| Acute myocardial infarction(AMI)is a severe type of cardiovascular disease with acute onset and poor prognosis,which is an important cause of death in cardiovascular diseases.To reduce the mortality of AMI,it is necessary to start with multi-dimensional and multi-level prevention and treatment from identification,prevention to treatment of high-risk groups,and reduction of treatment complications.Atherosclerosis(AS)is the main pathophysiological mechanism of cardiovascular diseases.Most AMI is caused by the collapse of unstable atherosclerotic plaques in coronary arteries,followed by hemorrhage and luminal thrombosis that causes lumen occlusion,finally rusult in death of myocardial cells due to long-term ischemia.Nowadays,the recognized risk factors for AMI include hypertension,smoking,dyslipidemia,diabetes,obesity/overweight,etc.With the continuous development and application of high-throughput sequencing technology,more and more studies have been conducted on genetic genes and diseases.At present,Single Nucleotide Polymorphisms(SNPs)have become a new generation of genetic markers for medical research on polygenic diseases.Abnormal lipid metabolism is an important factor for AMI,but it is not known whether genes related to lipid metabolism also affect AMI susceptibility.The key treatment of AMI is to restore the blood supply to the heart timely and effectively,that is,ischemia-reperfusion treatment.However,ischemia-reperfusion improves the blood supply to the heart,accompanied by a series of pathophysiological processes,including oxidative stress,inflammation,mitochondrial injury,endoplasmic reticulum stress,intracellular calcium overload,and finally irreversible cell death,apoptosis and necrosis,the extent of myocardial injury was further aggravated.Long chain noncoding RNAs(lncRNAs)is a kind of RNA with a length of more than 200 bases,but it lacks specific transcription and no function of coding protein,which plays an important role in many pathological mechanisms.Alpha-2-macroglobulin antisense RNA 1(A2M-AS1)is a long non coding RNA,and previous studies have shown that the expression of A2M-AS1 is down regulated in myocardial infarction,but its role in ischemia-reperfusion injury remains unclear.Based on the above background,this study first observed the correlation between SNPs related to lipid metabolism and AMI,aiming to find the possible susceptibility genes or protective genes for AMI.Then,in order to find a therapeutic target to reduce myocardial ischemia-reperfusion injury in the treatment of AMI,we established an in vitro cardiomyocyte hypoxia/reoxygenation(H/R)model.By measuring the expression level of A2M-AS1 and the activity of cardiomyocytes,we observed the effect of A2M-AS1 on ischemia-reperfusion injury.Finally,the effects of A2M-AS1 and IL1R2 on hypoxia/reoxygenated myocardial cell injury and the relationship between them were investigated for possible new therapeutic target for AMI in the treatment of post-ischemic reperfusion injury.This study was divided into three parts:Part one Relationship between gene polymorphisms related to lipid metabolism and acute myocardial infarctionObjective:To investigate the relationship between gene polymorphisms related to lipid metabolism and acute myocardial infarction(AMI).Method:A total of 336 patients with acute ST-segment elevation myocardial infarction and 270 controls were selected from Hebei General Hospital from January 2017 to June 2018.The information of 4 gene loci of lipoprotein lipase gene(LPL)rs320,low density lipoprotein receptor gene(LDLR)rs688,pre-protein invertase subtilysin 9 gene(PCSK9)rs505151 and transmembrane protein 6 superfamily member 2 gene(TM6SF2)rs58542926 in 2 groups were determined for genotyping,and general clinical data of patients were collected for analysis.Results:1.The distribution proportion of hypertension history(74.1%),diabetes mellitus(18.8%)and smoking history(56.5%)in the case group were higher than those in the healthy group(P<0.001).LDL(3.07±0.79)mmol/L,TG(1.69±1.11)mmol/L,Apo A1(1.26±0.27)mmol/L,blood glucose(6.89±2.75)mmol/L were higher than those in the control group,and HDL(1.12±0.25)mmol/L was lower than those in the control group,the difference was statistically significant.2.LPL rs320 T/G and GG/(GT+TT);LDLR rs688 C/T,CC/CT/TT and TT/(CC+ TC);TM6SF2 rs58542926 T/C and TT/TC/CC were significantly different between the control group and the case group(P<0.05).3.rs58542926 TT/CC was significantly correlated with AMI(P<0.05,OR=0.098,95%CI 0.024-0.392),which was a protective factor of AMI.Summary:1.The allele frequencies of rs688,rs320 and rs58542926 were different between the two groups,and the distribution of recessive model genotypes of rs320 and rs688 was significantly different between the two groups.2.The rs58542926 TT genotype was significantly associated with AMI and could reduce the risk of acute myocardial infarction.It was a protective genotype.Part two Expression of long non-coding RNA A2M-AS1 in hypoxic/reoxygenated cardiomyocytesObjective: To investigate the expression level of A2M-AS1 in myocardial injury cells of H/R model,and down regulate or up regulate the expression of A2M-AS1 by si RNA transfection and plasmid vector.Method:1.Human neonatal cardiomyocytes were used to construct an in vitro hypoxia/reoxygenation(H/R)model to simulate cardiomyocyte ischemia/reperfusion.The expression level of A2M-AS1 in cardiomyocytes was detected by q RT-PCR.2.si-A2M-AS1 and pc DNA3.1-A2M-AS1 were transfected into cardiomyocytes to make A2M-AS1 expression low or overexpressed,and the relative expression level of A2M-AS1 in transfected cells was detected by q RT-PCR.3.The cells were divided into five groups:(1)Control group: H/R treatment without transfection group;(2)Si-Con group:H/R transfection interference sequence control group;(3)Si-A2M-AS1:H/R transfection interference sequence group;(4)Vector(pc DNA3.1):H/R transfected vector control cells;(5)A2M-AS1: H/R transfected group,and CCK-8 assay was used to detect myocardial cell viability.Result:1.Compared with the control group without hypoxia/reoxygenation,the expression of A2M-AS1 was significantly decreased after H/R treatment(P<0.01).2.In H/R model cells,the expression of A2M-AS1 was down-regulated by small interfering RNA si-A2M-AS1,and the expression of A2M-AS1 was up-regulated by plasmid vector DNA3.1-A2M-AS1.The expression of A2M-AS1 was detected by q RT-PCR respectively.The results showed that the relative expression of A2M-AS1 after transfection of si-A2M-AS1 was significantly lower than that of si-Con group,while the expression of A2M-AS1 after transfection of pc DNA 3.1-A2M-AS1 was significantly higher than that of pc DNA3.1 vector group(P<0.01).3.Compared with empty vector or untreated cardiomyocytes,the overexpression of A2M-AS1 increased the optical density(OD)value(P<0.01).Downregulation of A2M-AS1 expression significantly decreased OD value compared with the interference control group or untreated cardiomyocytes(P<0.01).Summary:1.The expression of A2M-AS1 was decreased in H/R treated cardiomyocytes.2.In H/R model cells,si RNA was used to interfere the expression of A2M-AS1,and pc DNA3.1 up-regulated the expression of A2M-AS1.The expression of A2M-AS1 was detected by q RT-PCR respectively,and the H/R model of overexpression or inhibition of A2M-AS1 was successfully constructed.3.Inhibition of the expression of A2M-AS1 can further reduce the activity of cardiomyocytes after H/R treatment,while the activity of cardiomyocytes is significantly increased after the overexpression of A2M-AS1,which preliminarily confirmed that A2M-AS1 has a positive protective effect on H/R injury of cardiomyocytes.Part three Effects of long non-coding RNA A2M-AS1 and IL1R2 on injury of hypoxic/reoxygenated cardiomyocytesObjective:To investigate the effects of A2M-AS1 and IL1R2 on the injury of hypoxic/reoxygenated cardiomyocytes and the relationship between them.Method:1.Cell transfection was completed according to the experimental method in the previous part.The overexpressed parts were divided into groups according to the following tests:(1)Control group: untreated cells;(2)H/R: cells treated with H/R;(3)H/R+A2M-AS1: cells were transfected into A2M-AS1 group after H/R treatment;(4)H/R+A2M-AS1 +IL1R2: After H/R treatment,A2M-AS1 and IL1R2 were simultaneously transfected.The experimental groups with low expression were as follows:(1)Control group: untreated cells;(2)H/R: cells treated with H/R;(3)H/R+si-A2M-AS1:After H/R treatment,cells were transfected to interfere with A2M-AS1 sequence.(4)H/R+si-A2M-AS1+si-IL1R2:After H/R treatment,A2M-AS1 and IL1R2 were simultaneously transfected.(4)H/R+si-A2M-AS1+si-IL1R2:After H/R treatment,A2M-AS1 and IL1R2 were simultaneously transfected.2.The expression of IL1R2 in each group was determined by q RT-PCR and Western Blot.3.CCK-8 kit was used to detect the cell viability of the overexpressed/under-expressed groups.4.The apoptosis rate of each group was detected by flow cytometry.Result:1.Compared with the control group,the mRNA and protein expressions of IL1R2 in H/R treated cells were significantly increased(P < 0.01).Compared with the H/R group,the expression level of IL1R2 was further increased after A2M-AS1 knockout,while the mRNA expression of IL1R2 was significantly decreased after A2M-AS1 and IL1R2 gene knockout,the difference was statistically significant(P<0.01).On the contrary,the mRNA expression level of IL1R2 after the up-regulation of A2M-AS1 expression was significantly lower than that of H/R group(P < 0.01).When pc DNA3.1-IL1R2 was added to the H/R+A2M-AS1 group,that is both A2M-AS1 and IL1R2 genes were overexpressed,the mRNA expression level of IL1R2 was significantly higher than that of the H/R+ A2M-AS1 group(P<0.01).2.Compared with the control group,cell viability was significantly decreased after H/R treatment,and increased after overexpression of A2M-AS1(H/R+A2M-AS1 group)(P<0.01),while cell viability was reduced to the lowest level after knockout of A2M-AS1.When A2M-AS1 and IL1R2 were overexpressed simultaneously(H/R+A2M-AS1+IL1R2 group),the OD value was significantly lower than that of H/R+A2M-AS1 group(P<0.01).In the H/R model,the cell survival rate was significantly increased when IL1R2 and A2M-AS1 genes were knocked out simultaneously(H/R+si-A2M-AS1+si-IL1R2)compared with A2M-AS1 gene knockout only(H/R+si-A2M-AS1 group)(P< 0.01).3.Flow cytometry showed that compared with the control group(5.34±2.06),the apoptosis rate of cardiomyocytes after H/R treatment increased(12.64±1.38,P < 0.01).Overexpression of A2M-AS1 decreased the incidence of myocardial apoptosis(6.11±2.19,P<0.01),while knockout of A2M-AS1 significantly increased the rate of apoptosis(25.94±2.20,P<0.01).Compared with the overexpression of A2M-AS1 gene(6.11±2.19),the apoptosis rate of both A2M-AS1 and IL1R2 gene was increased by 2 times(13.07±1.36,P<0.01).Similarly,the apoptosis rate of A2M-AS1 gene knockout(25.94±2.20)was significantly higher than that of H/R group(12.64±1.38,P<0.01)and H/R+si-A2M-AS1+si-IL1R2 group(12.52±1.05,P< 0.01).Summary:1.The expression of A2M-AS1 was negatively correlated with IL1R2 in H/R treated cardiomyocytes.2.A2M-AS1 may negatively regulate IL1R2 to inhibit the apoptosis of cardiomyocytes in H/R injury and improve cell viability.Conclusions:1.rs58542926 is significantly associated with AMI,and TT genotype is a protective genotype that can reduce the risk of acute myocardial infarction.2.The expression of A2M-AS1 was decreased in H/R treated cardiomyocytes.3.A2M-AS1 has a positive protective effect on H/R injured cardiomyocytes.4.There was a negative correlation between the expression of A2M-AS1 and IL1R2 in H/R treated cardiomyocytes.5.A2M-AS1 may negatively regulate IL1R2 to inhibit the apoptosis of cardiomyocytes in H/R injury and improve cell viability. |
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