微小核糖核酸在白塞病發(fā)病中的作用及其臨床應(yīng)用前景

葉京芬(綜述) 管劍龍(審校)

(復(fù)旦大學(xué)附屬華東醫(yī)院免疫風(fēng)濕科 上海 200040)

微小核糖核酸在白塞病發(fā)病中的作用及其臨床應(yīng)用前景

葉京芬(綜述) 管劍龍△(審校)

(復(fù)旦大學(xué)附屬華東醫(yī)院免疫風(fēng)濕科 上海 200040)

微小核糖核酸(miRNA)是核糖核酸干擾的內(nèi)源性介質(zhì),通過誘導(dǎo)轉(zhuǎn)錄后靶基因表達(dá)沉默發(fā)揮生物學(xué)作用。miRNA對機(jī)體免疫細(xì)胞具有多種調(diào)控功能,參與固有免疫和適應(yīng)性免疫的調(diào)節(jié)。異常表達(dá)的miRNA通過調(diào)節(jié)免疫細(xì)胞炎性因子參與白塞病(Behcet’s disease,BD)的發(fā)病,可能成為BD診斷的標(biāo)志物和治療靶點(diǎn)。本文從miRNA調(diào)節(jié)自身免疫,部分miRNA參與BD發(fā)病的機(jī)制,miRNA潛在的臨床應(yīng)用價(jià)值等進(jìn)行綜述。

微小核糖核酸; 炎性因子; 白塞病

白塞病(Behcet’s disease,BD)是一種慢性、系統(tǒng)性、炎癥性疾病,其病理基礎(chǔ)是血管炎,可累及全身大小血管[1]。臨床上以復(fù)發(fā)性口腔潰瘍、生殖器潰瘍、葡萄膜炎和皮膚損害為特征,也可發(fā)生消化道潰瘍、心臟瓣膜病、靜脈血栓、動脈狹窄、動脈瘤、關(guān)節(jié)炎、血液和神經(jīng)系統(tǒng)損害。發(fā)病機(jī)制尚不完全明確,已知感染等環(huán)境因素作用于具有遺傳傾向的個(gè)體,導(dǎo)致免疫細(xì)胞炎性因子高表達(dá),參與BD的發(fā)病[2-3]。1990年,Napoli等[4]首次發(fā)現(xiàn)RNA干擾現(xiàn)象;隨后,Fire等[5]提出miRNA是RNA干擾的分子基礎(chǔ)。近年來miRNA作為新的生物學(xué)分子通過機(jī)體內(nèi)復(fù)雜的網(wǎng)絡(luò)調(diào)控體系,參與控制生物體內(nèi)一系列生物學(xué)進(jìn)程,在BD發(fā)病過程中發(fā)揮重要作用。本文就BD發(fā)病相關(guān)的miRNA和miRNA在BD診斷、治療方面的潛在臨床價(jià)值進(jìn)行闡述,為進(jìn)一步理解BD發(fā)病、明確診斷和治療提供新視角。

miRNA概述 miRNA是長度為19～22個(gè)核苷酸組成的非編碼RNA,基因轉(zhuǎn)錄后其與mRNAs互補(bǔ)結(jié)合誘導(dǎo)靶基因降解或抑制靶基因表達(dá)而發(fā)揮生物學(xué)作用。miRNA首先通過RNA聚合酶Ⅱ轉(zhuǎn)錄合成含有5’端7甲基鳥嘌呤帽子和3’端多聚核苷酸尾結(jié)構(gòu)的原始miRNA (pri-miRNA),經(jīng)過Dorsha和DGCR8兩個(gè)關(guān)鍵酶修飾,最終形成含有莖-環(huán)結(jié)構(gòu)的pre-miRNA,隨后,pre-miRNA被Exportin5和Dicer引導(dǎo)進(jìn)入含有Argonaute蛋白的RNA誘導(dǎo)復(fù)合體(RNA-induced silencing complex,RISC),結(jié)合到RISC的miRNA遵守堿基互補(bǔ)的原則指導(dǎo)RISC降解同源目標(biāo)mRNA,從而使靶基因沉默[6],進(jìn)一步影響蛋白質(zhì)的表達(dá),參與調(diào)節(jié)免疫細(xì)胞產(chǎn)生、分化和炎性因子的表達(dá)。研究表明,miRNA通過影響免疫細(xì)胞炎性因子表達(dá)參與BD發(fā)病與進(jìn)展(圖1)[6-12]。

Some miRNAs may be involved in this process by regulating the expression of target mRNAs and its coded protein.Studies showed that increased levels of inflammatory cytokines including IL-1β,IL-17,INF-γ,MCP-1,IL-6 and low level of IL-10 have been linked to altered expression of miRNAs,miRNA-155,miRNA-21,miR-23b and miRNA-196a.

圖1 miRNAs通過調(diào)節(jié)炎性因子表達(dá)參與BD發(fā)病

Fig 1 miRNAs involved in the pathogenesis of BD by regulating the expression of inflammatory cytokines

miRNA參與固有免疫調(diào)節(jié) 巨噬細(xì)胞(macrophages,mφ)是固有免疫重要的組成部分,根據(jù)mφ表面分子不同分為M1型和M2型。動物實(shí)驗(yàn)表明,M1、M2型mφ在BD的發(fā)病與進(jìn)展中起作用,疾病活動期主要依靠M1型mφ分泌的炎性因子IFN-γ、IL-6等實(shí)現(xiàn),而在恢復(fù)期BD中則表現(xiàn)為M2型mφ標(biāo)志物Arg1和MHC-Ⅱ表達(dá)升高,調(diào)節(jié)mφ表型可能是一種新的治療BD方法[13]。眾多miRNA在mφ極化中具有調(diào)控作用。miRNA-146a可通過抑制靶基因INHBA表達(dá)抑制M1型mφ極化、促進(jìn)M2型mφ極化[14]。相反,miRNA-155則通過靶向下調(diào)SOCS1表達(dá),參與并促進(jìn)mφ炎癥應(yīng)答,miRNA-155過表達(dá)顯著拮抗地塞米松對TNF-α、IL-6及NO等炎性介質(zhì)的抑制作用,是糖皮質(zhì)激素發(fā)揮抗炎效果的作用靶點(diǎn)及潛在增敏劑[15]。糖皮質(zhì)激素是BD常用治療藥物,進(jìn)一步探索BD炎癥應(yīng)答中相關(guān)miRNA的功能,調(diào)整內(nèi)源性miRNA的水平,可能為糖皮質(zhì)激素耐受BD患者提供新的治療思路。

miRNA參與適應(yīng)性免疫調(diào)節(jié) 初始CD4+T細(xì)胞活化誘導(dǎo)基因表達(dá)程序變化,進(jìn)一步分化為Th細(xì)胞、CTL細(xì)胞和Treg細(xì)胞,參與適應(yīng)性免疫應(yīng)答和免疫記憶。miRNA的核心蛋白Ago2影響T細(xì)胞中miRNA表達(dá),下調(diào)T細(xì)胞Ago2蛋白和miRNA表達(dá)能夠促進(jìn)初始CD4+T細(xì)胞向Th細(xì)胞分化[16]。Th細(xì)胞包括Th1細(xì)胞、Th2細(xì)胞、Th17細(xì)胞、Treg和Tfh細(xì)胞亞群,各亞群受不同miNRA調(diào)節(jié),維持Th細(xì)胞亞群之間的分化平衡[17];miRNA的異常表達(dá)將導(dǎo)致Th細(xì)胞亞群功能失調(diào),誘發(fā)自身免疫病。Qi等[7]發(fā)現(xiàn),BD患者CD4+T細(xì)胞中miRNA-23b表達(dá)水平下降,對Th1和Th17擴(kuò)增具有調(diào)控功能;抑制CD4+T細(xì)胞中miRNA-23b表達(dá),顯著增高Th1、Th17細(xì)胞數(shù)量及相應(yīng)細(xì)胞因子IFN-γ、IL-17表達(dá)。B細(xì)胞發(fā)揮免疫功能的過程同樣受到miRNA等微小分子的調(diào)節(jié),Dicer蛋白敲除后的祖B淋巴細(xì)胞中miRNA-17～92簇缺乏導(dǎo)致B細(xì)胞發(fā)育障礙[18]。而在小鼠成熟B淋巴細(xì)胞中敲除Dicer蛋白,發(fā)現(xiàn)小鼠自身抗體升高[19]。上述研究提示,miRNA在B淋巴細(xì)胞發(fā)育及免疫調(diào)節(jié)中發(fā)揮作用。正常免疫系統(tǒng)的形成和功能完善需要miRNA參與調(diào)節(jié),其表達(dá)動態(tài)調(diào)節(jié)免疫細(xì)胞分化及成熟免疫細(xì)胞發(fā)揮免疫功能。相反,miRNA異常表達(dá)將導(dǎo)致免疫紊亂、功能異常。

miRNA-23b與BD 早在2002年就有學(xué)者提出,Th1細(xì)胞是BD的主要致病細(xì)胞。近年來發(fā)現(xiàn)Th17細(xì)胞在BD發(fā)病中發(fā)揮作用,Th1、Th17分泌釋放IL-17、IL-6、IL-21、IL-8、IFN-γ及TNF-α等細(xì)胞因子參與BD的發(fā)病[3]。其中,Th17表達(dá)水平及相應(yīng)炎性因子IL-17與BD活動密切相關(guān)[2]。Notch1信號通路在Th17細(xì)胞分化中發(fā)揮著關(guān)鍵性作用[8]。Qi等[7]發(fā)現(xiàn),Notch1在活動期BD患者外周血單核細(xì)胞(peripheral blood mononuclear cell,PBMC)、CD4+T細(xì)胞中過度激活,抑制Notch1表達(dá)可以減輕Th1、Th17炎性反應(yīng);3種miRNAs能夠抑制靶蛋白Notch1的,即miRNA-23b、miRNA-21、miRNA-34a,在BD患者CD4+T細(xì)胞中進(jìn)行檢測發(fā)現(xiàn),僅miRNA-23b在活動性BD患者CD4+T細(xì)胞中低表達(dá);應(yīng)用miRNA-23b拮抗劑轉(zhuǎn)染CD4+T細(xì)胞發(fā)現(xiàn),Notch1信號通路過度活化、STAT3磷酸化水平升高;ELISA法檢測Th17相關(guān)細(xì)胞因子發(fā)現(xiàn),IL-17、IFN-γ表達(dá)上調(diào);結(jié)果表明,miRNA-23b在CD4+T細(xì)胞中低表達(dá)與Notch信號通路異常激活相關(guān),通過改變STAT3的磷酸化誘導(dǎo)Th17細(xì)胞分化,分泌IL-17、IFN-γ等炎性細(xì)胞因子導(dǎo)致BD患者的炎癥發(fā)生,促進(jìn)疾病進(jìn)展。

miRNA-155與BD Toll樣受體(Toll-like receptor,TLR)是固有免疫重要的抗原識別受體,經(jīng)配體刺激激活后,通過接頭分子MyD88 或TRIF 活化下游信號通路,啟動免疫反應(yīng)[20]。TGF-β活性激酶1靶蛋白2(TGF-β-activated kinase 1-binding protein 2,TAB2)是TLR/IL-1信號級聯(lián)反應(yīng)的鏈接蛋白,也是miRNA-155的直接靶點(diǎn)[21]。在成熟樹突狀細(xì)胞中,miRNA-155可通過作用于TAB2下調(diào)炎性因子,負(fù)反饋調(diào)節(jié)免疫反應(yīng)[22]。Zhou等[9]發(fā)現(xiàn)miRNA-155在活動期眼BD患者PBMC和未成熟樹突狀細(xì)胞(immature dendritic cell,imDC)中低表達(dá),而Vogt-小柳原田綜合征患者與健康對照組相比無差異;通過TargetScan和miRNABase等生物信息工具進(jìn)一步驗(yàn)證miRNA-155在BD發(fā)病中的作用機(jī)制,發(fā)現(xiàn)miRNA-155能夠與TAB2 mRNA的 3’端非翻譯區(qū)(untranslated region,UTR)互補(bǔ)結(jié)合,影響TLR/IL-1信號通路過程中級聯(lián)反應(yīng)的靶向蛋白TAB2表達(dá),抑制imDCs分泌IL-6 和IL-1β,促進(jìn)抗炎因子IL-10表達(dá);結(jié)果表明,miRNA-155在BD患者imDCs中低表達(dá),對促炎細(xì)胞因子的負(fù)向調(diào)控減弱而導(dǎo)致BD炎癥發(fā)生;下調(diào)imDC中miRNA-155水平,并將其與CD4+T細(xì)胞共培養(yǎng)發(fā)現(xiàn),IL-17表達(dá)顯著升高;相反,miRNA-155高表達(dá)則表現(xiàn)為IL-17表達(dá)下調(diào),結(jié)果提示,miRNA-155表達(dá)下調(diào)還可以通過影響imDC對T細(xì)胞的調(diào)節(jié)功能在BD中發(fā)揮作用。此外,Na等[10]發(fā)現(xiàn),活動性BD患者CD4+T細(xì)胞中miRNA-155高表達(dá),通過抑制Ets-1蛋白表達(dá)促使IL-17產(chǎn)生,參與BD發(fā)病,上述研究結(jié)果表明,miRNA-155在BD中可能同時(shí)影響多種炎癥通路從而促進(jìn)疾病發(fā)展。

miRNA-146a、miRNA-196a2基因多態(tài)性與BD相關(guān) miRNA-146a/rs2910164與BD的發(fā)病風(fēng)險(xiǎn)顯著相關(guān),在miRNA-146a/rs2910164 CC型BD患者PBMC中,miRNA-146a及IL-17、TNF-α、IL-1β等炎性因子表達(dá)顯著低于rs2910164 GG型,提示miRNA-146a基因多態(tài)性可能通過調(diào)控自身miRNA的成熟和炎性因子的表達(dá)與BD相關(guān)[23]。Qi等[11]發(fā)現(xiàn),pre-miRNA-196a2/rs11614913 TT型人群患BD的發(fā)病風(fēng)險(xiǎn)顯著升高;進(jìn)一步研究發(fā)現(xiàn),rs11614913 TT型BD患者PBMC中miRNA-196a表達(dá)下調(diào),可能通過靶基因Bach1高表達(dá),IL-1β和MCP-1等炎性因子水平上調(diào)與BD相關(guān)。miRNA不僅存在于細(xì)胞內(nèi),而且相對穩(wěn)定地存在于血液中,這使得其成為生物學(xué)標(biāo)志物的研究具有可行性。因此,進(jìn)一步研究miRNA-146a、miRNA-196a2及其基因多態(tài)性在BD中的作用,將會為臨床診斷及治療提供新的策略。

miRNA作為BD潛在生物學(xué)標(biāo)志物 miRNA不僅具有顯著的生物學(xué)相關(guān)性,還可能成為BD生物學(xué)標(biāo)志物。近來,Erre等[24]采用TaqMan探針進(jìn)行了基于低密度陣列miRNA表達(dá)譜分析,通過對750種人類miRNA研究,探索了5例BD患者和3例健康對照PBMC中miRNA表達(dá)譜,與健康人比較,BD患者13種miRNAs表達(dá)異常,包括miRNA-571、miRNA-127、miRNA-573、miRNA-770、miRNA-370、miRNA-139-3p、miRNA-720和miRNA-330表達(dá)上調(diào),miRNA-200b、miRNA-520c-3p、miRNA-199a-3p、miRNA-224和 miRNA-98表達(dá)下調(diào)。對miRNA-139-3p和miRNA-720進(jìn)行實(shí)時(shí)熒光定量PCR進(jìn)一步驗(yàn)證顯示,BD患者miRNA-139-3p和miRNA-720表達(dá)明顯上調(diào),但與BD活動性無關(guān),提示miRNA-139-3p和miRNA-720有可能成為BD診斷的標(biāo)志物。鑒于miRNA免疫調(diào)控網(wǎng)絡(luò)的復(fù)雜性和目前檢測技術(shù)的限制,miRNA作為血液學(xué)標(biāo)志物的臨床應(yīng)用尚待進(jìn)一步完善。

miRNA作為BD潛在治療靶點(diǎn) miRNA在BD患者免疫細(xì)胞中存在異常表達(dá),最終通過上調(diào)炎性因子水平參與BD的發(fā)病,可能有作為BD基因治療靶點(diǎn)的潛能。Choi等[12]發(fā)現(xiàn),miRNA-21在單純皰疹病毒誘導(dǎo)的鼠BD模型及BD患者外周血PBMC中高表達(dá)。對BD模型鼠分別應(yīng)用秋水仙堿和己酮可可堿治療后檢測出淋巴結(jié)中miRNA-21表達(dá)明顯下調(diào),其中經(jīng)過己酮可可堿治療的BD鼠皮膚癥狀有所改善;進(jìn)一步應(yīng)用miRNA-21拮抗劑驗(yàn)證體內(nèi)效應(yīng)發(fā)現(xiàn),miRNA-21的靶基因PDCD4、RhoB、PD-1和IL-12p35在具有miRNA-21拮抗效應(yīng)的PBMC中高表達(dá),通過下調(diào)相應(yīng)炎性因子IL-17和IL-6水平,改善BD鼠癥狀和評分。結(jié)果提示,BD患者PBMC中高表達(dá)的miRNA-21可能通過抑制靶基因表達(dá)上調(diào)IL-17和IL-6參與BD炎癥,miRNA-21拮抗劑對單純皰疹病毒誘導(dǎo)的鼠BD模型的治療是有效的,該研究成果為BD的治療提供了新的切入點(diǎn)。

結(jié)語 至今靶向miRNAs的藥物尚未從實(shí)驗(yàn)室向臨床應(yīng)用過渡,在這之前探索與BD密切相關(guān)的miRNAs及其作用機(jī)制,全面了解miRNAs在BD中特異性靶基因、靶蛋白及信號轉(zhuǎn)導(dǎo)通路將有助于進(jìn)一步了解BD的發(fā)病機(jī)制,為BD的預(yù)防、治療及判斷預(yù)后等臨床實(shí)踐提供理論基礎(chǔ)。

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The pathgenetic role and potential clinical application of miRNAs in patients with Behcet’s disease

YE Jing-fen, GUAN Jian-long△

(DepartmentofImmunologyandRheumatology,HuadongHospital,FudanUniversity,Shanghai200040,China)

As the endogenous mediators of RNA interference,miRNA play biological roles by inducing post-transcriptional silence of gene expression.Previous studies showed that miRNAs have multiple regulating potential to immune cells,involved in regulation of innate and adaptive immunity.Recently,aberrant expression of miRNAs have been confirmed to be relevant to the pathogenesis of Behcet’s disease (BD) by regulating the expression of inflammatory cytokines in immune cells,which may become of its diagnostic marker and therapeutic target in the future.In this review,we discuss how miRNAs regulate autoimmunity.Several miRNAs associate with the pathogenesis of BD,thus they have the potential clinical application value.

miRNA; inflammatory cytokines; Behcet’s disease

R593.1

B

10.3969/j.issn.1672-8467.2017.04.026

2016-08-22;編輯:段佳)

上海衛(wèi)生系統(tǒng)第二批重要疾病聯(lián)合攻關(guān)重點(diǎn)項(xiàng)目(2014ZYJB0010)

△Corresponding author E-mail:guanjianlong@medmail.com.cn

*This work was supported by Shanghai Health System Key Joint Research Project on Important Diseases of the Second Batch (2014ZYJB0010).