摘要
我国空气污染问题日趋严重，由此导致环境相关疾病（如心血管疾病）的发病率和死亡率增加，以及人体早期健康损害（如心率变异性下降）的增加。冠心病是世界上最常见，也是致死率最高的环境相关性疾病之一，我国每年有大约100万人死于冠心病,而急性心梗占了一半，是对人民生命和健康威胁最大的心血管疾病之一。为此，加强开展急性心梗的基础研究和人群防治工作，是保障我国经济可持续发展以及构建和谐社会的重大社会需求。急性心梗病因复杂，是环境与基因共同作用的结果。心率变异性反映中枢通过自主神经系统对心脏节律的调节。流行病学研究结果显示，空气污染可导致人群心率变异性水平下降,而心率变异性改变在预测急性心血管事件（如急性心梗)方面具有重要意义。
越来越多的研究表明，在一些疾病状态下，某些基因的序列不发生改变，只是基因的修饰加工和表达发生异常，这就是表观遗传学的主要研究内容。表观遗传学是连接外在环境因素和内在遗传因素的桥梁和纽带，表观遗传学调节异常可产生多种疾病，如心血管疾病等。microRNA(miRNA)是表观遗传调控机制中最重要的组成部分之一，在心血管疾病的发生与发展中发挥重要的作用。许多研究表明，miRNA参与调节心血管系统的生长、发育以及血管生成等生理过程。同时，miRNAs也可稳定存在于外周血液循环系统中（包括血浆和血清），而那些在某疾病状态下具有特征性表达谱的循环miRNA有望成为该疾病的生物标记物，因此具有潜在的临床价值。然而，关于血浆miRNA与急性心梗发病风险之间的关系，尚缺乏流行病学证据。
体内外研究结果显示，miRNA可调节空气污染物暴露（包括吸烟）引起的氧化应激和免疫炎症反应等。焦炉逸散物是一种由煤燃烧释放出来的空气污染物，含有许多有毒有害的化学物质，可经呼吸道进入人体。本实验室之前的研究表明，焦炉工人心率变异性下降可能与焦炉逸散物中多环芳烃的暴露水平有关。然而，目前尚未有研究表明空气颗粒物暴露是否可以导致人体血浆miRNA表达谱改变；而miRNA作为一种重要的基因表达调控机制，其血浆水平与HRV之间是否存在关联性，也未有文献报道。
鉴于miRNA在心血管疾病以及空气污染致人体早期健康损害中的作用，本研究开展了如下几个方面的工作：（1）将20名急性心梗病人或20名年龄性别匹配的健康对照人群血浆混合后提取总RNA进行Solexa测序，筛选在两组中有表达差异的miRNA，用实时荧光定量PCR的方法初步验证这些miRNA的表达量，接着在两个独立的大样本病例对照人群中对表达水平有差异的miRNA再次进行验证，并分析吸烟和miRNA的交互作用对于急性心梗发病风险的影响（2）对与急性心梗发病风险有显著关联性的miRNA进行功能探讨（3）根据本实验室前期测序结果和文献报道选择若干与心血管疾病密切相关的miRNA，在暴露于焦炉逸散物的焦炉工人中检测其血浆miRNA表达水平，并对焦炉工人尿羟基PAHs浓度、血浆miRNA水平以及心率变异性三者之间的关系进行关联性分析。
第一部分血浆microRNA与急性心梗发病风险的关联性研究
本研究用于Solexa测序的发现阶段人群是从验证人群I中挑选出来的20名急性心梗病人和20名健康对照。验证人群I包括从2008年到2010年在武汉同济医院和协和医院两个医院进行住院治疗的178例急性心梗病人，以及来自于武汉社区，年龄和性别频率与病例组匹配的198名社区居民（健康对照）。验证人群II包括从2008年到2010年在武汉武钢医院进行住院治疗的150例急性心梗病人和随机选择的150例同一时期收集的年龄和性别频率与病例组匹配的武汉社区健康对照人群。验证人群II主要用于验证人群I得到的阳性结果。血浆MiRNA表达水平的检测采用实时荧光定量PCR的方法。同时，我们还利用ROC曲线计算了血浆miRNA作为急性心梗诊断标记物的曲线下面积。
Solexa测序结果显示，共有77个miRNA的表达水平在急性心梗和对照组间存在显著差异。我们共选择5个miRNA用qRT-PCR的方法首先在测序人群中进行验证。miRNA选择标准为：（1）在病例组或对照组至少一组中miRNA的拷贝数>50;(2)在这两组样本间的表达差异倍数>4。最终，我们选择了同时满足这两个条件，且差异倍数最大的5个miRNA（分别为miR-483-5p, miR-106b, miR-25，miR-125b和miR-320b）。结果显示，miR-125b和miR-320b的表达水平在急性心梗组中显著低于对照人群，与测序结果一致，而其他3个miRNA的表达水平在两组间无显著差异。因此，我们选择在验证阶段I和验证阶段II人群中进一步验证这个结果。
验证阶段I结果显示，miR-125b和miR-320b在急性心梗人群中的相对表达量显著低于健康对照。验证阶段II结果显示，miR-320b和miR-125b在急性心梗人群中的相对表达量也显著低于对照人群。当我们把验证人群I和验证人群II合并分析时，结果显示：低表达水平的miR-320b与急性心梗发病风险的增加有显著关联性（校正后OR=4.15，95%CI为2.71-6.34，P<0.0001）。同样地，低表达水平的miR-125b与急性心梗发病风险的增加也有显著关联性（校正后OR=3.89，95%CI为2.57-5.86，P<0.0001）。这两个血浆miRNA对急性心梗的发病风险也有累积作用。与携带高表达水平miRNA的人群相比，随着携带低表达水平的miRNA的个数的增加，个体急性心梗发病风险也逐渐增加（携带一个低表达miRNA的人校正后OR=3.46,95%CI为1.87-6.40；携带两个低表达miRNA的人校正后OR=7.79,95%CI为4.47-13.57；趋势检验Ptrend<0.0001）。然而，吸烟和这两个miRNA的交互作用无统计学意义。混合人群的ROC曲线分析结果显示，miR-125b的曲线下面积为0.72（95%置信区间为0.68-0.75），miR-320b的曲线下面积为0.75（95%置信区间为0.71-0.79）。两个miRNA合并分析结果显示曲线下面积为0.76（95%置信区间为0.72-0.80）。
急性心梗的发生常常是由于冠状动脉粥样斑块破裂导致血栓形成，从而使冠脉闭塞心肌缺血坏死所致。许多研究表明，血浆miRNA也参与动脉粥样硬化的发生发展过程，而血管内皮细胞往往是血浆miRNA发挥生物学作用的重要受体细胞。由于血浆miR-125b和miR-320b的细胞来源未明，因此，我们选择在人脐静脉内皮细胞（HUVEC）中对这两个miRNA进行初步的功能探讨。我们将miR-320mimics或者miR-125b mimics转染到HUVEC中，利用基因芯片的方法筛选HUVEC转染前后表达改变的基因。结果显示，miR-320b mimics转染组中共1042个基因的表达水平发生了改变，而miR-125b mimics转染组中共2737个基因的表达水平发生了改变。对这些表达水平发生了改变的基因进行生物信息学分析，结果显示，miR-320b调节与翻译、细胞增殖、细胞迁移、转化生长因子-beta信号通路和炎症免疫反应有关的基因的表达，而这些基因也富集在与心血管疾病病理机制有关的信号通路上，比如转化生长因子-beta和细胞因子-细胞因子受体作用通路。miR-125b调节与翻译、细胞凋亡、血小板激发和炎症反应相关的基因的表达，这些基因也富集在与心血管疾病相关的信号通路上，包括凋亡通路和细胞因子-细胞因子受体作用通路。我们接着在100个健康对照、100个稳定冠心病病人和100个急性心梗病人的血浆中检测了这两个miRNA的表达水平，发现血浆miR-125b和miR-320b的表达水平在健康对照、稳定冠心病和急性心梗三组中有显著下降趋势。
总之，我们的实验结果表明，miR-320b和miR-125b在急性心梗病人血浆中的表达水平显著低于健康对照人群，并且低表达水平的miR-125b和miR-320b与急性心梗发病风险增加有显著关联性。同时，这两个miRNA也调节血管内皮细胞中与冠心病发病机制有关的一系列信号通路中的基因的表达。
第二部分血浆microRNA与焦炉工心率变异性改变的关联性研究
我们选取了365名在目前的岗位上至少工作了一年的健康男性焦炉工人作为研究对象。采用气相色谱-质谱联用仪检测焦炉工人尿中各羟基多环芳烃代谢产物的浓度，采用Holter测量工人的心率变异性，并用实时荧光定量PCR的方法检测了血浆miRNA的表达水平。
尿羟基多环芳烃与miRNA的剂量反应关系结果显示：在校正了年龄、工龄、吸烟、吸烟包年、饮酒和体重指数后，miR-24、miR-27a、miR-320b和miR-142-5p的表达水平与4-羟基菲浓度呈显著负相关，而miR-150的表达水平与1-羟基萘、2-羟基萘、2-羟基菲和羟基总多环芳烃均呈显著正相关。miR-126表达水平与尿羟基多环芳烃之间无显著相关性。
miRNA与心率变异性的关联性分析结果显示：在校正了年龄、工龄、吸烟、吸烟包年、饮酒、体重指数和锻炼后，miR-24和miR-27a的表达水平与rMSSD呈显著负相关。miR-320b的表达水平与SDNN和rMSSD指标都呈显著负相关。miR-142-5p的表达水平也与SDNN和rMSSD指标呈显著负相关。而miR-150的表达水平与HRV各指标之间都无显著关联性。尽管血浆miR-126表达水平并不受PAH暴露的影响，然而，我们发现，血浆miR-126表达水平与工人的rMSSD指标之间呈显著负相关。总之，本部分实验结果表明，空气PAH暴露与焦炉工人血浆miRNA的表达水平之间呈一定的剂量反应关系，且血浆miRNA的表达水平与心率变异性之间也有显著关联性。因此，血浆miRNA可能是一种潜在的空气污染致人体早期健康损害的生物标志物。
综上所述，本研究主要探讨了急性心梗患者和健康对照人群血浆中的miRNA表达谱差异，并系统分析了差异表达的miRNA与急性心梗发病风险之间的关系；同时，还对与急性心梗发病风险有显著关联性的血浆miRNA进行了初步的功能探讨。另外，我们还在暴露于焦炉逸散物的焦炉工人中探讨了血浆miRNA表达水平与焦炉逸散物致心率变异性改变之间的关系。总的来说，我们的研究结果表明：
1)在急性心梗病人和健康对照人群血浆中，共77个miRNA存在表达差异，且miR-125b和miR-320b的表达水平在急性心梗病人中显著低于健康对照人群；
2)低表达水平的血浆miR-125b和miR-320b与急性心梗发病风险的增加有显著关联性，ROC曲线显示这两个miRNA作为急性心梗诊断标记物时具有较好的准确度；
3) miR-125b和miR-320b能调节血管内皮细胞中与冠心病发病机制密切相关的一系列信号通路中的基因的表达；
4)血浆miR-125b和miR-320b在健康对照、稳定冠心病和急性心梗人群中表达水平呈显著下降趋势；
5)血浆miR-24、miR-27a、miR-320b和miR-142-5p的表达水平不仅与空气PAH暴露有剂量反应关系，而且还与焦炉工人的心率变异性有显著关联性。本研究的创新之处：
1)本研究发现血浆miR-125b和miR-320b表达水平与急性心梗的发病风险之间有显著关联性；
2)发现miR-125b和miR-320b可能参与调节血管内皮细胞中与动脉粥样硬化发病机制相关的一系列基因的表达；
3)血浆miR-125b和miR-320b可能作为疾病进程的生物标记物，可能在评价冠心病预后方面具有重要意义
4)发现血浆miR-24、miR-27a、miR-320b和miR-142-5p的表达水平与焦炉工的空气多环芳烃暴露水平和心率变异性改变之间存在显著关联性，揭示血浆miRNA可能是潜在的空气污染致人体早期健康损害的生物标志物
然而，尽管本研究已经取得了一些重要的发现，但由于时间的限制，尚有许多不足有待更为深入的研究，包括：
1)第一部分研究中只选择了5个miRNA在大样本人群中进行验证
2)队列研究仍处于起步阶段，还没有进行空气污染致心率变异性改变以及急性心梗发病的前瞻性研究；
3)环境因素以及生活方式与miRNA的交互作用对急性心梗易感性的影响，需要进一步研究
The air pollution problem becomes worse in recent years, and it has resulted inincreaing morbidity and mortality of cardiovascular diseases and varieties of adverse healtheffects, such as decreased heart rate variability. Coronary heart disease (CHD) is the mostcommon environment-related disease with the leading cause of morbidity and mortalityworldwide. The WHO declared that about one million people would die from CHD, andhalf of them were diagnosed as acute myocardial infarction (AMI), which will be a seriousthreat to people’s health and a heavy burden for the society and families. To improve healthcare of the chronic non-infectious diseases, the Healthy China2020programme has beenrecently announced by Chinese government with the goal to strengthen public health andmedicine, 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 andenvironmental factors. Heart rate variability (HRV) reflects the cardiac rhythm regulationby the autonomic nervous system. Epidemiologic studies suggest that alteration of cardiacautonomic function as measured by HRV is considered to be one of the pathophysiologicalpathways through which air pollution influences the cardiovascular system, whileconsistent associations between decreased HRV and myocardial infarction have alreadybeen observed in previous studies.
An increasing body of evidence has suggested that the epigenetic changes of genesalso associate with cardiovascular diseases. Recently, microRNAs (miRNAs) have attractedextensive interests among researchers in the exploration of the field of cardiovasculardiseases. miRNAs are short, endogenous, non-coding RNAs that regulate gene expressionsat the posttranscriptional level by binding to the3' untranslated regions (3' UTRs) of theirtarget mRNAs. MiRNAs have now emerged as key regulators of cardiac growth, vasculardevelopment, and angiogenesis. Recent studies demonstrated that miRNAs can be detectedin circulating blood and may be useful as disease biomarkers. The levels and identities ofcirculating 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 inAMI remain to be determined.
Recently, an increasing body of evidence in vitro and in vivo has also shown thatexposure to environmental insults can result in a modification of miRNAs expression,which are related to the oxidative stress, immune and inflammatory status. Coke ovenemissions (COEs) are complex coal-combustion related air pollutants which containvarious toxic chemical substances, such as polycyclic aromatic hydrocarbons (PAHs). Ourprevious study suggested that occupational exposure to COEs is associated with adose-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 profilesbetween20AMI patients and20healthy controls by Solexa sequencing. Differentiallyexpressed miRNAs were validated in178AMI patients and198controls, and furtherreplicated in150AMI patients and150controls. A preliminary functional study was alsoperformed in human umbilical vein endothelial cells (HUVECs) overexpressing miR-320bor miR-125b. Moreover, six candidate miRNAs, which were identified by Solexasequencing in one of our previous studies (unpublished data) and were related to thecardiovascular diseases by literature review, were detected in the plasma of365coke ovenworkers 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 myocardialinfarction in Chinese population
An initial screening of genome-wide plasma miRNA expression profiles (discoverystage) followed by two independent case-control studies (validation stage I and validationstage II) were performed. AMI patients in discovery stage and validation stage I wereconsecutively 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 ascontrols 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 werefurther valiated in a second case-control study (validation stage II). The expression levls ofplasma miRNAs in the validation stages were detected by q RT-PCR assays.
In the discovery stage, seventy-seven miRNAs were found to be differentiallyexpressed between AMI patients and controls. We selected five miRNAs for q RT-PCRassays 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 expressionbetween the two pooled samples. Results show that the expression levels of miR-125b andmiR-320b were lower in AMI patients compared with controls. These two miRNAs werefurther detected in the validation stage I.
Results in validation stage I suggest that levels of plasma miR-320b and miR-125bwere both significantly lower in AMI patients than controls. Results in validation stage IIsuggest that levels of plasma miR-320b and miR-125b were consistently lower in AMIpatients compared with controls. Pooled analysis of the two validation populations showsthat 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 twodecreased 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 twodecreased miRNAs, ptrend <0.0001) compared with those with high levels of miRNAs. Nosignificant interaction was found between smoking and the two miRNAs in predicting AMIrisk. Moreover, miR-125b and miR-320b were able to discriminate AMI cases fromcontrols 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 miRNAsmay also act as extracellular communicators. AMI is a thrombosis-related disease, andmiRNAs have been implicated in the development and progression of atherosclerosis.Moreover, the vascular endothelial cells are found to be the most common recipient cellsfor the circulating miRNAs. Therefore, we performed gene expression profiling in HUVECcells transfected with miRNA mimics to search for genes with altered expression. Genechip results suggest that1042and2737genes were deregulated in HUVECs transfectedwith miR-320b or miR-125b, respectively. GO mapping analysis shows that besides thetranslation process, miR-320b also regulated genes relevant to the process of cellproliferation, cell migration, transforming growth factor (TGF)-beta signaling, immune andinflammatory response. The differentially expressed genes are enriched in thecardiovascular disease-related pathways including TGF-beta signaling (p=0.0007) andcytokine-cytokine receptor interaction (p=0.02). Likewise, up-regulation of miR-125b inHUVECs interfered with the expression of genes involved in apoptosis, platelet activation,and inflammatory response. The differentially expressed genes are enriched in thecardiovascular disease-related pathways including apoptosis (p<0.0001) andcytokine-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 agradual decrease in the plasma levels of miR-320b and miR-125b across categories ofhealthy control, stable CHD and AMI.
In this study we found that levels of plasma miR-320b and miR-125b were lower inAMI patients compared with controls, and lower levels of miR-320b and miR-125b wereconsistently associated with the occurrence of AMI in Chinese populations. Thepreliminary functional study further suggested that miR-320b and miR-125b could regulatethe expression profiles of genes enriched in several signal transduction pathways criticalfor CHD in human vascular endothelial cells.
Section2Associations of plasma miRNAs with the alteration of heart ratevariability in coke oven workers
We recruited365healthy male coke oven workers from different work places in a cokeoven plant (Wuhan, China). All the workers had been working in their current job positionfor at least one year. The internal airborne PAHs exposure levels of the workers weredetected by urinary PAHs metabolites using gas chromatography-mass spectrometry. HRVindices of the workers were measured by the three-channel digital Holter monitory. PlasmamiRNAs levels were measured by quantitative reverse transcriptase polymerase chainreaction.
The results of the association between PAH exposure and miRNA expression suggestthat the expression of miR-24, miR-27a, miR-320b, and miR-142-5p showedconcentration-dependent decrease for urinary4-OHPHE. The expression of miR-150elicited concentration-dependent increase for urinary1-OHNAP,2-OHNAP,2-OHPHE,and ΣOH-PAHs. However, the expression of miR-126was not significantly associated withany 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 andmiR-142-5p were both negatively associated with SDNN and rMSSD. No association wasfound between miR-150expression and any of the HRV indices. Although miR-126wasnot associated with any of the PAH metabolites, we found that the levels of miR-126werenegatively associated with rMSSD of the workers.
In this study, we evaluated the effect of airborne PAHs exposure on the plasma levelsof 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-dependentdecrease for urinary PAHs metabolites, while the expression of miR-150was positivelyassociated with the urinary PAHs metabolites. Moreover, inverse associations were alsofound between four miRNAs and HRV indices of the workers, including SDNN andrMSSD.
To summarize, our results suggest:
1) Seventy-seven miRNAs were found to be differentially expressed between AMIpatients and controls. Levels of miR-125b and miR-320b were found to be lower inAMI patients compared with controls.
2) Lower levels of two miRNAs (miR-320b and miR-125b) were associated with AMIafter adjustment for traditional risk factors, and ROC curve analysis showed thatmiR-125b and miR-320b were able to discriminate AMI from controls with modestdiagnostic accuracy.
3) These two miRNAs may also regulate the expression of genes related to CHD invascular endothelial cells.
4) There was a gradual decrease in the plasma levels of miR-125b and miR-320b acrosscategories of healthy control, stable CHD and AMI.
5) Four miRNAs (including miR-24, miR-27a, miR-320b and miR-142-5p) wereassociated 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 patientscompared with controls, and their plasma levels were significantly associated with theoccurrence 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 AMI
3) Plasma miR-125b and miR-320b may hold prognostic value for CHD
4) The relationship between plasma miRNAs, HRV, and the occurrence of AMI are novelfindings, which highlights a critical role of miRNAs in regulating environmentalfactor-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 studyis 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 investigatedin future studies.
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