【作者】 朱国富；

【导师】 杨丽霞；

【作者基本信息】 第三军医大学 ， 内科学， 2013， 博士

【摘要】 研究背景及目的：动脉粥样硬化性心血管疾病是全球发病率和死亡率居首位的疾病。大量的证据表明炎症和免疫反应在动脉粥样硬化(atherosclerosis,AS)的发生发展中有举足轻重的作用，从脂纹的形成到最终急性冠脉综合征(acute coronarysyndromes,ACS)的发生。小分子RNA(MicroRNAs,miRNAs)已被证明参与炎症和免疫反应。 miRNAs是长度为18-22个核苷酸的非编码RNA分子，通过抑制蛋白翻译或引起mRNA降解，从而在转录后水平调控基因的表达。在众多的miRNA，炎症相关的miRNA microRNA-155(miR-155)已被发现参与动脉粥样硬化。目前认为AS为体内外多种因素综合作用的结果，凋亡的增加为其中的因素之一，而凋亡增加确切的分子机制尚不明确。巨噬细胞凋亡已被证实为进展期动脉粥样硬化斑块的一个突出特点。进展期动脉粥样硬化病变的大量病理研究提示：巨噬细胞凋亡和大的坏死核心密切相关，并最终促进斑块破裂和急性血管事件。氧化型低密度脂蛋白(Oxidized low-density lipoprotein,OxLDL)是一个重要的动脉粥样硬化致病因子，也是一个潜在的细胞凋亡诱导剂。以前的研究已经表明，OxLDL诱导了多种组织和细胞的凋亡，包括内皮细胞，平滑肌细胞和巨噬细胞。一个由死亡受体家族，主要涉及Fas/FasL的信号和下游的caspase-3，介导的外源性凋亡途径，已被认为是OxLDL介导的细胞凋亡的机制之一。由于巨噬细胞凋亡在动脉粥样硬化和斑块不稳定中起着重要作用，细胞凋亡的调节可对进展期动脉粥样硬化斑块破裂提供显著的保护。越来越多的证据表明，细胞凋亡可由miRNA调控。作为一个多功能的miRNA，miR-155被证实参与免疫反应和细胞凋亡通路的调节。miR-155和动脉粥样硬化疾病的进展之间的关系目前还不清楚。这项研究首先旨在研究miR-155与冠状动脉狭窄病变的严重程度及斑块稳定性的关系。此外，鉴于其在凋亡途径中的重要作用，我们推测miR-155在OxLDL诱导的巨噬细胞凋亡中扮演重要角色。因此，本研究的主要目的是为了证明miR-155在OxLDL诱导的巨噬细胞凋亡中的作用及其具体机制。研究方法：(1)我们收集了110例疑似冠心病(coronary heart disease,CHD)患者，行冠状动脉CTA及冠状动脉造影检查冠脉病变情况，根据冠状动脉造影结果进行Gensini评分，评估冠状动脉狭窄病变的严重程度和范围；根据冠状动脉CTA评估斑块稳定性采用实时定量PCR方法检测了新鲜分离的外周血单个核细胞(peripheral bloodmonouclear cell,PBMCs)和血浆中miR-155的表达。(2)用不同浓度及时间梯度的OxLDL处理RAW264.7细胞，采用实时定量PCR方法检测miR-155的表达水平。(3)miR-155的模拟物，miR-155的抑制剂和阴性对照被瞬时转染入RAW264.7细胞或HEK-293细胞以实现miR-155的表达水平的上调或下调。或FADD表达载体被用来感染RAW264.7细胞以实现FADD表达水平的上调。(4)我们采用细胞活力实验(细胞计数试剂盒-8)检测oxLDL处理过的RAW264.7细胞的活力。(5)采用TUNEL法、双染色流式法(Annexin V/PI)和caspase-3的活化片段(17kDa)的蛋白表达等方法评估巨噬细胞凋亡。(6)采用生物信息学技术预测miR-155的潜在靶标。(7)采用荧光素酶实验来验证假定的miR-155靶标。(8)免疫印迹法(WB)用于检测caspase-3和FADD的蛋白表达水平。研究结果：(1)56例冠心病患者PBMCs及血浆中miR-155的表达水平明显低于54例非冠心病患者(P <0.01)。(2)不稳定性心绞痛组(unstable angina pectoris, UAP,n=31)和急性心梗组(acute myocardial infarction,AMI,n=24)患者PBMCs及血浆中miR-155的水平明显低于胸痛综合征组(chest pain syndrome,CPS，n=31)组，而稳定性心绞痛组(stable angina pectoris,SAP，n=24)和CPS组患者之间无统计学差异。(3)Spearman相关分析表明，血浆中miR-155的表达水平与PBMCs中miR-155的表达水平呈正相关(4)有2支或大于等于3支病变血管患者的miR-155水平明显低于0和1支病变血管的患者。0支和1支病变血管的患者之间miR-155的水平没有显着差异。(5)miR-155水平与Gensini积分呈负相关(r=-0.663，P <0.001)。(6)软斑块组miR-155的表达水平较纤维斑块组及钙化斑块组降低(P<0.05)。(7)miR-155的表达水平与年龄(r=-0.227)、高血压(R=-0.440)、总胆固醇(R=0.239)、高密度脂蛋白(HDL)胆固醇(R=0.280)、低密度脂蛋白(LDL)胆固醇(r=-0.315)、吸烟(R=-0.363)、血管紧张素转换酶抑制剂(R=-0.250)、他汀类药物(r=-0.368)，高敏C-反应蛋白(hs-CRP)(r=-0.515)等相关。(8)OxLDL以剂量和时间依赖方式增加RAW264.7细胞中miR-155的表达。(9)OxLDL能显著增加RAW264.7细胞凋亡，miR-155模拟物能抑制此作用，而miR-155的拮抗剂能抵消miR-155的抗凋亡作用。(10)生物信息学分析显示FADD可能为miR-155的直接靶标。(11)萤光素酶报告基因检测和WB实验进一步证实miR-155直接通过作用于FADD的3’-UTR区域从而抑制FADD表达。(12)miR-155抑制oxLDL诱导的细胞凋亡，但FADD过表达限制了miR-155的抗凋亡活性。研究结论：miR-155的表达水平与复杂的致动脉粥样硬化的代谢危险因素、冠状动脉狭窄病变的严重程度(由Gensini积分评估)、斑块稳定性(由冠脉CTA评估)呈负相关，提示miR-155在动脉粥样硬化进展中起保护作用。此外，我们的研究结果表明，miR-155通过作用于其靶标FADD，抑制oxLDL诱导的RAW264.7细胞凋亡，可望提供一种新的动脉粥样硬化的治疗方式。更多还原

【Abstract】 Background and Objective: Atherosclerotic cardiovascular disease is the leadingcause of morbidity and mortality worldwide. A large amount of evidence supports a pivotalrole of inflammation and immune responses in all phases of atherosclerosis(AS), frominitiation of the fatty streak to final breakout of acute coronary syndromes (ACS).MicroRNAs (miRNAs) have been demonstrated to be associated with inflammation andimmune responses. miRNAs are18-22nucleotide long non-coding RNA molecules thatregulate gene expression posttranscriptionally by triggering either translation repression orRNA degradation. Among various miRNA, inflammation-related miRNAs-microRNA-155(miR-155) have been found to be involved in the atherogenesis.The etiology of AS is ascribed to various factors, one of which is the increase inapoptosis. The underlying molecular mechanisms of the increase in apoptosis remainlargely unknown. Macrophage apoptosis has been identified as a prominent feature ofadvanced atherosclerotic plaques. Pathological studies of advanced atherosclerotic lesionshave revealed a strong correlation between macrophage apoptosis and large necrotic cores,thought to promote plaque rupture and acute vascular events. Oxidized low-densitylipoprotein (OxLDL) is an important pathogenetic factor in atherosclerosis and a potentialinducer of cell apoptosis. Previous studies have demonstrated OxLDL-induced apoptosis ina variety of tissues and cells, including endothelial cells, smooth muscle cells (SMCs) andmacrophages. An extrinsic pathway mediated by the death receptor family, mainlyinvolving Fas/FasL signaling and down-stream caspase-3, has been defined as theunderlying mechanism in OxLDL-mediated apoptosis. Since macrophage apoptosis playsan important role in atherogenesis and plaque destabilization, modulation of cell death mayprovide significant protection against advanced atherosclerotic plaque rupture. Accumulating evidence indicates that the apoptotic machinery is regulated by miRNAs. Asa multi-functional miRNA, miR-155was previously shown to participate in the regulationof immunological responses and apoptotic pathways.The relationship between miR-155and the progression of atherosclerotic diseases isunclear. This study first aimed to investigate the association between miR-155and theseverity and extent of coronary stenotic lesions and atherosclerotic plaques stability.Moreover,given its crucial involvement in apoptotic pathways, we hypothesized thatmiR-155plays a role in oxLDL-induced macrophage apoptosis. Accordingly, the primaryaim of the current study was to address the specific role of miR-155in oxLDL-inducedmacrophage apoptosis and clarify the underlying mechanisms.Methods:(1) We measured the miR-155expression in both freshly isolated peripheralblood monouclear cell (PBMCs) and plasma by real-time PCR in110consecutive patientsundergoing coronary computed tomography angiography (CTA) and coronary angiographyfor suspected coronary heart disease (CHD). The severity and extent of coronary stenoticlesions were evaluated on coronary angiography findings by Gensini score and the plaquesstability was evaluated on coronary CTA.(2) qRT-PCR was performed to detect miR-155expression in oxLDL-treated RAW264.7cells.(3) miR-155mimics, miR-155inhibitor,andnegative control were transiently transfected into RAW264.7cells or HEK-293cells to up-or down-regulate the miR-155expression level. Or the fas-associated deathdomain-containing protein (FADD)-expressing vector was used to infect RAW264.7cellsto up-regulate the FADD expression level.(4)Cell viability assays (Cell Counting Kit-8)was performed in oxLDL-treated RAW264.7cells,with or without transfection of miR-155mimics/inhibitor.(5) TUNEL assay, Annexin V/propidium iodide (PI) staining,andcleaved caspase-3(17kDa) protein expression were used to assess the macrophageapoptosis.(6) Bioinformatics technique was used to predict the potential target of miR-155.(7) Luciferase assays was performed to validate the putative miR-155target.(8) Westernblotting (WB) was used to detect caspase-3and FADD protein expression.Results:(1)The miR-155expression was significantly lower in56patients with CHDthan those in54controls (p <0.01).(2) The level of miR-155in PBMCs or plasma waslower in patients with unstable angina pectoris (UAP,n=31) and acute myocardial infarction(AMI,n=24) than in patients with chest pain syndrome (CPS,n=31), while no statistically significant differences were observed between patients with stable angina pectoris(SAP,n=24) and CPS.(3) Spearman’s correlation analysis showed that the expression ofmiR-155in plasma positively correlated with the expression in PBMCs.(4) The levels ofmiR-155in the subjects with diseased vessels of2and>=3were significantly lower thanthose with diseased vessel of0and1. The levels of miR-155were not significantlydifferent among groups with diseased vessels of0and1.(5) miR-155were negativelyassociated with Gensini scores(r=-0.663,p<0.001).(6) The miR-155level in patiens of softplaque is significantly lower than those with calcified plaque and fibrous plaque.(7) ThemiR-155expression was significantly correlated to age (r=-0.227), hypertension (r=-0.440),total cholesterol (r=0.239), high-density lipoprotein (HDL) cholesterol (r=0.280),low-density lipoprotein(LDL) cholesterol (r=-0.315), tobacco (r=-0.363),ACEI (r=-0.250),statins(r=-0.368)，high sensitivity C-reactive protein (hs-CRP)(r=-0.515).(8) Constitutiveexpression of miR-155in RAW264.7cells was increased following stimulation withOxLDL in a dose-and time-dependent manner.(9) OxLDL-treated RAW264.7cells showeda marked increase in apoptosis, which was suppressed in the presence of miR-155mimicsand increased with antagonists of miR-155.(10) Bioinformatics analysis revealed FADD asa putative target of miR-155.(11) Luciferase reporter assay and WB further disclosed thatmiR-155inhibits FADD expression by directly targeting the3’-UTR region.(12) miR-155inhibits oxLDL-induced apoptosis, but overexpression of FADD limits this anti-apoptoticactivity.Conclusion: miR-155expression is inversely associated with complicatedproatherogenic metabolic risk factors, and the severity of coronary stenotic lesionscalculated by Gensini scores, supporting a protective role for miR-155against theprogression of atherosclerosis. Moreover,our results collectively suggest that miR-155attenuates apoptosis of OxLDL-mediated RAW264.7cells by targeting FADD, supporting apossible therapeutic role in atherosclerosis.更多还原