Associations Of Plasma MicroRNAs With The Occurrence Of Acute Myocardial Infarction And The Alteration Of Heart Rate Variability In Coke Oven Workers

The air pollution problem becomes worse in recent years, and it has resulted in increaing morbidity and mortality of cardiovascular diseases and varieties of adverse health effects, such as decreased heart rate variability. Coronary heart disease (CHD) is the most common environment-related disease with the leading cause of morbidity and mortality worldwide. The WHO declared that about one million people would die from CHD, and half of them were diagnosed as acute myocardial infarction (AMI), which will be a serious threat to people’s health and a heavy burden for the society and families. To improve health care of the chronic non-infectious diseases, the Healthy China2020programme has been recently announced by Chinese government with the goal to strengthen public health and medicine, which is critical in the process of constructing a harmonious society of China. The development of AMI is a complex process caused by multiple genetic and environmental factors. Heart rate variability (HRV) reflects the cardiac rhythm regulation by the autonomic nervous system. Epidemiologic studies suggest that alteration of cardiac autonomic function as measured by HRV is considered to be one of the pathophysiological pathways through which air pollution influences the cardiovascular system, while consistent associations between decreased HRV and myocardial infarction have already been observed in previous studies. An increasing body of evidence has suggested that the epigenetic changes of genes also associate with cardiovascular diseases. Recently, microRNAs (miRNAs) have attracted extensive interests among researchers in the exploration of the field of cardiovascular diseases. miRNAs are short, endogenous, non-coding RNAs that regulate gene expressions at the posttranscriptional level by binding to the3’untranslated regions (3’UTRs) of their target mRNAs. MiRNAs have now emerged as key regulators of cardiac growth, vascular development, and angiogenesis. Recent studies demonstrated that miRNAs can be detected in circulating blood and may be useful as disease biomarkers. The levels and identities of circulating miRNAs in cardiovascular diseases have been evaluated in a series of studies. However, the sample sizes of these studies were small, and the roles of plasma miRNAs in AMI remain to be determined.Recently, an increasing body of evidence in vitro and in vivo has also shown that exposure to environmental insults can result in a modification of miRNAs expression, which are related to the oxidative stress, immune and inflammatory status. Coke oven emissions (COEs) are complex coal-combustion related air pollutants which contain various toxic chemical substances, such as polycyclic aromatic hydrocarbons (PAHs). Our previous study suggested that occupational exposure to COEs is associated with a dose-response decrease in HRV. However, the associations between plasma miRNAs, occupational PAHs exposure levels and the alteration of HRV are unknown.In this study, we explored the difference in plasma miRNA expression profiles between20AMI patients and20healthy controls by Solexa sequencing. Differentially expressed miRNAs were validated in178AMI patients and198controls, and further replicated in150AMI patients and150controls. A preliminary functional study was also performed in human umbilical vein endothelial cells (HUVECs) overexpressing miR-320b or miR-125b. Moreover, six candidate miRNAs, which were identified by Solexa sequencing in one of our previous studies (unpublished data) and were related to the cardiovascular diseases by literature review, were detected in the plasma of365coke oven workers with exposre to COEs, and the associations between urinary PAH metabolites, plasma miRNA expressions and HRV indices were analyzed.Section Ⅰ. Plasma miRNAs and the occurrence of acute myocardial infarction in Chinese populationAn initial screening of genome-wide plasma miRNA expression profiles (discovery stage) followed by two independent case-control studies (validation stage I and validation stage Ⅱ) were performed. AMI patients in discovery stage and validation stage I were consecutively recruited from Wuhan Tongji and Union Hospital between2008and2010. Patients in validation stage Ⅱ were recruited from Wugang hospital between2008and2010. Healthy subjects without medical history of cardiovascular diseases were selected as controls during a physical health examination at hospital, and they were matched by age, sex, and area of residence with AMI patients. The miRNAs assocaited with AMI were further valiated in a second case-control study (validation stage Ⅱ). The expression levls of plasma miRNAs in the validation stages were detected by q RT-PCR assays.In the discovery stage, seventy-seven miRNAs were found to be differentially expressed between AMI patients and controls. We selected five miRNAs for q RT-PCR assays first in the discovery stage according to the following criteria:1) having at least50copies in either AMI or control group;2) and showing at least four-fold altered expression between the two pooled samples. Results show that the expression levels of miR-125b and miR-320b were lower in AMI patients compared with controls. These two miRNAs were further detected in the validation stage Ⅰ.Results in validation stage Ⅰ suggest that levels of plasma miR-320b and miR-125b were both significantly lower in AMI patients than controls. Results in validation stage Ⅱ suggest that levels of plasma miR-320b and miR-125b were consistently lower in AMI patients compared with controls. Pooled analysis of the two validation populations shows that lower levels of plasma miR-320b and miR-125b were consistently associated with AMI (adjusted OR=4.15,95%CI2.71to6.34, p<0.0001; and OR=3.89,95%CI2.57to5.86, p<0.0001respectively). Additionally, participants carrying one or both of the two decreased miRNAs had significantly increased occurrence of AMI (adjusted OR=3.46,95%CI1.87to6.40for one decreased miRNA and OR=7.79,95%CI4.47to13.57for two decreased miRNAs, ptrend<0.0001) compared with those with high levels of miRNAs. No significant interaction was found between smoking and the two miRNAs in predicting AMI risk. Moreover, miR-125b and miR-320b were able to discriminate AMI cases from controls with an AUC of0.72(95%CI,0.68-0.75) and0.75(95%CI,0.71-0.79) respectively. Combined determination of miR-125b and miR-320b showed an AUC of0.76(95%CI,0.72-0.80).Accumulating evidence has suggested that besides as biomarkers, circulating miRNAs may also act as extracellular communicators. AMI is a thrombosis-related disease, and miRNAs have been implicated in the development and progression of atherosclerosis. Moreover, the vascular endothelial cells are found to be the most common recipient cells for the circulating miRNAs. Therefore, we performed gene expression profiling in HUVEC cells transfected with miRNA mimics to search for genes with altered expression. Gene chip results suggest that1042and2737genes were deregulated in HUVECs transfected with miR-320b or miR-125b, respectively. GO mapping analysis shows that besides the translation process, miR-320b also regulated genes relevant to the process of cell proliferation, cell migration, transforming growth factor (TGF)-beta signaling, immune and inflammatory response. The differentially expressed genes are enriched in the cardiovascular disease-related pathways including TGF-beta signaling (p=0.0007) and cytokine-cytokine receptor interaction (p=0.02). Likewise, up-regulation of miR-125b in HUVECs interfered with the expression of genes involved in apoptosis, platelet activation, and inflammatory response. The differentially expressed genes are enriched in the cardiovascular disease-related pathways including apoptosis (p<0.0001) and cytokine-cytokine receptor interaction pathway (p=0.0008) by KEGG pathway analysis. We also detected the plasma levels of miR-125b and miR-320b in100healthy controls,100 stable CHD patients and100AMI patients, and the results suggested that there was a gradual decrease in the plasma levels of miR-320b and miR-125b across categories of healthy control, stable CHD and AMI.In this study we found that levels of plasma miR-320b and miR-125b were lower in AMI patients compared with controls, and lower levels of miR-320b and miR-125b were consistently associated with the occurrence of AMI in Chinese populations. The preliminary functional study further suggested that miR-320b and miR-125b could regulate the expression profiles of genes enriched in several signal transduction pathways critical for CHD in human vascular endothelial cells.Section2Associations of plasma miRNAs with the alteration of heart rate variability in coke oven workersWe recruited365healthy male coke oven workers from different work places in a coke oven plant (Wuhan, China). All the workers had been working in their current job position for at least one year. The internal airborne PAHs exposure levels of the workers were detected by urinary PAHs metabolites using gas chromatography-mass spectrometry. HRV indices of the workers were measured by the three-channel digital Holter monitory. Plasma miRNAs levels were measured by quantitative reverse transcriptase polymerase chain reaction.The results of the association between PAH exposure and miRNA expression suggest that the expression of miR-24, miR-27a, miR-320b, and miR-142-5p showed concentration-dependent decrease for urinary4-OHPHE. The expression of miR-150elicited concentration-dependent increase for urinary1-OHNAP,2-OHNAP,2-OHPHE, and SOH-PAHs. However, the expression of miR-126was not significantly associated with any of the PAHs metabolites.The results of the association between miRNA expression and HRV indices suggest that miR-24and miR-27a were both negatively associated with rMSSD. miR-320b and miR-142-5p were both negatively associated with SDNN and rMSSD. No association was found between miR-150expression and any of the HRV indices. Although miR-126was not associated with any of the PAH metabolites, we found that the levels of miR-126were negatively associated with rMSSD of the workers.In this study, we evaluated the effect of airborne PAHs exposure on the plasma levels of six cardiovascular disease-related miRNAs (miR-24, miR-27a, miR-126, miR-150, miR-320b, and miR-142-5p). We found that the expression levels of four miRNAs, including miR-24, miR-27a, miR-320b, and miR-142-5p, showed concentration-dependent decrease for urinary PAHs metabolites, while the expression of miR-150was positively associated with the urinary PAHs metabolites. Moreover, inverse associations were also found between four miRNAs and HRV indices of the workers, including SDNN and rMSSD.To summarize, our results suggest:1) Seventy-seven miRNAs were found to be differentially expressed between AMI patients and controls. Levels of miR-125b and miR-320b were found to be lower in AMI patients compared with controls.2) Lower levels of two miRNAs (miR-320b and miR-125b) were associated with AMI after adjustment for traditional risk factors, and ROC curve analysis showed that miR-125b and miR-320b were able to discriminate AMI from controls with modest diagnostic accuracy.3) These two miRNAs may also regulate the expression of genes related to CHD in vascular endothelial cells.4) There was a gradual decrease in the plasma levels of miR-125b and miR-320b across categories of healthy control, stable CHD and AMI.5) Four miRNAs (including miR-24, miR-27a, miR-320b and miR-142-5p) were associated with the urinary PAH metabolites and the HRV of coke oven workers. Noteworthy strengths of this study are:1) We showed that plasma levels of miR-125b and miR-320b were lower in AMI patients compared with controls, and their plasma levels were significantly associated with the occurrence of AMI, which was validated in two independent case-control studies;2) We suggested that the two miRNAs may also be involved in the pathogenesis of AMI3) Plasma miR-125b and miR-320b may hold prognostic value for CHD4) The relationship between plasma miRNAs, HRV, and the occurrence of AMI are novel findings, which highlights a critical role of miRNAs in regulating environmental factor-induced adverse health effect or diseases.However, several potential limitations should also be addressed in the present study. First, limited miRNAs were selected for validation in Section I study. Second, cohort study is clearly needed to evaluate the effect of air pollution on HRV and the onset of AMI. Third, the interaction between environmental factors and plasma miRNAs needs to be investigated in future studies.