MicroRNA:POPs诱导动脉粥样硬化的潜在调控者

黄风尘,王静,单秋丽,杜宇国

中国科学院生态环境研究中心环境化学与生态毒理学国家重点实验室,北京100085

持久性有机污染物(persistent organic pollutants,简称POPs)是指在环境中具有持久性、生物富集性、半挥发性,可通过各种环境介质对生物体健康产生危害的有机化合物。研究表明POPs可通过在环境中长距离的运输至未被使用和生产的地方,导致人体通过食用被污染的食物,如鱼、乳制品和肉类而暴露于POPs的污染中[1]。POPs具有较强的脂溶性,能够储存在生物体的脂质组织中,脂质组织能释放POPs至血液,成为POPs的慢性暴露源[2]。目前在全世界许多国家居民的脂质组织中都检测到了POPs残留。

动脉粥样硬化(atherosclerosis,AS)是以血管内皮损伤和脂质代谢紊乱为特征的慢性炎症疾病。心血管疾病的发生也通常由于AS的发展引起,是全球范围内导致成人死亡的首要原因。AS通常是由内皮细胞受到某些因素如高血压、高血脂等刺激发生损伤而引起功能紊乱,血液中的脂质在皮下沉积,随后单核细胞黏附在损伤处进入内皮,吞噬脂质成为泡沫细胞,形成脂肪斑。血小板也逐渐聚集并黏附于内皮的损伤处[3]。巨噬细胞、内皮细胞及血小板释放生长因子刺激平滑肌细胞进入内膜,增生并合成胶原纤维,脂肪斑演变成纤维斑块[4-5]。此时脂质进一步沉积加重巨噬细胞黏附、血小板聚集和炎性因子的释放。随着这一过程的发展,脂质不断沉积、炎性细胞逐渐浸润、纤维帽渐渐变薄,慢慢演变为不稳定斑块。不稳定斑块可发生破裂造成血栓,导致严重不良后果。

大规模的流行病学调查证实POPs与心血管疾病显著相关。在研究1976年意大利塞维索严重暴露二噁英的人群中发现,冠心病和高血压疾病明显增加[6]。Henning等人发现,居住在受多氯联苯(polychlorinated biphenyls,PCBs)污染地区的居民,因冠心病和急性心肌梗塞的住院率明显上升[7]。研究原捷克斯洛伐克暴露于二噁英(2,3,7,8-tetrachlorodibenzop-dioxin,TCDD)的工人发现,高血脂症、缺血性心脏病和AS均有显著的高发率[8]。除了流行病学的调查研究,后续研究也表明POPs会引发内皮细胞损伤、脂代谢紊乱和慢性炎症,与AS的发生发展关系密切。有研究表明PCBs能够激活氧化应激信号通路,损害内皮细胞的正常功能,诱发AS的发生发展[9-10]。多种POPs对生物体的危害都伴有脂质紊乱,即使在没有肥胖表型的生物体中,二噁英类POPs也可导致脂毒性和血脂异常。如PCB-77能导致ApoE-/-小鼠血清内极低密度脂蛋白(very low-density lipoprotein,VLDL)的上升[11]。TCDD可通过芳香烃受体(aryl hydrocarbon receptor,AhR)导致AS斑块中胆固醇的含量增高[12]。最近一项流行病学调查显示,加拿大第一民族社区居民暴露于较高水平的POPs,体内细胞因子也有明显上升,说明POPs能引起机体的炎症反应[13]。体外实验也表明,PCB-77的暴露能增加内皮细胞和脂肪细胞中促炎性因子的表达[11-14]。TCDD还可通过AhR的激活,显著增加白细胞介素8(Interleukin-8,IL-8)表达,促进巨噬细胞分化为泡沫细胞[12-15]。

目前我国尚未见POPs暴露与心血管疾病的流行病学及实验报道。然而,我国不同地区POPs的污染状况已有不少研究。我国是世界上POPs生产、使用和排放大国,尽管多氯联苯、有机氯农药已禁用多年,但化工、钢铁和冶金等行业非有意生产的POPs如TCDD等总量也非常可观。据报道,我国境内水体、底泥、土壤等环境介质及农作物、家禽家畜和人体组织、乳汁和血液中均有POPs被检出[16]。而AS在我国有相当高的发病率,我国总死亡病因分析中,每5个死亡人中就有2人死于心血管病,心血管病死亡占总死亡原因的41%,居各种死因的首位[17]。因此,POPs污染可能导致AS及其相关的健康毒性,成为我国环境健康研究人员迫切需要解决的问题。microRNA是一类在进化上高度保守的非编码小分子单链RNA(～22 nt),在基因表达调控中发挥至关重要的作用。近来有研究表明miRNAs在调节与AS病变密切相关的血管新生、炎症反应和脂质代谢等方面发挥了重要作用[18-19]。本文将从内皮细胞损伤、免疫应答和脂质代谢异常这3个主要方面探讨miRNAs对POPs诱导AS的潜在调控机制(图1)。

1 MicroRNA的生物学基础

microRNA是一类在进化上高度保守的非编码小分子单链RNA(～22 nt),在基因表达调控中发挥至关重要的作用。1993年,Lee等[20]在秀丽隐杆线虫(C.elegans)中发现了第1个可时序调控胚胎后期发育的基因lin-4,但它并不编码任何蛋白,最后证明是一对小分子RNA。直至2001年,不同国家的3个研究小组[21]在线虫、果蝇和人体中发现了近百个这样的小分子RNA。国际上将这类小分子RNA统一命名为microRNA(miRNA),其研究成为新的热点。

图1 miRNAs对POPs诱导AS的潜在调控机制Fig.1 Potential regulation mechanism of microRNAs in atherosclerosis induced by POPs

miRNAs编码基因以单拷贝、多拷贝或基因簇等形式存在基因组中,通常位于基因间或内含子区域。在细胞核,RNA聚合酶Ⅱ或RNA聚合酶Ⅲ[22]从miRNAs编码基因,转录出约几千个碱基长的初始miRNAs(pri-miRNAs),pri-miRNAs拥有颈环结构[23](miRNAs生物合成过程见图2)。随后,核酸内切酶Drosha与DGCR8组成复合物将pri-miRNAs剪切成长约70～100 nt、带茎环结构的前体 miRNAs(precursor miRNAs,pre-miRNAs)[24]。Pre-miRNAs通过 Ran-GTP依赖的核受体蛋白Exportin-5由细胞核转运至胞浆[25]。在胞浆中,pre-miRNAs被属于RNA酶Ⅲ家族的Dicer酶识别并处理成长度为19～25 nt的双链RNA(miRNA:miRNA*)。其中 miRNAs*链被很快降解,另一miRNAs链与蛋白作用形成RNA诱导沉默复合体(RNA-induced silencing complex,RISC)。与RISC结合的miRNAs,通过特异的碱基配对方式结合到靶基因mRNA的3'端非编码区,也有不少miRNAs结合到靶基因的5'端编码区或非编码区。miRNAs可以以不完全互补配对方式与靶mRNAs作用,但其中miRNAs的5'端第2至第8个核苷酸(即种子区,seed region)要与靶mRNA位点序列完全配对[26]。这一重要特征让一个miRNA能调节多个基因,使其成为基因的潜在管理者,可调节10～30%的基因表达[27]。miRNAs能够诱导靶mRNA的降解或抑制其翻译,从而在转录后水平下调靶基因表达。miRNAs参与调控范围涉及细胞分化、增殖、凋亡、代谢以及生长发育等多个过程,被广泛应用于基因功能的基础研究和人类疾病的模型研究。

2 MicroRNAs参与动脉粥样硬化的潜在机制

2.1 内皮细胞损伤

内皮细胞除了参与完成血液和组织液的代谢交换外,还提供天然的物理屏障,在保持血管稳态上发挥重要作用。血管内皮细胞损伤是动脉粥样硬化的启动因素,同时内皮细胞移行增殖形成新的血管,参与损伤血管的修复。研究显示miRNAs可调节内皮细胞结构和功能的完整性,如对细胞黏附分子表达和对细胞增殖、迁移及参与血管形成能力的调节等。

图2 microRNA生物合成过程［28］Fig.2 The canonical pathway of microRNA processing

miR-126在人的内皮细胞中特异性高表达,其功能和作用研究最为详尽深入。体外实验证实,miR-126广泛调节内皮细胞的多种功能,包括细胞迁移、细胞骨架构型、毛细血管网络完整性以及细胞存活。miR-126可通过靶向内皮细胞粘附因子-1(vascular cell adhesion molecule-1,VCAM-1)调节白细胞的粘附,从而控制血管炎症[29]。Wang等[30]在体内敲除小鼠的miR-126,结果造成血管完整性丧失以及内皮细胞增殖、迁移和血管形成能力的缺陷。miR-126能直接抑制血管内皮生长因子(vascular endothelial growth factor,VEGF)通路的负调控因子SPRED1和PIK3R2/p85β表达,促进内皮细胞的血管生成[30-31]。Zerneck等[32]人研究发现,内皮细胞损伤后,在AS斑块的凋亡小体中检测到miR-126,并被传递给周边细胞,因此增加抗免疫因子CXCL12的表达,从而减少AS的发展。AS过程中miR-21也扮演了重要角色,与非AS的动脉相比,miR-21在AS斑块中的表达显著上升[33],miR-21的表达异常可引起血管新生内膜病变和急性心肌梗塞。当内皮细胞暴露于单向剪切力时,miR-21的表达量显著增加并通过靶向PTEN增加一氧化氮合酶磷酸化和细胞内一氧化氮含量,从而减少内皮细胞凋亡[34]。另有研究表明内皮细胞中过表达miR-21能够抑制过氧化物酶体增殖物激活受体α(peroxisome proliferator-activated receptor-alpha,PPARα),从而增强VCAM-1和单核细胞趋化因子-1(monocyte chemotactic protein-1,MCP-1)的 表 达[35]。Minami等人[36]发现,冠心病人内皮祖细胞(endothelial progenitor cells,EPC)中的 miR-221和miR-222表达明显增高,且miR-221和miR-222的表达水平与EPC数量呈负相关。此外,研究者还发现miR-221和miR-222可通过负调控干细胞因子受体c-kit或eNOS,抑制内皮细胞迁移、增殖和血管新生[37-38]。在AS斑块中miR-222的低表达能上调信号转导和转录激活因子5A(signal transducer and activator of transcription 5A,STAT5A),从而调节内皮细胞增殖和迁移[39]。

miR-365在经氧化型低密度脂蛋白(oxidized lowdensity lipoprotein,ox-LDL)处理的内皮细胞中表达上调,直接作用于凋亡蛋白Bcl-2,引起内皮细胞凋亡,促进AS的发展[40]。另外,在内皮细胞中过表达miR-34a,可抑制沉默信息调节因子1(silent information regulator 1,SirT1)的表达,引起内皮细胞老化并抑制增殖[41]。而miR-125a/125b-5p则可抑制由ox-LDL引起内皮素-1(endothelin-1,ET-1)的表达,抵抗AS的发展从而保护机体[42]。

2.2 免疫应答

AS与炎症反应和自身免疫密切相关,二者贯穿AS发生发展的始终。miRNAs可调控参与AS炎性反应的细胞功能,诱导炎性因子表达,进而影响AS的发生和发展。miRNAs调控炎症反应的机制研究正逐渐成为心血管研究领域新的热点。ox-LDL是动脉粥样硬化发展中一个重要的脂蛋白,能够促进单核细胞向血管内皮下聚集,抑制巨噬细胞胆固醇外流,在泡沫细胞的形成中起到非常重要的作用。有研究表明,TCDD可以像ox-LDL一样诱导泡沫细胞的形成[43]。在加拿大的一项研究表明,体内PCBs含量与ox-LDL有良好的相关性,PCBs有可能促进LDL氧化修饰成ox-LDL[44]。在ox-LDL诱导泡沫细胞形成过程中,miRNAs也参与调控过程。miR-125a-5p在泡沫细胞形成中发挥重要作用,能够在经ox-LDL刺激的单核细胞中高表达,参与调控脂类摄取和炎性因子的变化,抑制AS的发生发展[45]。Takahashi[46]等发现,miR-146a和TLR4在冠心病人外周血中高表达。miR-146a能够抑制ox-LDL聚集,并抑制TLR4引起的免疫应答[47]。

miR-155是免疫系统中一个重要的调节者。Yao等[48]发现,冠心病人单核细胞中miR-155的表达量下调近60%。体外沉默巨噬细胞中的miR-155之后,发现脂质摄取显著增加,清道夫受体(scarenger receptor,SR)的表达上调,并且促进多种细胞因子如IL-6、IL-8和TNF-α的释放[49]。Tili[50]等人则发现,miR-155的表达上调可参与Toll样受体(Toll-like receptor,TLR)和及其相关通路的多个环节,促进TNF-α的产生;反之,miR-155也可在炎性因子的诱导下,在单核细胞中高表达。miR-125b可诱导血管平滑肌细胞中炎性因子如IL-6和MCP-1等的表达增加[51]。TLRs尤其是TLR4,与斑块的形成和稳定性有关。Th1细胞在AS发展中也发挥着重要作用。Guo[52]等人发现在单核细胞过表达miR-146a可上调Th1细胞功能,而miR-146a能够诱导 TNF-α、MCP-1 和 NF-κB p65 的表达而促进炎症发展。在巨噬细胞中,多种相关刺激则可引起miR-147对TLRs相关信号通路的负向调节[53]。

2.3 脂质代谢异常

大量临床和流行病学研究证实,血液中胆固醇含量增高是AS最重要的危险因素之一,且胆固醇和胆固醇酯还是AS斑块脂质核心的组成成分。miR-122是肝脏miRNAs表达谱中含量最为丰富的一种miRNA[54]。研究发现,miR-122可参与调控胆固醇的合成过程。在小鼠体内抑制miR-122后,胆固醇合成的限速酶3-羟基-3-甲基戊二酰基-辅酶 A还原酶(HMGCR)活性下降约45%,血浆胆固醇水平下降40%[55]。在高脂喂养的小鼠体内拮抗miR-122的表达则可显著改善脂肪肝,并减少肝脏中甘油三酯含量和增加脂肪酸的氧化。miR-122的这些功能可以作为治疗脂肪肝或动脉粥样硬化的潜在靶标[56]。固醇反应元件结合蛋白(sterol-response element binding protein,SREBP)是胆固醇合成的转录调节因子,能够调节参与细胞胆固醇运输基因的表达,此基因内含子可编码 miR-33。miR-33能够抑制 ABCA1(ATP binding cassette A1)的表达,从而减少胆固醇流出至脂蛋白A1。在小鼠巨噬细胞中,miR-33可以靶向ABCA1,减少胆固醇流出到初期的HDL。沉默miR-33的表达,能显著增加肝脏中ABCA1的表达和血浆中HDL的水平[57]。此外,miR-33还能直接作用于胰岛素受体底物2(insulin receptor substrate 2,IRS2),影响胰岛素信号通路[58]。另有文献报道miR-758也能靶向ABCA1的3'端非编码区,过表达miR-758则可抑制人和小鼠巨噬细胞及干细胞中ABCA1的表达,减少胆固醇流出到脂蛋白A1[59]。表1对miRNAs在AS发生发展中的作用进行了总结。

表1 microRNAs在动脉粥样硬化中作用Table 1 Role of microRNAs in atherosclerosis

PPARα 内皮细胞功能紊乱Endothelial cells dysfunction[35]miR-221/222 c-kite/NOS/STAT5A 抑制内皮细胞迁移、增殖和血管新生Migration,proliferation of endothelial cells and angiogenesis [32][33][39]miR-365 Bcl-2 促内皮细胞凋亡Promote apoptosis of endothelial cells [40]miR-34a SirT1 促进内皮细胞老化和抑制增殖Promote aging of endothelial cells and inhibit proliferation [41]miR-125a/125b-5p ET-1 抑制内皮细胞功能紊乱Inhibit endothelial cells dysfunction [42]Immune response miR-155 MyD88 抑制免疫响应Inhibit immune response免疫应答[49]FADD 调节脂多糖信号通路Regulate signaling pathway of lipopolysaccharide [50]miR-125a-5p ORP9 抑制细胞因子分泌Inhibit secretion of cytokine[45]miR-125b IL-6 MCP-1诱导炎症因子表达Induce expression of inflammatory factor [51]miR-146a TLR4 抑制Toll样受体免疫应答Inhibit immune response of Toll-like receptors [47]TNF-α/MCP-1/NF-κB诱导促炎症细胞因子表达Induce expression of pro-inflammatory cytokine [52]miR-147 TLR2/TLR3/TLR4 负向调控Toll样受体Negative regulate Toll-like receptors [53]Abnormal lipid metabolism miR-122 HMGCR 抑制胆固醇合成Inhibit synthesis of cholesterol脂代谢异常[55]miR-33 SREBP 调节胆固醇合成Regulate synthesis of cholesterol [57]ABCA1 ABCG1抑制胆固醇流出Inhibit cholesterol efflux[57]IRS2 调节胰岛素信号通路Regulate insulin signaling pathway [58]miR-785 ABCA1 抑制胆固醇流出Inhibit cholesterol efflux[59]

3 展望

虽然miRNAs生物学功能的研究越来越多,POPs也显示可引起生物体内 miRNAs的表达紊乱[60],然而POPs暴露与AS有关的miRNAs的研究尚属空白。研究POPs暴露与AS有关的miRNAs,寻找POPs引致AS机制中可能的miRNAs,可将miRNAs作为预防和治疗POPs相关AS疾病的生物靶标。当然如果想达到这一目标,还有一系列的问题有待解决。

首先,miRNAs表达分析的检测技术受到限制。先进的检测技术能准确有效地检测miRNAs,从而使miRNAs作为环境污染物相关疾病的指示物成为可能。目前主要的miRNA研究技术有Northern印迹分析、miRNA芯片、实时定量PCR(Quantitative realtime PCR)。Northern印迹分析操作繁琐、耗时费力、灵敏度低;实时定量PCR虽具有较高的灵敏度和特异性,但不适于大规模筛选;miRNA芯片可进行大规模的筛选,但无法清楚的区分序列差异很小的miRNAs。所以目前尚无哪一种方法能够达到高通量、灵敏及准确,在miRNAs的检测方法上还有相当长的路要走。

其次,miRNAs的作用靶点及其功能尚未明确。随着检测技术的进步,越来越多的miRNAs被检测出来,但是这些miRNAs作用的靶基因以及生物学功能尚未完全明确。由于miRNA与靶mRNA序列不完全配对便可发挥作用,因此一个miRNA可以实现多个靶标mRNA的调节。目前对于miRNA靶基因的预测只能通过在线分析软件,根据算法的不同又分为第一代和第二代测序软件。第一代预测软件大多是从种子区互补这一规则出发设计的算法,如Mi-Randa[61]、MIRBase[62]、DIANA LAB[63]等。第二代预测软件更倾向于机器学习方法训练参数对靶基因的预测,如 PicTar[64]、RNA22[65]和 microTar[66]等。但是不同的算法所得到的预测结果不一致并且不能预测所有的miRNA,还需要利用荧光定量PCR及Western blot分别检测转染或抑制miRNA后细胞中靶基因mRNA水平及蛋白水平的变化,从而确定miRNA与靶基因的对应关系,进一步还可通过荧光素酶报告基因法来鉴定miRNA的靶位点。通过实验研究确定和证实更多的靶基因有助于理解miRNA的生物学功能。

最后,与环境疾病相关的特异性miRNA尚待发现。miRNA作为疾病的检测和治疗靶标已经研究多年。血浆中的miR-210和miR-141分别是胰腺癌和前列腺癌临床诊断的指标[67-68]。miR-9和miR-223可以作为卵巢癌复发的标记[69]。目前对于miRNA与AS之间的研究还集中于正常条件和病理条件下miRNA表达图谱的分析及其功能的研究。发现与环境污染物特异性的miRNAs不仅有利于对环境疾病机理的理解,还可作为一种新兴的生物监测和疾病预防的靶标。因此研究与环境疾病相关的miRNAs和其调节的基因非常重要,对miRNAs在AS中的调节机制以及应用还需深入探索。

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