鄭 皖,馮湘玲,楊 飛,陳繼華,2*

(1.中南大學(xué)湘雅公共衛(wèi)生學(xué)院營養(yǎng)與食品衛(wèi)生學(xué)系,中國湖南長沙410078;2.湖南省植物功能成分利用協(xié)同創(chuàng)新中心,中國湖南長沙410128)

活性氧類(reactive oxygen species,ROS)是一類具有很強(qiáng)氧化能力的含氧自由基的統(tǒng)稱。在生理狀態(tài)下,低濃度的ROS可作為信號分子激活細(xì)胞漿中的第二信使,參與調(diào)控細(xì)胞信號轉(zhuǎn)導(dǎo)、細(xì)胞分化等過程,對機(jī)體生理功能發(fā)揮有益作用[1];當(dāng)細(xì)胞受到環(huán)境毒物、高氧、低氧等外界刺激時會產(chǎn)生過量的ROS,高濃度的ROS可通過脂質(zhì)過氧化、DNA損傷和蛋白質(zhì)破壞等方式,引起氧化應(yīng)激損傷,造成機(jī)體生理功能紊亂,導(dǎo)致皮膚病變、衰老以及多種慢性病的發(fā)生,如帕金森病、阿爾茨海默病、癌癥、心血管疾病、糖尿病等[2,3]。

研究發(fā)現(xiàn),多條通路上的關(guān)鍵分子參與調(diào)控機(jī)體內(nèi)的氧化還原穩(wěn)態(tài)。其中,核因子E2相關(guān)因子2(nuclear factor erythroid 2 related factor 2,NFE2L2或Nrf2)是調(diào)節(jié)胞內(nèi)眾多抗氧化基因表達(dá)的關(guān)鍵性因子,可識別并結(jié)合抗氧化反應(yīng)元件(antioxidant responsive element,ARE),誘導(dǎo)下游抗氧化蛋白質(zhì)的表達(dá),清除細(xì)胞內(nèi)產(chǎn)生的過量ROS,從而對抗細(xì)胞內(nèi)的氧化損傷,在疾病預(yù)防和治療中具有積極的意義。Nrf2-ARE信號通路是目前國內(nèi)外研究較多的抗氧化熱點通路。

微RNA(microRNAs,miRNAs)是真核生物體中廣泛存在的一類長度約為22個核苷酸的非編碼小分子RNA。根據(jù)人類基因組計劃測序結(jié)果預(yù)測出人體內(nèi)miRNAs的數(shù)量約占人類基因組的1%～3%[4]。在miRBase v.21中,包含了35 828個成熟的miRNA序列,其中有2 588個人類的成熟miRNAs[5]。研究表明,60%的蛋白質(zhì)編碼基因受到miRNAs的調(diào)控[6]。miRNAs通過與靶基因信使RNA(mRNA)的堿基配對結(jié)合,降解靶基因或抑制靶基因表達(dá),參與轉(zhuǎn)錄后基因表達(dá)的調(diào)控[7,8],與機(jī)體多種生理和病理過程密切相關(guān)。

1Nrf2-ARE信號通路

Nrf2-ARE通路主要由轉(zhuǎn)錄因子Nrf2、Nrf2抑制因子以及ARE元件構(gòu)成。Nrf2在通路中起到開關(guān)作用,是機(jī)體內(nèi)調(diào)控細(xì)胞對抗外來異物和氧化損傷的重要轉(zhuǎn)錄因子。

在正常穩(wěn)態(tài)水平下,Nrf2定位于細(xì)胞質(zhì)中,并與其胞漿抑制蛋白質(zhì)Kelch樣ECH相關(guān)蛋白1(kelch-like ECH-associated protein 1,Keap1)或β-TrCP(β-transducin repeat-containing protein)結(jié)合,且在泛素激活酶E1、結(jié)合酶E2和連接酶E3的協(xié)同作用下不斷被泛素化,泛素化的復(fù)合體在26S蛋白酶體作用下降解,以此維持Nrf2的低水平無活性狀態(tài)[9～12]。當(dāng)機(jī)體接觸外源性物質(zhì)如藥物、毒物、致癌物等或受到氧化應(yīng)激刺激時,主要通過Keap1依賴或Keap1非依賴的方式穩(wěn)定Nrf2,如Keap1構(gòu)象發(fā)生改變或β-TrCP發(fā)生磷酸化,引起Nrf2與Keap1或β-TrCP發(fā)生解離,從而使Nrf2免于泛素化降解[10,12]。

未被泛素化的Nrf2進(jìn)入核內(nèi)與小Maf蛋白(small Maf protein,sMaf)結(jié)合形成異源二聚體,并與基因啟動子區(qū)核苷酸序列5′-TGANNNNGC-3′(即ARE元件)結(jié)合,繼而激活這類基因的轉(zhuǎn)錄(圖1)。研究發(fā)現(xiàn)這類基因的編碼產(chǎn)物主要有Ⅱ相解毒酶或抗氧化酶,如谷胱甘肽S轉(zhuǎn)移酶(glutathione S-transferase,GST)、NAD(P)H 醌氧化還原酶1(NAD(P)H quinone oxidoreductase-1,NQO1)、血紅素加氧酶-1(heme oxygenase-1,HO-1)、谷氨酸半胱氨酸連接酶催化亞基(glutamate-cysteine ligase catalytic subunit,GCLC)、谷氨酸半胱氨酸連接酶修飾亞基(glutamate-cysteine ligase modifier subunit,GCLM)等[13]。Nrf2通過識別并結(jié)合ARE元件,上調(diào)上述Ⅱ相解毒酶或抗氧化酶基因的表達(dá)[14,15],發(fā)揮清除過量ROS的抗氧化作用[16](圖1),保護(hù)細(xì)胞免受氧化應(yīng)激的損傷,發(fā)揮解毒作用或抵御氧化損傷[17～21]。此外,越來越多的研究提示,眾多miRNAs同樣參與調(diào)控了Nrf2-ARE通路,下文將會就此展開詳細(xì)介紹。

2 miRNAs的生物合成及作用機(jī)制

2.1 miRNAs的生成

細(xì)胞核內(nèi)包含miRNAs的DNA片段,該片段首先在RNA聚合酶Ⅱ(RNA polymeraseⅡ)的作用下轉(zhuǎn)錄形成初級miRNAs(primary miRNAs,primiRNAs),后者經(jīng)核酸酶Drosha剪切后,生成大約70 nt長且具有發(fā)夾結(jié)構(gòu)的前體miRNAs(precursor miRNAs,pre-miRNAs),隨后進(jìn)一步在轉(zhuǎn)運受體Exp-5(exportin-5)的作用下從細(xì)胞核轉(zhuǎn)運至細(xì)胞質(zhì),被胞質(zhì)中的Dicer酶剪切為22 nt左右,最終被加工成成熟的miRNAs[22](圖2)。

圖1 Nrf2-ARE通路及相關(guān)miRNAsFig.1 Nrf2-ARE pathway and related miRNAs

2.2 miRNAs的作用機(jī)制

miRNAs參與基因表達(dá)的調(diào)控,與細(xì)胞分化、凋亡、生長發(fā)育、腫瘤發(fā)生等密切相關(guān)[4]。通常,一種miRNAs可以調(diào)控多種靶基因,而一種基因同時又可以受幾種miRNAs的調(diào)控。目前,已知miRNAs與靶mRNA的作用方式主要有以下幾種(圖2):當(dāng)miRNAs非核心序列與基因的轉(zhuǎn)錄因子[23]或啟動子區(qū)[24,25]結(jié)合時,抑制基因轉(zhuǎn)錄,導(dǎo)致轉(zhuǎn)錄水平基因沉默;當(dāng)miRNAs核心序列與靶mRNA某段序列完全互補(bǔ)配對時,靶mRNA分子斷裂,導(dǎo)致mRNA水平基因沉默,使其失去生物學(xué)作用[6];當(dāng)miRNAs核心序列與靶mRNA的3′-非翻譯區(qū)(3′-UTR)、5′-非翻譯區(qū)(5′-UTR)或基因編碼區(qū)不完全互補(bǔ)配對時,則阻斷靶基因轉(zhuǎn)錄后的翻譯過程,抑制靶基因表達(dá)[26～30]。除了抑制靶基因表達(dá)外,也有文獻(xiàn)報道m(xù)iRNAs能夠與靶mRNA的5′-UTR序列結(jié)合,增強(qiáng)蛋白質(zhì)翻譯,從而上調(diào)基因表達(dá)[31～33]。因此,miRNAs調(diào)節(jié)目的基因的方式與miRNAs的結(jié)合序列有關(guān)。

3 靶向調(diào)控Nrf2-ARE通路的miRNAs

Nrf2-ARE通路是重要的內(nèi)源性抗氧化應(yīng)激通路,研究發(fā)現(xiàn),多種miRNAs可以直接或間接作用于該通路,調(diào)控Nrf2-ARE信號通路上相關(guān)基因的表達(dá),影響機(jī)體的氧化還原穩(wěn)態(tài)[34～36],進(jìn)而參與介導(dǎo)機(jī)體的生理活動或病理變化。

圖2 miRNAs的合成及作用方式Fig.2 miRNAs biogenesis and mode of action

3.1 靶向Nrf2的miRNAs

Nrf2是Nrf2-ARE抗氧化通路中最為核心的因子,目前的研究表明,多個miRNAs參與調(diào)控Nrf2的表達(dá)變化,從而調(diào)控通路下游各基因的表達(dá)或活性,參與機(jī)體的抗氧化反應(yīng)。

miR-153是一類與神經(jīng)元相關(guān)的微小RNA分子,與神經(jīng)保護(hù)作用有關(guān)。研究發(fā)現(xiàn),miR-153[37,38]和miR-93[39]均能靶向Nrf2(圖1),抑制Nrf2蛋白的表達(dá),使Nrf2/HO-1信號失活,氧化應(yīng)激誘導(dǎo)神經(jīng)損傷、凋亡,從而使神經(jīng)元因缺血再灌注損傷。以上研究說明miR-153、miR-93在體內(nèi)神經(jīng)元細(xì)胞自我保護(hù)過程中,發(fā)揮拮抗機(jī)體抗氧化應(yīng)激反應(yīng)的作用。

此外,研究發(fā)現(xiàn),miR-34a、miR-153、miR-27a、miR-142-5p和miR-144等在SH-SY5Y神經(jīng)元細(xì)胞中能以Nrf2的mRNA 3′-UTR為靶點(圖1),直接下調(diào)其蛋白質(zhì)表達(dá),導(dǎo)致大腦中ARE/谷胱甘肽(glutathione,GSH)通路失調(diào),擾亂氧化還原穩(wěn)態(tài),進(jìn)而導(dǎo)致帕金森病的發(fā)生[40～42]。

在老年大鼠的腦血管內(nèi)皮細(xì)胞中miR-144表達(dá)上升,Nrf2表達(dá)下降,而且無論在幼年還是老年大鼠的腦血管內(nèi)皮細(xì)胞中,miR-144的過表達(dá)均能顯著降低Nrf2的表達(dá)。經(jīng)進(jìn)一步分析證明miR144可通過與Nrf2的3′-UTR結(jié)合,從而抑制Nrf2基因表達(dá)[43](圖1)。在此之前,也有學(xué)者在研究鐮刀型細(xì)胞貧血癥病人的血液細(xì)胞時,應(yīng)用計算機(jī)靶區(qū)掃描分析發(fā)現(xiàn),人Nrf2基因的3′-UTR有2個潛在的miR-144結(jié)合區(qū)域,即nt265-271和nt370-377,miR-144能夠以Nrf2 mRNA為靶點,下調(diào)其表達(dá),減少紅細(xì)胞對氧化應(yīng)激的耐受性,導(dǎo)致貧血嚴(yán)重程度增加[44]。

抗氧化應(yīng)激能力隨著年齡的增長逐漸減弱,在大鼠肝細(xì)胞中Nrf2蛋白含量隨著年齡的增長逐漸降低,然而Nrf2 mRNA的量并未隨年齡的改變而變化,究其原因主要是miR-146a的高表達(dá),miR-146a能與Nrf2 mRNA結(jié)合,阻礙Nrf2轉(zhuǎn)錄后的翻譯,使其蛋白質(zhì)含量下降,影響Nrf2-ARE通路的激活,導(dǎo)致下游抗氧化酶的表達(dá)下調(diào),進(jìn)而降低機(jī)體抗氧化應(yīng)激能力[45]。

在正常細(xì)胞中,Nrf2對于癌癥化學(xué)預(yù)防和腫瘤抑制起到關(guān)鍵作用。但另一方面,在腫瘤形成和腫瘤保護(hù)中,Nrf2也起到主要作用。因此,miRNAs可以通過調(diào)控Nrf2的表達(dá),發(fā)揮抑癌和致癌的雙重作用。

據(jù)報道,在乳腺癌細(xì)胞中過表達(dá)miR-153、miR-93、miR-28均能顯著降低Nrf2及下游基因的表達(dá)(圖1);同時,在乳腺上皮細(xì)胞中miR-153能夠減少凋亡、增加克隆形成。這些研究結(jié)果說明,miR-153、miR-93、miR-28 通過作用于Nrf2 在乳腺癌發(fā)生發(fā)展過程中起到致癌因子的作用[46～48]。然而,在神經(jīng)膠質(zhì)瘤細(xì)胞的研究中發(fā)現(xiàn),miR-153通過靶向Nrf2基因,抑制其蛋白質(zhì)表達(dá),從而促進(jìn)凋亡、抑制細(xì)胞增殖,發(fā)揮抑癌作用[49]。

化療是臨床上治療全身性腫瘤及已經(jīng)轉(zhuǎn)移的中晚期腫瘤的主要方案,但是藥物抵抗常導(dǎo)致化療效果差、預(yù)后不良。研究表明,Nrf2的過表達(dá)能夠?qū)е掳┘?xì)胞對化療藥物的抵抗性增加。Shi等[50]發(fā)現(xiàn)在對化療藥物順鉑耐受的HepG2細(xì)胞中,miR-340的表達(dá)顯著下調(diào),Nrf2表達(dá)上調(diào)。生物信息學(xué)和熒光素酶報告基因?qū)嶒灧治霭l(fā)現(xiàn)Nrf2是miR-340的直接靶基因。因此,上調(diào)miR-340,可以抑制Nrf2-依賴的抗氧化通路,從而增加HepG2細(xì)胞對順鉑的敏感性,成為靶向治療的依據(jù)。

在順鉑誘導(dǎo)的急性腎損傷模型中,研究發(fā)現(xiàn),miR-140-5p可通過靶向Nrf2 mRNA的3′-UTR,激活Nrf2-ARE信號通路,增加抗氧化蛋白HO-1、NQO1的表達(dá),從而降低順鉑對腎功能的氧化損傷[51]。

綜上研究可知,眾多miRNAs可以直接靶向Nrf2的表達(dá),從而下調(diào)Nrf2-ARE信號通路(表1)。不同細(xì)胞類型中調(diào)控Nrf2的miRNAs種類不同,miRNAs的表達(dá)是否與細(xì)胞類型有關(guān)、是否受Nrf2水平的影響還需進(jìn)一步研究。Nrf2-ARE通路與腫瘤的發(fā)生發(fā)展密切相關(guān),而miRNAs可以調(diào)控該通路,研究是否可以通過靶向miRNAs預(yù)防腫瘤形成或促進(jìn)腫瘤凋亡具有重要的現(xiàn)實指導(dǎo)意義。

3.2 靶向Keap1的miRNAs

Nrf2活性受到胞漿中抑制性蛋白質(zhì)Keap1的嚴(yán)格調(diào)控,多種miRNAs也可以通過作用Keap1來調(diào)控Nrf2-ARE信號通路。如miR-29可通過靶向Keap1來調(diào)節(jié)Nrf2-ARE信號通路(圖1)。研究表明,在高糖誘導(dǎo)的腎小管上皮細(xì)胞中,miR-29的表達(dá)下降,同時Keap1的表達(dá)增加,使得Nrf2泛素化增加,從而降低Nrf2的含量,引起腎小管上皮細(xì)胞的氧化損傷,導(dǎo)致腎功能紊亂[52]。但另一方面,也有研究指出Nrf2能夠調(diào)控miR-29家族的基因表達(dá)[53]。由此可見,miR-29與Nrf2-ARE信號通路之間存在著非常緊密的聯(lián)系,但兩者之間是否存在反饋調(diào)節(jié)以及具體調(diào)控機(jī)制還需進(jìn)一步探討。

研究發(fā)現(xiàn)在人視網(wǎng)膜色素上皮細(xì)胞和視網(wǎng)膜神經(jīng)節(jié)細(xì)胞中,miR-141可以通過下調(diào)Keap1(圖1),引起Nrf2穩(wěn)定性增強(qiáng),促進(jìn)Nrf2向核內(nèi)轉(zhuǎn)移,從而激活Nrf2-ARE信號通路,啟動多種抗氧化基因的轉(zhuǎn)錄,如HMOX1、NQO1、GCLC等,最終保護(hù)細(xì)胞免受紫外線誘導(dǎo)的氧化應(yīng)激損傷[54]。相關(guān)的腫瘤耐藥機(jī)制研究發(fā)現(xiàn),miR-141通過下調(diào)Keap1的表達(dá),激活Nrf2-ARE抗氧化通路,增加肝癌細(xì)胞對抗癌藥物5-氟尿嘧啶的抵抗性[55],因此,研發(fā)抑制miR-141表達(dá)的藥物,將有望增加肝癌細(xì)胞的藥物敏感性,為臨床治療提供新思路。

在缺血缺氧性心肌細(xì)胞中,過表達(dá)miR-200a能顯著增加Nrf2的核轉(zhuǎn)移以及下游抗氧化酶基因的表達(dá);抑制miR-200a的表達(dá)顯示相反的效果,進(jìn)一步的實驗證實miR-200a通過與Keap1的3′-UTR結(jié)合(圖1),下調(diào)Keap1蛋白的表達(dá)。由此說明miR-200a可通過抑制Keap1來增加Nrf2,激活Nrf2-ARE信號通路,保護(hù)心肌細(xì)胞免受低氧和ROS誘導(dǎo)的損傷,發(fā)揮細(xì)胞保護(hù)作用[56]。此外,miR-200a在肝星形細(xì)胞中也可以通過靶向Keap1,激活Nrf2-ARE通路,抵抗氧化應(yīng)激,預(yù)防肝細(xì)胞纖維化病變[57]。

研究發(fā)現(xiàn)在食管鱗狀細(xì)胞和乳腺上皮細(xì)胞中,miR-200a可直接結(jié)合Keap1 mRNA的3′-UTR使其降解(圖1),抑制Keap1表達(dá),誘導(dǎo)Nrf2向核內(nèi)轉(zhuǎn)移,增強(qiáng)ARE啟動子活性,激活Nrf2-ARE抗氧化通路,發(fā)揮腫瘤的化學(xué)預(yù)防作用[58,59]。而Akdemir等[60]研究發(fā)現(xiàn),miR-432-3p通過直接結(jié)合Keap1編碼區(qū),下調(diào)Keap1表達(dá),增強(qiáng)Nrf2活性,誘導(dǎo)Ⅱ相解毒酶表達(dá),導(dǎo)致食管鱗狀細(xì)胞癌對順鉑的敏感性降低。

此外,有研究發(fā)現(xiàn)腦細(xì)胞中高度表達(dá)的miR-7在人神經(jīng)母細(xì)胞瘤細(xì)胞中也能夠與Keap1 mRNA的3′-UTR結(jié)合(圖1),抑制Keap1表達(dá),進(jìn)而導(dǎo)致Nrf2活性增加,促進(jìn)其向核內(nèi)轉(zhuǎn)移,使下游靶基因HMOX1、GCLM等表達(dá)增加,減少ROS誘導(dǎo)的細(xì)胞死亡[61]。

盡管目前已發(fā)現(xiàn)多種miRNAs可以通過直接靶向Keap1的表達(dá),上調(diào)Nrf2-ARE信號通路,參與調(diào)節(jié)機(jī)體的氧化還原反應(yīng)(表1),但是否還有其他miRNAs調(diào)控Keap1仍需進(jìn)一步研究。

3.3 靶向通路中部分E3泛素化酶的miRNAs

在正常生理狀況下,Nrf2與Keap1結(jié)合,不斷被Cul3(Cullin 3)-E3泛素連接酶泛素化后,通過蛋白酶體途徑降解,使Nrf2保持較低水平。因此,Cul3的水平可以影響Nrf2的泛素化,從而影響Nrf2的表達(dá),調(diào)控Nrf2-ARE信號通路。

有文獻(xiàn)報道,miRNAs可以直接調(diào)控Cul3。研究發(fā)現(xiàn),低氧可以誘導(dǎo)miR-101的表達(dá),進(jìn)一步通過實驗證實miR-101能夠與Cul3 mRNA的3′-UTR結(jié)合,抑制Cul3蛋白表達(dá),從而抑制蛋白酶體降解途徑,增加Nrf2的穩(wěn)定性。此外,有研究顯示,過表達(dá)miR-101能夠促進(jìn)Nrf2向核內(nèi)轉(zhuǎn)移,促進(jìn)HMOX1、血管內(nèi)皮生長因子的基因表達(dá),對于血管生成、血管重建以及血液脊髓屏障的完整性起到重要作用[62,63]。以上研究提示miR-101可以作為血管生成和血管重建的潛在治療靶點。

目前,miRNAs調(diào)控E3泛素化酶的研究尚少,關(guān)于其在泛素化酶系統(tǒng)中的作用尚需進(jìn)一步研究。

3.4 靶向HMOX1的miRNAs

血紅素加氧酶-1(HO-1)是Nrf2-ARE信號通路調(diào)控的主要抗氧化酶之一,由HMOX1基因編碼,是血紅素降解過程中的關(guān)鍵限速酶,可將血紅素降解為膽綠素、一氧化碳和二價鐵離子,具有抗氧化、抗炎、抗凋亡、抗纖維化等多種生物學(xué)作用。

miR-155是與免疫調(diào)節(jié)最密切相關(guān)的一種miRNA,可以調(diào)控T細(xì)胞的成熟、分化,維持內(nèi)環(huán)境穩(wěn)態(tài),廣泛參與炎癥性疾病和腫瘤的形成。相關(guān)研究發(fā)現(xiàn),在耗竭性CD4+T細(xì)胞中,miR-155表達(dá)增加;通過基因芯片對比篩選出了miR-155的潛在靶基因HMOX1,并進(jìn)一步通過雙熒光素酶報告基因和RNA免疫共沉淀實驗發(fā)現(xiàn),miR-155 可以與HMOX1 mRNA 3′-UTR 結(jié)合(圖1),導(dǎo)致HO-1表達(dá)降低[64],使機(jī)體免疫保護(hù)及免疫監(jiān)視等功能發(fā)生障礙,最終可能會導(dǎo)致自身免疫性疾病、慢性感染性疾病的發(fā)生。

然而,另有文獻(xiàn)報道,HO-1的表達(dá)是通過miR-155間接調(diào)控,而非直接調(diào)控。在人臍靜脈內(nèi)皮細(xì)胞中,腫瘤壞死因子-α(tumor necrosis factor-α,TNF-α)通過 NF-κB 通路誘導(dǎo) miR-155 的表達(dá),miR-155抑制轉(zhuǎn)錄因子BACH1的蛋白質(zhì)表達(dá),但對胞漿和核內(nèi)Nrf2的蛋白質(zhì)表達(dá)無影響。BACH1是HMOX1基因的抑制因子,研究進(jìn)一步發(fā)現(xiàn)HMOX1的mRNA和蛋白質(zhì)表達(dá)增加。熒光素酶報告基因?qū)嶒烇@示,miR-155能夠降低含有BACH1 mRNA 3′-UTR的報告基因活性,并且預(yù)測出4個結(jié)合位點。以上結(jié)果說明,miR-155通過靶向BACH1的表達(dá),降低其活性,從而間接上調(diào)HO-1的表達(dá)[65],提高細(xì)胞的抗炎能力。

大多數(shù)miRNAs與靶基因的作用方式主要是通過其5′端的種子序列與mRNA的3′-UTR結(jié)合,抑制基因表達(dá)。然而,有文獻(xiàn)報道在人非小細(xì)胞肺癌中,miR-1254可以通過其種子序列和非種子序列對HMOX1發(fā)揮雙重調(diào)控作用(圖1)。其一,miR-1254通過其種子序列直接與HMOX1 3′-UTR結(jié)合,抑制HO-1蛋白翻譯;其二,miR-1254通過其非種子序列與HMOX1的轉(zhuǎn)錄激活因子AP2A結(jié)合,抑制HMOX1基因轉(zhuǎn)錄。miR-1254通過以上兩種方式抑制HO-1表達(dá),誘導(dǎo)細(xì)胞凋亡和細(xì)胞周期阻滯,從而抑制人非小細(xì)胞肺癌生長[23]。

HO-1參與調(diào)控炎癥反應(yīng)、腫瘤發(fā)展,目前關(guān)于miRNAs調(diào)控HMOX1的報道較少,因此,還需進(jìn)一步研究發(fā)現(xiàn)更多調(diào)控HMOX1的miRNAs,從而為免疫性疾病、慢性感染性疾病及腫瘤的防治提供更多的基礎(chǔ)數(shù)據(jù)。

3.5 靶向GCL的miRNAs

γ-谷氨酸半胱氨酸連接酶(γ-glutamyl-cysteine ligase,GCL)是Nrf2-ARE信號通路下游的重要抗氧化酶,包括兩個亞基:催化亞基(GCLC)和修飾亞基(GCLM),是谷胱甘肽(GSH)生物合成過程的限速酶,能夠調(diào)控細(xì)胞的氧化還原穩(wěn)態(tài),維持機(jī)體正常的生理功能。

研究發(fā)現(xiàn),在人臍靜脈血管內(nèi)皮細(xì)胞中過表達(dá)miR-433可下調(diào)GCLC、GCLM和GSH的水平,但對Nrf2的表達(dá)無影響。更進(jìn)一步研究發(fā)現(xiàn)miR-433可以直接作用于GCLC、GCLM的mRNA 3′-UTR(圖1),影響其蛋白質(zhì)表達(dá),增加血管內(nèi)皮細(xì)胞的氧化損傷,而這種直接調(diào)控作用不依賴于Nrf2 的水平[66]。

除了miR-433外,目前尚未查到其他文獻(xiàn)報道直接調(diào)控GCL的相關(guān)miRNAs,因此,還需進(jìn)一步探索以GCL為靶基因的miRNAs。

綜上可知,Nrf2-ARE通路參與調(diào)控多種生理病理途徑,與多種疾病的發(fā)生密切相關(guān),主要包括腫瘤、氧化損傷性疾病、免疫相關(guān)性疾病等。除靶向通路相關(guān)因子的miRNAs外,還有許多miRNAs間接參與調(diào)控該通路中相關(guān)因子的表達(dá)或活性,本文主要列舉了靶向調(diào)控Nrf2-ARE通路相關(guān)的miRNAs 序列及靶基因[17,23,37～52,54～62,64～70](表1)。

4 展望

關(guān)于miRNAs調(diào)控Nrf2-ARE信號通路中關(guān)鍵因子Nrf2、Keap1的研究較多,但是miRNAs調(diào)控該通路中其他因子的報道較少,并且尚未查到可以直接作用sMaf蛋白,從而間接影響Nrf2-ARE通路的miRNAs,因此,仍需要進(jìn)一步研究miRNAs與Nrf2-ARE通路的調(diào)控關(guān)系,以便進(jìn)一步了解該通路活化或失活的分子機(jī)制,為疾病預(yù)防提供新的思路。

表1 靶向調(diào)控Nrf2-ARE通路的miRNAsTable1 miRNAs targeting the regulation of Nrf2-ARE pathway

(接上表)

隨著miRNAs研究的不斷進(jìn)展,將會發(fā)現(xiàn)更多與疾病發(fā)生發(fā)展有關(guān)的miRNAs,可以此為生物標(biāo)志物,應(yīng)用于臨床檢測;同時為研發(fā)以miRNAs為治療靶點的藥物提供依據(jù),為將來疾病的診斷、防治提供新策略。

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