黃風(fēng)塵,王靜,單秋麗,杜宇國

中國科學(xué)院生態(tài)環(huán)境研究中心環(huán)境化學(xué)與生態(tài)毒理學(xué)國家重點實驗室,北京100085

持久性有機(jī)污染物(persistent organic pollutants,簡稱POPs)是指在環(huán)境中具有持久性、生物富集性、半揮發(fā)性,可通過各種環(huán)境介質(zhì)對生物體健康產(chǎn)生危害的有機(jī)化合物。研究表明POPs可通過在環(huán)境中長距離的運輸至未被使用和生產(chǎn)的地方,導(dǎo)致人體通過食用被污染的食物,如魚、乳制品和肉類而暴露于POPs的污染中[1]。POPs具有較強(qiáng)的脂溶性,能夠儲存在生物體的脂質(zhì)組織中,脂質(zhì)組織能釋放POPs至血液,成為POPs的慢性暴露源[2]。目前在全世界許多國家居民的脂質(zhì)組織中都檢測到了POPs殘留。

動脈粥樣硬化(atherosclerosis,AS)是以血管內(nèi)皮損傷和脂質(zhì)代謝紊亂為特征的慢性炎癥疾病。心血管疾病的發(fā)生也通常由于AS的發(fā)展引起,是全球范圍內(nèi)導(dǎo)致成人死亡的首要原因。AS通常是由內(nèi)皮細(xì)胞受到某些因素如高血壓、高血脂等刺激發(fā)生損傷而引起功能紊亂,血液中的脂質(zhì)在皮下沉積,隨后單核細(xì)胞黏附在損傷處進(jìn)入內(nèi)皮,吞噬脂質(zhì)成為泡沫細(xì)胞,形成脂肪斑。血小板也逐漸聚集并黏附于內(nèi)皮的損傷處[3]。巨噬細(xì)胞、內(nèi)皮細(xì)胞及血小板釋放生長因子刺激平滑肌細(xì)胞進(jìn)入內(nèi)膜,增生并合成膠原纖維,脂肪斑演變成纖維斑塊[4-5]。此時脂質(zhì)進(jìn)一步沉積加重巨噬細(xì)胞黏附、血小板聚集和炎性因子的釋放。隨著這一過程的發(fā)展,脂質(zhì)不斷沉積、炎性細(xì)胞逐漸浸潤、纖維帽漸漸變薄,慢慢演變?yōu)椴环€(wěn)定斑塊。不穩(wěn)定斑塊可發(fā)生破裂造成血栓,導(dǎo)致嚴(yán)重不良后果。

大規(guī)模的流行病學(xué)調(diào)查證實POPs與心血管疾病顯著相關(guān)。在研究1976年意大利塞維索嚴(yán)重暴露二噁英的人群中發(fā)現(xiàn),冠心病和高血壓疾病明顯增加[6]。Henning等人發(fā)現(xiàn),居住在受多氯聯(lián)苯(polychlorinated biphenyls,PCBs)污染地區(qū)的居民,因冠心病和急性心肌梗塞的住院率明顯上升[7]。研究原捷克斯洛伐克暴露于二噁英(2,3,7,8-tetrachlorodibenzop-dioxin,TCDD)的工人發(fā)現(xiàn),高血脂癥、缺血性心臟病和AS均有顯著的高發(fā)率[8]。除了流行病學(xué)的調(diào)查研究,后續(xù)研究也表明POPs會引發(fā)內(nèi)皮細(xì)胞損傷、脂代謝紊亂和慢性炎癥,與AS的發(fā)生發(fā)展關(guān)系密切。有研究表明PCBs能夠激活氧化應(yīng)激信號通路,損害內(nèi)皮細(xì)胞的正常功能,誘發(fā)AS的發(fā)生發(fā)展[9-10]。多種POPs對生物體的危害都伴有脂質(zhì)紊亂,即使在沒有肥胖表型的生物體中,二噁英類POPs也可導(dǎo)致脂毒性和血脂異常。如PCB-77能導(dǎo)致ApoE-/-小鼠血清內(nèi)極低密度脂蛋白(very low-density lipoprotein,VLDL)的上升[11]。TCDD可通過芳香烴受體(aryl hydrocarbon receptor,AhR)導(dǎo)致AS斑塊中膽固醇的含量增高[12]。最近一項流行病學(xué)調(diào)查顯示,加拿大第一民族社區(qū)居民暴露于較高水平的POPs,體內(nèi)細(xì)胞因子也有明顯上升,說明POPs能引起機(jī)體的炎癥反應(yīng)[13]。體外實驗也表明,PCB-77的暴露能增加內(nèi)皮細(xì)胞和脂肪細(xì)胞中促炎性因子的表達(dá)[11-14]。TCDD還可通過AhR的激活,顯著增加白細(xì)胞介素8(Interleukin-8,IL-8)表達(dá),促進(jìn)巨噬細(xì)胞分化為泡沫細(xì)胞[12-15]。

目前我國尚未見POPs暴露與心血管疾病的流行病學(xué)及實驗報道。然而,我國不同地區(qū)POPs的污染狀況已有不少研究。我國是世界上POPs生產(chǎn)、使用和排放大國,盡管多氯聯(lián)苯、有機(jī)氯農(nóng)藥已禁用多年,但化工、鋼鐵和冶金等行業(yè)非有意生產(chǎn)的POPs如TCDD等總量也非?？捎^。據(jù)報道,我國境內(nèi)水體、底泥、土壤等環(huán)境介質(zhì)及農(nóng)作物、家禽家畜和人體組織、乳汁和血液中均有POPs被檢出[16]。而AS在我國有相當(dāng)高的發(fā)病率,我國總死亡病因分析中,每5個死亡人中就有2人死于心血管病,心血管病死亡占總死亡原因的41%,居各種死因的首位[17]。因此,POPs污染可能導(dǎo)致AS及其相關(guān)的健康毒性,成為我國環(huán)境健康研究人員迫切需要解決的問題。microRNA是一類在進(jìn)化上高度保守的非編碼小分子單鏈RNA(～22 nt),在基因表達(dá)調(diào)控中發(fā)揮至關(guān)重要的作用。近來有研究表明miRNAs在調(diào)節(jié)與AS病變密切相關(guān)的血管新生、炎癥反應(yīng)和脂質(zhì)代謝等方面發(fā)揮了重要作用[18-19]。本文將從內(nèi)皮細(xì)胞損傷、免疫應(yīng)答和脂質(zhì)代謝異常這3個主要方面探討miRNAs對POPs誘導(dǎo)AS的潛在調(diào)控機(jī)制(圖1)。

1 MicroRNA的生物學(xué)基礎(chǔ)

microRNA是一類在進(jìn)化上高度保守的非編碼小分子單鏈RNA(～22 nt),在基因表達(dá)調(diào)控中發(fā)揮至關(guān)重要的作用。1993年,Lee等[20]在秀麗隱桿線蟲(C.elegans)中發(fā)現(xiàn)了第1個可時序調(diào)控胚胎后期發(fā)育的基因lin-4,但它并不編碼任何蛋白,最后證明是一對小分子RNA。直至2001年,不同國家的3個研究小組[21]在線蟲、果蠅和人體中發(fā)現(xiàn)了近百個這樣的小分子RNA。國際上將這類小分子RNA統(tǒng)一命名為microRNA(miRNA),其研究成為新的熱點。

圖1 miRNAs對POPs誘導(dǎo)AS的潛在調(diào)控機(jī)制Fig.1 Potential regulation mechanism of microRNAs in atherosclerosis induced by POPs

miRNAs編碼基因以單拷貝、多拷貝或基因簇等形式存在基因組中,通常位于基因間或內(nèi)含子區(qū)域。在細(xì)胞核,RNA聚合酶Ⅱ或RNA聚合酶Ⅲ[22]從miRNAs編碼基因,轉(zhuǎn)錄出約幾千個堿基長的初始miRNAs(pri-miRNAs),pri-miRNAs擁有頸環(huán)結(jié)構(gòu)[23](miRNAs生物合成過程見圖2)。隨后,核酸內(nèi)切酶Drosha與DGCR8組成復(fù)合物將pri-miRNAs剪切成長約70～100 nt、帶莖環(huán)結(jié)構(gòu)的前體 miRNAs(precursor miRNAs,pre-miRNAs)[24]。Pre-miRNAs通過 Ran-GTP依賴的核受體蛋白Exportin-5由細(xì)胞核轉(zhuǎn)運至胞漿[25]。在胞漿中,pre-miRNAs被屬于RNA酶Ⅲ家族的Dicer酶識別并處理成長度為19～25 nt的雙鏈RNA(miRNA:miRNA*)。其中 miRNAs*鏈被很快降解,另一miRNAs鏈與蛋白作用形成RNA誘導(dǎo)沉默復(fù)合體(RNA-induced silencing complex,RISC)。與RISC結(jié)合的miRNAs,通過特異的堿基配對方式結(jié)合到靶基因mRNA的3'端非編碼區(qū),也有不少miRNAs結(jié)合到靶基因的5'端編碼區(qū)或非編碼區(qū)。miRNAs可以以不完全互補(bǔ)配對方式與靶mRNAs作用,但其中miRNAs的5'端第2至第8個核苷酸(即種子區(qū),seed region)要與靶mRNA位點序列完全配對[26]。這一重要特征讓一個miRNA能調(diào)節(jié)多個基因,使其成為基因的潛在管理者,可調(diào)節(jié)10～30%的基因表達(dá)[27]。miRNAs能夠誘導(dǎo)靶mRNA的降解或抑制其翻譯,從而在轉(zhuǎn)錄后水平下調(diào)靶基因表達(dá)。miRNAs參與調(diào)控范圍涉及細(xì)胞分化、增殖、凋亡、代謝以及生長發(fā)育等多個過程,被廣泛應(yīng)用于基因功能的基礎(chǔ)研究和人類疾病的模型研究。

2 MicroRNAs參與動脈粥樣硬化的潛在機(jī)制

2.1 內(nèi)皮細(xì)胞損傷

內(nèi)皮細(xì)胞除了參與完成血液和組織液的代謝交換外,還提供天然的物理屏障,在保持血管穩(wěn)態(tài)上發(fā)揮重要作用。血管內(nèi)皮細(xì)胞損傷是動脈粥樣硬化的啟動因素,同時內(nèi)皮細(xì)胞移行增殖形成新的血管,參與損傷血管的修復(fù)。研究顯示miRNAs可調(diào)節(jié)內(nèi)皮細(xì)胞結(jié)構(gòu)和功能的完整性,如對細(xì)胞黏附分子表達(dá)和對細(xì)胞增殖、遷移及參與血管形成能力的調(diào)節(jié)等。

圖2 microRNA生物合成過程［28］Fig.2 The canonical pathway of microRNA processing

miR-126在人的內(nèi)皮細(xì)胞中特異性高表達(dá),其功能和作用研究最為詳盡深入。體外實驗證實,miR-126廣泛調(diào)節(jié)內(nèi)皮細(xì)胞的多種功能,包括細(xì)胞遷移、細(xì)胞骨架構(gòu)型、毛細(xì)血管網(wǎng)絡(luò)完整性以及細(xì)胞存活。miR-126可通過靶向內(nèi)皮細(xì)胞粘附因子-1(vascular cell adhesion molecule-1,VCAM-1)調(diào)節(jié)白細(xì)胞的粘附,從而控制血管炎癥[29]。Wang等[30]在體內(nèi)敲除小鼠的miR-126,結(jié)果造成血管完整性喪失以及內(nèi)皮細(xì)胞增殖、遷移和血管形成能力的缺陷。miR-126能直接抑制血管內(nèi)皮生長因子(vascular endothelial growth factor,VEGF)通路的負(fù)調(diào)控因子SPRED1和PIK3R2/p85β表達(dá),促進(jìn)內(nèi)皮細(xì)胞的血管生成[30-31]。Zerneck等[32]人研究發(fā)現(xiàn),內(nèi)皮細(xì)胞損傷后,在AS斑塊的凋亡小體中檢測到miR-126,并被傳遞給周邊細(xì)胞,因此增加抗免疫因子CXCL12的表達(dá),從而減少AS的發(fā)展。AS過程中miR-21也扮演了重要角色,與非AS的動脈相比,miR-21在AS斑塊中的表達(dá)顯著上升[33],miR-21的表達(dá)異?？梢鹧苄律鷥?nèi)膜病變和急性心肌梗塞。當(dāng)內(nèi)皮細(xì)胞暴露于單向剪切力時,miR-21的表達(dá)量顯著增加并通過靶向PTEN增加一氧化氮合酶磷酸化和細(xì)胞內(nèi)一氧化氮含量,從而減少內(nèi)皮細(xì)胞凋亡[34]。另有研究表明內(nèi)皮細(xì)胞中過表達(dá)miR-21能夠抑制過氧化物酶體增殖物激活受體α(peroxisome proliferator-activated receptor-alpha,PPARα),從而增強(qiáng)VCAM-1和單核細(xì)胞趨化因子-1(monocyte chemotactic protein-1,MCP-1)的 表 達(dá)[35]。Minami等人[36]發(fā)現(xiàn),冠心病人內(nèi)皮祖細(xì)胞(endothelial progenitor cells,EPC)中的 miR-221和miR-222表達(dá)明顯增高,且miR-221和miR-222的表達(dá)水平與EPC數(shù)量呈負(fù)相關(guān)。此外,研究者還發(fā)現(xiàn)miR-221和miR-222可通過負(fù)調(diào)控干細(xì)胞因子受體c-kit或eNOS,抑制內(nèi)皮細(xì)胞遷移、增殖和血管新生[37-38]。在AS斑塊中miR-222的低表達(dá)能上調(diào)信號轉(zhuǎn)導(dǎo)和轉(zhuǎn)錄激活因子5A(signal transducer and activator of transcription 5A,STAT5A),從而調(diào)節(jié)內(nèi)皮細(xì)胞增殖和遷移[39]。

miR-365在經(jīng)氧化型低密度脂蛋白(oxidized lowdensity lipoprotein,ox-LDL)處理的內(nèi)皮細(xì)胞中表達(dá)上調(diào),直接作用于凋亡蛋白Bcl-2,引起內(nèi)皮細(xì)胞凋亡,促進(jìn)AS的發(fā)展[40]。另外,在內(nèi)皮細(xì)胞中過表達(dá)miR-34a,可抑制沉默信息調(diào)節(jié)因子1(silent information regulator 1,SirT1)的表達(dá),引起內(nèi)皮細(xì)胞老化并抑制增殖[41]。而miR-125a/125b-5p則可抑制由ox-LDL引起內(nèi)皮素-1(endothelin-1,ET-1)的表達(dá),抵抗AS的發(fā)展從而保護(hù)機(jī)體[42]。

2.2 免疫應(yīng)答

AS與炎癥反應(yīng)和自身免疫密切相關(guān),二者貫穿AS發(fā)生發(fā)展的始終。miRNAs可調(diào)控參與AS炎性反應(yīng)的細(xì)胞功能,誘導(dǎo)炎性因子表達(dá),進(jìn)而影響AS的發(fā)生和發(fā)展。miRNAs調(diào)控炎癥反應(yīng)的機(jī)制研究正逐漸成為心血管研究領(lǐng)域新的熱點。ox-LDL是動脈粥樣硬化發(fā)展中一個重要的脂蛋白,能夠促進(jìn)單核細(xì)胞向血管內(nèi)皮下聚集,抑制巨噬細(xì)胞膽固醇外流,在泡沫細(xì)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一樣誘導(dǎo)泡沫細(xì)胞的形成[43]。在加拿大的一項研究表明,體內(nèi)PCBs含量與ox-LDL有良好的相關(guān)性,PCBs有可能促進(jìn)LDL氧化修飾成ox-LDL[44]。在ox-LDL誘導(dǎo)泡沫細(xì)胞形成過程中,miRNAs也參與調(diào)控過程。miR-125a-5p在泡沫細(xì)胞形成中發(fā)揮重要作用,能夠在經(jīng)ox-LDL刺激的單核細(xì)胞中高表達(dá),參與調(diào)控脂類攝取和炎性因子的變化,抑制AS的發(fā)生發(fā)展[45]。Takahashi[46]等發(fā)現(xiàn),miR-146a和TLR4在冠心病人外周血中高表達(dá)。miR-146a能夠抑制ox-LDL聚集,并抑制TLR4引起的免疫應(yīng)答[47]。

miR-155是免疫系統(tǒng)中一個重要的調(diào)節(jié)者。Yao等[48]發(fā)現(xiàn),冠心病人單核細(xì)胞中miR-155的表達(dá)量下調(diào)近60%。體外沉默巨噬細(xì)胞中的miR-155之后,發(fā)現(xiàn)脂質(zhì)攝取顯著增加,清道夫受體(scarenger receptor,SR)的表達(dá)上調(diào),并且促進(jìn)多種細(xì)胞因子如IL-6、IL-8和TNF-α的釋放[49]。Tili[50]等人則發(fā)現(xiàn),miR-155的表達(dá)上調(diào)可參與Toll樣受體(Toll-like receptor,TLR)和及其相關(guān)通路的多個環(huán)節(jié),促進(jìn)TNF-α的產(chǎn)生;反之,miR-155也可在炎性因子的誘導(dǎo)下,在單核細(xì)胞中高表達(dá)。miR-125b可誘導(dǎo)血管平滑肌細(xì)胞中炎性因子如IL-6和MCP-1等的表達(dá)增加[51]。TLRs尤其是TLR4,與斑塊的形成和穩(wěn)定性有關(guān)。Th1細(xì)胞在AS發(fā)展中也發(fā)揮著重要作用。Guo[52]等人發(fā)現(xiàn)在單核細(xì)胞過表達(dá)miR-146a可上調(diào)Th1細(xì)胞功能,而miR-146a能夠誘導(dǎo) TNF-α、MCP-1 和 NF-κB p65 的表達(dá)而促進(jìn)炎癥發(fā)展。在巨噬細(xì)胞中,多種相關(guān)刺激則可引起miR-147對TLRs相關(guān)信號通路的負(fù)向調(diào)節(jié)[53]。

2.3 脂質(zhì)代謝異常

大量臨床和流行病學(xué)研究證實,血液中膽固醇含量增高是AS最重要的危險因素之一,且膽固醇和膽固醇酯還是AS斑塊脂質(zhì)核心的組成成分。miR-122是肝臟miRNAs表達(dá)譜中含量最為豐富的一種miRNA[54]。研究發(fā)現(xiàn),miR-122可參與調(diào)控膽固醇的合成過程。在小鼠體內(nèi)抑制miR-122后,膽固醇合成的限速酶3-羥基-3-甲基戊二酰基-輔酶 A還原酶(HMGCR)活性下降約45%,血漿膽固醇水平下降40%[55]。在高脂喂養(yǎng)的小鼠體內(nèi)拮抗miR-122的表達(dá)則可顯著改善脂肪肝,并減少肝臟中甘油三酯含量和增加脂肪酸的氧化。miR-122的這些功能可以作為治療脂肪肝或動脈粥樣硬化的潛在靶標(biāo)[56]。固醇反應(yīng)元件結(jié)合蛋白(sterol-response element binding protein,SREBP)是膽固醇合成的轉(zhuǎn)錄調(diào)節(jié)因子,能夠調(diào)節(jié)參與細(xì)胞膽固醇運輸基因的表達(dá),此基因內(nèi)含子可編碼 miR-33。miR-33能夠抑制 ABCA1(ATP binding cassette A1)的表達(dá),從而減少膽固醇流出至脂蛋白A1。在小鼠巨噬細(xì)胞中,miR-33可以靶向ABCA1,減少膽固醇流出到初期的HDL。沉默miR-33的表達(dá),能顯著增加肝臟中ABCA1的表達(dá)和血漿中HDL的水平[57]。此外,miR-33還能直接作用于胰島素受體底物2(insulin receptor substrate 2,IRS2),影響胰島素信號通路[58]。另有文獻(xiàn)報道m(xù)iR-758也能靶向ABCA1的3'端非編碼區(qū),過表達(dá)miR-758則可抑制人和小鼠巨噬細(xì)胞及干細(xì)胞中ABCA1的表達(dá),減少膽固醇流出到脂蛋白A1[59]。表1對miRNAs在AS發(fā)生發(fā)展中的作用進(jìn)行了總結(jié)。

表1 microRNAs在動脈粥樣硬化中作用Table 1 Role of microRNAs in atherosclerosis

PPARα 內(nèi)皮細(xì)胞功能紊亂Endothelial cells dysfunction[35]miR-221/222 c-kite/NOS/STAT5A 抑制內(nèi)皮細(xì)胞遷移、增殖和血管新生Migration,proliferation of endothelial cells and angiogenesis [32][33][39]miR-365 Bcl-2 促內(nèi)皮細(xì)胞凋亡Promote apoptosis of endothelial cells [40]miR-34a SirT1 促進(jìn)內(nèi)皮細(xì)胞老化和抑制增殖Promote aging of endothelial cells and inhibit proliferation [41]miR-125a/125b-5p ET-1 抑制內(nèi)皮細(xì)胞功能紊亂Inhibit endothelial cells dysfunction [42]Immune response miR-155 MyD88 抑制免疫響應(yīng)Inhibit immune response免疫應(yīng)答[49]FADD 調(diào)節(jié)脂多糖信號通路Regulate signaling pathway of lipopolysaccharide [50]miR-125a-5p ORP9 抑制細(xì)胞因子分泌Inhibit secretion of cytokine[45]miR-125b IL-6 MCP-1誘導(dǎo)炎癥因子表達(dá)Induce expression of inflammatory factor [51]miR-146a TLR4 抑制Toll樣受體免疫應(yīng)答Inhibit immune response of Toll-like receptors [47]TNF-α/MCP-1/NF-κB誘導(dǎo)促炎癥細(xì)胞因子表達(dá)Induce expression of pro-inflammatory cytokine [52]miR-147 TLR2/TLR3/TLR4 負(fù)向調(diào)控Toll樣受體Negative regulate Toll-like receptors [53]Abnormal lipid metabolism miR-122 HMGCR 抑制膽固醇合成Inhibit synthesis of cholesterol脂代謝異常[55]miR-33 SREBP 調(diào)節(jié)膽固醇合成Regulate synthesis of cholesterol [57]ABCA1 ABCG1抑制膽固醇流出Inhibit cholesterol efflux[57]IRS2 調(diào)節(jié)胰島素信號通路Regulate insulin signaling pathway [58]miR-785 ABCA1 抑制膽固醇流出Inhibit cholesterol efflux[59]

3 展望

雖然miRNAs生物學(xué)功能的研究越來越多,POPs也顯示可引起生物體內(nèi) miRNAs的表達(dá)紊亂[60],然而POPs暴露與AS有關(guān)的miRNAs的研究尚屬空白。研究POPs暴露與AS有關(guān)的miRNAs,尋找POPs引致AS機(jī)制中可能的miRNAs,可將miRNAs作為預(yù)防和治療POPs相關(guān)AS疾病的生物靶標(biāo)。當(dāng)然如果想達(dá)到這一目標(biāo),還有一系列的問題有待解決。

首先,miRNAs表達(dá)分析的檢測技術(shù)受到限制。先進(jìn)的檢測技術(shù)能準(zhǔn)確有效地檢測miRNAs,從而使miRNAs作為環(huán)境污染物相關(guān)疾病的指示物成為可能。目前主要的miRNA研究技術(shù)有Northern印跡分析、miRNA芯片、實時定量PCR(Quantitative realtime PCR)。Northern印跡分析操作繁瑣、耗時費力、靈敏度低;實時定量PCR雖具有較高的靈敏度和特異性,但不適于大規(guī)模篩選;miRNA芯片可進(jìn)行大規(guī)模的篩選,但無法清楚的區(qū)分序列差異很小的miRNAs。所以目前尚無哪一種方法能夠達(dá)到高通量、靈敏及準(zhǔn)確,在miRNAs的檢測方法上還有相當(dāng)長的路要走。

其次,miRNAs的作用靶點及其功能尚未明確。隨著檢測技術(shù)的進(jìn)步,越來越多的miRNAs被檢測出來,但是這些miRNAs作用的靶基因以及生物學(xué)功能尚未完全明確。由于miRNA與靶mRNA序列不完全配對便可發(fā)揮作用,因此一個miRNA可以實現(xiàn)多個靶標(biāo)mRNA的調(diào)節(jié)。目前對于miRNA靶基因的預(yù)測只能通過在線分析軟件,根據(jù)算法的不同又分為第一代和第二代測序軟件。第一代預(yù)測軟件大多是從種子區(qū)互補(bǔ)這一規(guī)則出發(fā)設(shè)計的算法,如Mi-Randa[61]、MIRBase[62]、DIANA LAB[63]等。第二代預(yù)測軟件更傾向于機(jī)器學(xué)習(xí)方法訓(xùn)練參數(shù)對靶基因的預(yù)測,如 PicTar[64]、RNA22[65]和 microTar[66]等。但是不同的算法所得到的預(yù)測結(jié)果不一致并且不能預(yù)測所有的miRNA,還需要利用熒光定量PCR及Western blot分別檢測轉(zhuǎn)染或抑制miRNA后細(xì)胞中靶基因mRNA水平及蛋白水平的變化,從而確定miRNA與靶基因的對應(yīng)關(guān)系,進(jìn)一步還可通過熒光素酶報告基因法來鑒定miRNA的靶位點。通過實驗研究確定和證實更多的靶基因有助于理解miRNA的生物學(xué)功能。

最后,與環(huán)境疾病相關(guān)的特異性miRNA尚待發(fā)現(xiàn)。miRNA作為疾病的檢測和治療靶標(biāo)已經(jīng)研究多年。血漿中的miR-210和miR-141分別是胰腺癌和前列腺癌臨床診斷的指標(biāo)[67-68]。miR-9和miR-223可以作為卵巢癌復(fù)發(fā)的標(biāo)記[69]。目前對于miRNA與AS之間的研究還集中于正常條件和病理條件下miRNA表達(dá)圖譜的分析及其功能的研究。發(fā)現(xiàn)與環(huán)境污染物特異性的miRNAs不僅有利于對環(huán)境疾病機(jī)理的理解,還可作為一種新興的生物監(jiān)測和疾病預(yù)防的靶標(biāo)。因此研究與環(huán)境疾病相關(guān)的miRNAs和其調(diào)節(jié)的基因非常重要,對miRNAs在AS中的調(diào)節(jié)機(jī)制以及應(yīng)用還需深入探索。

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