up,Kaplan-Meier plots were generated using the cut-off values of lnc RNA MALAT1 level,and the Log-rank test was used to compare the resulting curves.（3）Using CD31 antibody to detect the purity of the human umbilical vein endothelial cells（HUVECs）by flow cytometry;To explore the optimal conditions of the vascular endothelial cell injury induced by oxidative stress using CCK-8 and flow cytometry,and to detect the expression of lnc RNA MALAT1 by q RT-PCR.To clarify the relationship between lnc RNA MALAT1 and hsa-mi R-205-5p using bioinformatics prediction,dual luciferase reporter gene and RNA immunoprecipitation（RIP）experiments.To detect the location of lnc RNA in cells through nuclear separation experiments,and using chromatin immunoprecipitation（Ch IP）and RIP experiments to explore the relationship among lnc RNA MALAT1,mi R-205 and the transcription factor E2F1;In the gain and loss experiments,to detect the expression of lnc RNA MALAT1,mi R-205 and E2F1 by q RT-PCR,and apoptotic protein（Bcl-2,Bax）by Western blot.Results:（1）（1）The rs600231 distribution frequencies of AA,AG,GG genotypes in ACS patients were314（33.8%）,469（50.5%）,146（15.7%）,in control subjects were 412（39.1%）,486（46.2%）,155（14.7%）,were significantly different between ACS patients and control subjects（P<0.05）.（2）In ACS patients,the AG+GG genotype was significantly higher than in controls（66.2%vs.60.9%,P<0.05）.（3）The frequencies of A and G alleles were significantly different between ACS patients and control subjects,the frequency of G allele was significantly higher in ACS than that in healthy controls（41.0%vs.37.8%,P<0.05）.（4）Logistic regression analysis showed that the dominant model of rs600231 was association with the incidence of ACS,and the dominant model of rs600231 was a risk factor for ACS（OR=1.267,95%CI 1.024-1.567,P=0.030）.（5）Compared with ACS patients without complication of diabetes,the proportion in AG+GG genotype was higher in ACS patients with diabetes（72.0%vs.64.3%,P<0.05）.（6）In ACS patients,the creatine kinase（CK）and creatine kinase MB（CK-MB）in the AG+GG genotype were higher than in AA genotype（P<0.05）.（2）（1）The relative expression of lnc RNA MALAT1 in ACS patients was higher than that in controls（P<0.05）.Among ACS patients,the relative expression in the group of ST-elevation myocardial infraction（STEMI）was highest,the relative expression was 2.14（0.40-5.88）vs.0.69（0.18-1.72）,the group of non-ST-elevation myocardial infraction（NSTEMI）was 1.86（0.53-4.25）vs.0.69（0.18-1.72）.（2）Logistic regression analysis showed that the relative expression of lnc RNA MALAT1 was association with the incidence of ACS,and the lnc RNA MALAT1 was a risk factor for ACS（OR=1.193,95%CI 1.037-1.372,P<0.05）.（3）ROC of lnc RNA MALAT1 for the ability of diagnosis in ACS patients was used.The area under the ROC curve for lnc RNA MALAT1 was 0.664.The recommended cut-off value for lnc RNA MALAT1 based on the maximum of Youden’s index on the ROC curve was 0.816 and it had 70.0%sensitivity and 58.0%specificity.（4）During the follow-up,Kaplan-Meier curves showed that the prevalence of major adverse cardiovascular events（MACE）was significantly higher in patients with high-MALAT1 levels than those with low-MALAT1levels（P<0.05）.（3）（1）The best condition during the vascular endothelial cell injury by oxidative stress was 400umol/L H₂O₂,induced for 12h.（2）In HUVEC,the relative expression of lnc RNA MALAT1 in the vascular endothelial cell injury model induced by oxidative stress was increased compared with the control,however,the relative expression of mi R-205 was decreased（P<0.05）.（3）Compared with the group of MALAT1-WT1+mi R-NC,the group of MALAT1-WT1+mi R-205 mimic,the value of Luc/Rluc was decreased（P<0.05）.（4）Compared with the control,the Bcl-2 protein was increased and the Bax protein was decreased in the group of loss of lnc RNA MALAT1,moreover,the effect was opposite in the gain of lnc RNA MALAT1（P<0.05）.（5）In the group of mi R-205 mimic,the Bcl-2 protein was increased and the Bax protein was decreased compared with the group of mi R-NC,moreover,the effect was opposite in the mi R-205Inhibitor（P<0.05）.（6）Compared with sh-NC,the relative expression of mi R-205 was increased in the group of sh-MALAT1-1（P<0.05）.（7）The results of nucleoplasm isolation experiment showed that 92.7%of lnc RNA MALAT1 was in the nucleus,the rest was in the cytoplasma.（8）Ch IP experiment confirmed that E2F1 could bind to the upper region of the MIR205 gene.（9）Compared with the sh-E2F1-1,the relative expression of mi R-205 was increased,however,the effect of mi R-205 was decreased compared with the pc DNA3.1-E2F1（P<0.05）.Compared with the sh-MALAT1-1 or pc DNA3.1-MALAT1,the expression of E2F1 was no different（P>0.05）.（10）RIP experiment showed that E2F1could bind to lnc RNA MALAT1.Compared with the sh-NC,the binding with MIR205gene was decreased in the loss of lnc RNA MALAT1 by Ch IP.Conclusion:（1）rs600231of MALAT1 gene was association with the incidence of ACS and traditional myocardial injury biomarkers（CK,CK-MB）,the dominant model of rs600231 was the risk factor for ACS,especially ACS with complication of diabetes.（2）The relative expression of lnc RNA MALAT1 in ACS patients was increased,and could serve as a biomarker for early diagnosis and predictive prognosis.（3）In the vascular endothelial cell injury model induced by oxidative stress,the relative expression of lnc RNA MALAT1 was increased.In the cytoplasm,lnc RNA MALAT1 could act as“sponge molecule”to bind mi R-205 to regulate apoptosis.In the nucleus,lnc RNA MALAT1 could bind the transcription factor E2F1 to regulate the mi R-205 on apoptosis at the transcription level.