| Objective:To explore the mechanism and role long non-coding RNA Metastasis associated in lung adenocarcinoma transcript 1（LncRNA MALAT1）in myocardial infarction（MI）:1)At the animal level,to investigate the altered expression of LncRNA MALAT1 in myocardial tissue after myocardial infarction;2)At the cellular level,to investigate the molecular mechanism of LncRNA MALAT1-mediated apoptosis;3)At the population level,to explore relationship between MALAT1 gene polymorphisms and MI,and the association between genetic polymorphisms of MALAT1 and prognosis of MI.Methods:Part Ⅰ:C57BL/6J male mice were divided into MI and control groups,and myocardial infarction models were constructed by ligating the anterior descending branches of the coronary arteries,and heart tissues were collected at 1,3 and 7 days after surgery.Transmission electron microscopy was used to observe mitochondrial ultrastructural changes in myocardial tissues,TUNEL staining to observe apoptotic changes,Western Blot to detect apoptosis-related proteins and AKT pathway-related proteins,cardiac ultrasound to assess the ejection capacity of the heart,HE staining and Masson staining to observe myocardial histopathological damage and fibrosis,and the use of online database combined with Venn diagram Real-time quantitative PCR was performed to detect the expression of LncRNA MALAT1 and miR-205-5p in myocardial ischemic infarcts.Part Ⅱ:Isolation and culture of primary cardiomyocytes from mice.Antisense oligonucleotides（ASO）,miRNA mimic（mimic）and miRNA inhibitor（inhibitor）were used to regulate the expression of LncRNA MALAT1 and miR-205-5p.Real-time fluorescence quantitative PCR was used to detect the expression levels of LncRNA MALAT1 and miR-205-5p,flow cytometry was used to detect changes in apoptosis rate,Western Blot was used to detect the expression of apoptosis-related proteins and AKT pathway-related proteins,dual luciferase reporter gene assay was used to clarify the binding of LncRNA MALAT1 and miR-205-5p.Mito Traker was used to detect mitochondrial morphology.Part Ⅲ:Case-control studies were used,549 patients with MI and 557 healthy controls were included,and the MALAT1 gene polymorphism was typed using the SNPscanTM technique.Logistic regression was used to analyze the association of MALAT1 gene polymorphism with MI.Kaplan-Meier survival analysis was used to assess the occurrence of major adverse cardiac and cerebrovascular event（MACCE）in carriers of different genotypes and Log-rank test for comparison of survival rates between groups.COX proportional risk regression model was used to observe the correlation between MALAT1 gene polymorphism and the occurrence of MACCE in MI patients.Results:Part I:The changes of LncRNA MALAT1 expression in mice after the occurrence of myocardial infarction were observed.1)The expression level of LncRNA MALAT1 in the tissue of myocardial infarction area increased with the increase of infarction time in the early stage of myocardial infarction in mice（P<0.05）.2)Online database analysis suggested that miR-205-5p might be a target molecule of LncRNA MALAT1,and the miR-205-5p expression level decreased with the increase of infarct time in the infarct zone tissues in the early stage of infarction（P<0.05）.3)After myocardial infarction in mice,myocardial tissues showed increased expression of the pro-apoptotic protein Bax and decreased expression of the anti-apoptotic protein Bcl-2,with a significant increase in the Bax/Bcl-2 ratio（2.28±0.58 vs.0.91±0.20,P=0.033）,increased expression of the AKT pathway-related protein PTEN（1.60±0.16 vs.0.86±0.18,P=0.013）,and a significant decrease in the p-AKT/AKT ratio（0.39±0.07 vs.1.00±0.15,P=0.007）.Part II:To explore the molecular mechanism of LncRNA MALAT1 regulating cardiomyocyte apoptosis at the cellular level.1)Establishing a cellular hypoxia model,compared with the control group,the apoptosis rate increased in the hypoxia group（apoptosis rate:9.90±2.09%vs.5.70±0.32%,P<0.001）,the expression level of the pro-apoptotic protein Bax increased,while the expression level of the anti-apoptotic protein Bcl-2 decreased,the Bax/Bcl-2 ratio increased（Bax/Bcl-2:1.64±0.11 vs.1.04±0.13,P=0.017）.The intracellular LncRNA MALAT1 expression level was significantly increased（23.61±2.00 vs.0.98±0.11,P<0.001）,however,miR-205-5p expression level was decreased（0.42±0.39 vs.1.07±0.23,P=0.027）.2)Comparing with the hypoxic group,the apoptosis rate of cardiomyocytes in the silenced LncRNA MALAT1 group was decreased（apoptosis rate:7.52±1.57%vs.19.90±2.09%,P=0.003）,the expression level of the pro-apoptotic protein Bax was downregulated,the Bax/Bcl-2 ratio was decreased（Bax/Bcl-2:1.16±0.13 vs.1.64±0.11,P=0.019）and increased expression levels of miR-205-5p（1.92±0.19 vs.0.51±0.16,P<0.001）;compared to the LncRNA MALAT1 silencing group,the combined LncRNA MALAT1and miR-205-5p silencing group had an increased apoptosis rate in cardiomyocytes（apoptosis rate:12.64±1.88%vs.7.52±1.57%,P=0.038）and an increased Bax/Bcl-2ratio（Bax/Bcl-2:2.18±0.22 vs.1.16±0.13,P=0.005）.3)The sea kidney-Firefly dual luciferase reporter gene assay suggested that LncRNA MALAT1 had a binding site to miR-205-5p.4)Comparing with the hypoxic group,silencing LncRNA MALAT1 group showed down-regulation of AKT pathway-related protein PTEN protein expression in cardiomyocytes（PTEN:0.75±0.08 vs.1.30±0.07,P=0.028）,up-regulation of p-AKT protein expression level,and increase in p-AKT/AKT ratio（p-AKT/AKT:1.41±0.21 vs.0.74±0.14,P=0.039）;In comparison to the LncRNA MALAT1 silencing group alone,the combined LncRNA MALAT1 and miR-205-5p silencing group had elevated PTEN expression levels in cardiomyocytes（PTEN:1.73±0.13 vs.0.75±0.08,P=0.004）,p-AKT protein expression level decreased and p-AKT/AKT ratio decreased（p-AKT/AKT:0.36±0.18 vs.1.41±0.21,P=0.008）.5)Mitotraker was used to observe the mitochondrial morphology of cells and found that compared with the control group,hypoxia induced an increase in mitochondrial division;silencing of LncRNA MALAT1 group increased the average length of mitochondria while decreasing the number of mitochondria and inhibited mitochondrial division（P<0.05）;silencing of both LncRNA MALAT1 and miR-205-5p group decreased the average length of mitochondria while increasing the number of mitochondria and promoted mitochondrial division（P<0.05）.The average length of mitochondria in the MALAT1 and miR-205-5p groups decreased while the number of mitochondria increased,and mitochondrial division was promoted（P<0.05）.Part III:To explore the association of MALAT1 gene polymorphism with the onset and prognosis of MI at the population level.1)The distribution frequency of rs3200401CT+TT genotype of MALAT1 gene was significantly higher in MI patients compared with controls（33.39%vs.28.62%,P=0.039）.2)Logistic regression suggested that after adjusting for confounders the MALAT1 gene rs3200401 polymorphism was associated with the development of MI,and the CT+TT genotype was a risk factor for the development of MI（OR:1.601,95%CI:1.196-2.144,P=0.002）.3)Kaplan-Meier analysis showed that the incidence of MACCE was significantly higher in MI patients carrying the rs3200401 CT+TT genotype than in CC genotype carriers（Log-rank P=0.001）.COX regression analysis showed that the risk of MACCE was 1.554 times that in CT+TT genotype carriers than in CC carriers（HR:1.554,95%CI:1.060-2.277,P=0.024）.Conclusion:（1）After myocardial infarction in mice,the expression level of LncRNA MALAT1 in infarct area tissues increased with increasing infarction time,while the expression level of miR-205-5p decreased with increasing infarction time.（2）After hypoxia in cardiomyocytes,LncRNA MALAT1 could adsorb miR-205-5p through sponge action and attenuate the inhibitory effect of miR-205-5p on PTEN,thus inhibiting AKT pathway,promoting apoptosis and aggravating ischemic injury in cardiomyocytes.Silencing LncRNA MALAT1,which promotes the expression of miR-205-5p,inhibits PTEN expression,promotes AKT protein phosphorylation,inhibits apoptosis and attenuates ischemic injury in cardiac myocytes.（3）MALAT1 gene rs3200401 gene polymorphism is correlated with the occurrence of myocardial infarction,and its CT+TT genotype may be a risk factor for MI.And MI patients carrying this genotype are more likely to develop MACCE. |
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