| [Background and objective]Acute Myocardial Infarction (AMI) has become an important reason for the increasing mortality in cardiovascular disease. Heart failure is a common complication of myocardial infarction, is the main cause of death. Therefore, at the early stage of acute myocardial infarction, timely and accurate diagnosis, and proactive assessment of the disease, is to take positive and effective treatment measures to ensure timely reperfusion in heart, a critical condition for reducing mortality. Cardiomyocyte apoptosis after myocardial infarction and ventricular remodeling are the intermediate process of heart failure, but also the whole lesion repair and compensatory important pathophysiological process.Newly discovered non-coding RNA over the past decade-the occurrence, development and prognosis of a variety of diseases are closely linked with microRNA(miRNA). miRNA is stable in blood. miRNA can participate in the apoptosis of myocardial remodeling process, the growth or apoptosis of myocardial cells and extracellular matrix remodeling by inhibiting the target gene expression. Meanwhile, miRNA may suggest the occurrence of heart failure. A variety of circulating miRNA expression abnormalities can occur during acute myocardial infarction.The latest study has found that miR-499a is abundant in cardiac myocytes and plays important roles in anti-apoptotic cardiomyocytes after AMI, reducing infarct size, and recovery of cardiac function. miRNA is expected as an important biomarker for the diagnosis of AMI and prognosis of patients. The methods of detecting miRNA mainly include miRNA chip, Northern blots, and qPCR.The purpose of this study was to examine the dynamics change of serum miRNA-499a level in acute myocardial infarction patients over the time, to explore the relationship between miRNA-499a, biomarkers for cardiac injury and cardiac function, and to understand the prediction value of cardiac dysfunction in AMI patients using this novel marker. In addition, we further study the potential molecular mechanism of miRNA-499a expression change in human cardiac myocytes in vitro. This may provide evidence to improve the diagnosis of AMI and better predict patient outcome in the future.[Methods]Serum samples collected from patients with acute myocardial infarction in Yangpu District Central Hospitaler during2013-2014. A total of20cases are enrolled. Blood samples were collected in each patient at24hour,48hour,72hour and96hour after the onset of AMI. All the patients were monitored for cardiac biomarkers, cardiac function, biochemistry, ECG and clinical symptoms/signs as well as the prognosis since admission. Another20cases of healthy people were selected as control group. Inclusion criteria for healthy controls were no history of cardiovascular disease, normal electrocardiogram and chest X-ray, normal liver and kidney function and excluding infection and maliganancy.293T cells used for lentivirus packaging were cultured in DMEM medium; HEK293cells used for dual luciferase reporter assay were cultured in DMEM medium; human myocardiocytes were cultured in cardiac myocyte medium. miRNA was extracted from serum sample using miRNeasy miRNA extraction kit. The serum levels of miRNA-499a were quantified by the method of qPCR. The lentivirus with overexpressed and knock-dow miRNA-499a expression was prepared for in vitro study and the condition of transfection of primary human cardiac mycytes (HCM) by lentivirus was optimized. The HCM models with overexpressed miRNA-499a expression or knock-down miRNA-499a expression were built up through using prepared lentivirus with specific expression of miRNA-499a to be transfected into primary HCM. Target genes of miR-499a-5p were predicted by MiRNA online databases MICRORNA.ORG, MIRDB and TargetScan. Candidate genes of SOX5and SOX6were finally selected as target genes to be valiadated by taking the intersection of predictions and manual interpretation. SOX5-3’UTR, SOX6-3’UTR reporter gene plasmids and corresponding SOX5-3’UTR-mut and SOX6-3’UTR-mut reporter gene plasmids were constructed. Dual luciferase reporter gene assay was used to verify the regulatory relations between miRNA-499a and SOX5or SOX6.HCMs were transfected using miRNA-499a over-expression or knock-down lentivirus. miRNA-499a expression in the cell models were detected by qPCR. Meanwhile, the protein expression of SOX6was dectected by Western blot. Gene expression was semi-quantified by calculating the ratio of expression of miRNA-499a versus control using formula. The relative fluorescence intensity was calculated by using the mean value of Firefly fluorescence intensity divided by the mean value of fluorescence intensity Renilla in dual luciferase reporter gene assay. In Western blot part, the gray image of each band from protein electrophoresis was quantified by Gel-Pro analyzer software. The value of each protein band was divided by the value of beta-actin as internal control so as to calculate the relative expression of the target protein.[Results]1. Circulating miR-499a levels in20AMI patients have shown a pattern of initial increase (highest at24h) and then decrease afterwards. The serum miR-499a from healthy control group was appeared as consistently relative low level, see table4.2. In dual luciferase reporter gene assay, the blank control group and negative control group showed unchanged fluorescence intensity given increased expression of miRNA-499a which indiacted the stability of our test good. When increasing expression of miRNA-499a, SOX5wild-type and mutant3’UTR group3’UTR did not exhibit changes in relative fluorescence intensity. When increasing expression of miRNA-499a, SOX6exhibited decreased relative fluorescence intensity in wild-type3’UTR while the mutant3’UTR group did not show any change inj relative fluorescence intensity.3. Using the Western blot method to detect the protein level of SOX6in HCM with miRNA-499a up-regulated and knock-down, it has shown that SOX6protein expression was markedly reduced in HCM with miRNA-499a up-regulated, on the contrary, SOX6protein expression was significantly increased in HCM with miR-499a knock-down.4. The pattern of circulating miR-499a was similar to that of serum CK-MB. At24h the level of circulating miR-499a was almost statistically correlated with the level of CK-MB.5. There was significantly lower level of circulating miR-499a in AMI patients with heart failure than those without heart failure. The analysis of AUC at24h (0.86) indicated that circulating miR-499a may be a sensitive marker for differentiation of patients with or without heart failure in early stage.[Conclusion]Circulating miRNA-499a is a potential marker of monitoing the cardiac injury and predicating prognosis in patients after acute myocardial infarction since the change of miR-499a is similar to that of serum CK-MB and has lower expression in patients with heart failure. miRNA-499a can regulate the expression of SOX6. This indicates that miRNA-499a may play important role in the apoptosis of cardiac myocytes and ventricular remodeling after myocardial infarction via SOX6modulation and cascading downstream pathway further. |
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