靶向調控Nrf2-ARE通路的miRNAs研究進展

鄭 皖,馮湘玲,楊 飛,陳繼華,2*

(1.中南大學湘雅公共衛(wèi)生學院營養(yǎng)與食品衛(wèi)生學系,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胞漿中的第二信使,參與調控細胞信號轉導、細胞分化等過程,對機體生理功能發(fā)揮有益作用[1];當細胞受到環(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可通過脂質過氧化、DNA損傷和蛋白質破壞等方式,引起氧化應激損傷,造成機體生理功能紊亂,導致皮膚病變、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),多條通路上的關鍵分子參與調控機體內(nèi)的氧化還原穩(wěn)態(tài)。其中,核因子E2相關因子2(nuclear factor erythroid 2 related factor 2,NFE2L2或Nrf2)是調節(jié)胞內(nèi)眾多抗氧化基因表達的關鍵性因子,可識別并結合抗氧化反應元件(antioxidant responsive element,ARE),誘導下游抗氧化蛋白質的表達,清除細胞內(nèi)產(chǎn)生的過量ROS,從而對抗細胞內(nèi)的氧化損傷,在疾病預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果預測出人體內(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%的蛋白質編碼基因受到miRNAs的調控[6]。miRNAs通過與靶基因信使RNA(mRNA)的堿基配對結合,降解靶基因或抑制靶基因表達,參與轉錄后基因表達的調控[7,8],與機體多種生理和病理過程密切相關。

1Nrf2-ARE信號通路

Nrf2-ARE通路主要由轉錄因子Nrf2、Nrf2抑制因子以及ARE元件構成。Nrf2在通路中起到開關作用,是機體內(nèi)調控細胞對抗外來異物和氧化損傷的重要轉錄因子。

在正常穩(wěn)態(tài)水平下,Nrf2定位于細胞質中,并與其胞漿抑制蛋白質Kelch樣ECH相關蛋白1(kelch-like ECH-associated protein 1,Keap1)或β-TrCP(β-transducin repeat-containing protein)結合,且在泛素激活酶E1、結合酶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]。當機體接觸外源性物質如藥物、毒物、致癌物等或受到氧化應激刺激時,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進入核內(nèi)與小Maf蛋白(small Maf protein,sMaf)結合形成異源二聚體,并與基因啟動子區(qū)核苷酸序列5′-TGANNNNGC-3′(即ARE元件)結合,繼而激活這類基因的轉錄(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通過識別并結合ARE元件,上調上述Ⅱ相解毒酶或抗氧化酶基因的表達[14,15],發(fā)揮清除過量ROS的抗氧化作用[16](圖1),保護細胞免受氧化應激的損傷,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同樣參與調控了Nrf2-ARE通路,下文將會就此展開詳細介紹。

2 miRNAs的生物合成及作用機制

2.1 miRNAs的生成

細胞核內(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)夾結構的前體miRNAs(precursor miRNAs,pre-miRNAs),隨后進一步在轉運受體Exp-5(exportin-5)的作用下從細胞核轉運至細胞質,被胞質中的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miRNAsFig.1 Nrf2-ARE pathway and related miRNAs

2.2 miRNAs的作用機制

miRNAs參與基因表達的調控,與細胞分化、凋亡、生長發(fā)育、腫瘤發(fā)生等密切相關[4]。通常,一種miRNAs可以調控多種靶基因,而一種基因同時又可以受幾種miRNAs的調控。目前,已知miRNAs與靶mRNA的作用方式主要有以下幾種(圖2):當miRNAs非核心序列與基因的轉錄因子[23]或啟動子區(qū)[24,25]結合時,抑制基因轉錄,導致轉錄水平基因沉默;當miRNAs核心序列與靶mRNA某段序列完全互補配對時,靶mRNA分子斷裂,導致mRNA水平基因沉默,使其失去生物學作用[6];當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)不完全互補配對時,則阻斷靶基因轉錄后的翻譯過程,抑制靶基因表達[26～30]。除了抑制靶基因表達外,也有文獻報道m(xù)iRNAs能夠與靶mRNA的5′-UTR序列結合,增強蛋白質翻譯,從而上調基因表達[31～33]。因此,miRNAs調節(jié)目的基因的方式與miRNAs的結合序列有關。

3 靶向調控Nrf2-ARE通路的miRNAs

Nrf2-ARE通路是重要的內(nèi)源性抗氧化應激通路,研究發(fā)現(xiàn),多種miRNAs可以直接或間接作用于該通路,調控Nrf2-ARE信號通路上相關基因的表達,影響機體的氧化還原穩(wěn)態(tài)[34～36],進而參與介導機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參與調控Nrf2的表達變化,從而調控通路下游各基因的表達或活性,參與機體的抗氧化反應。

miR-153是一類與神經(jīng)元相關的微小RNA分子,與神經(jīng)保護作用有關。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,使Nrf2/HO-1信號失活,氧化應激誘導神經(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)元細胞自我保護過程中,發(fā)揮拮抗機體抗氧化應激反應的作用。

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)元細胞中能以Nrf2的mRNA 3′-UTR為靶點(圖1),直接下調其蛋白質表達,導致大腦中ARE/谷胱甘肽(glutathione,GSH)通路失調,擾亂氧化還原穩(wěn)態(tài),進而導致帕金森病的發(fā)生[40～42]。

在老年大鼠的腦血管內(nèi)皮細胞中miR-144表達上升,Nrf2表達下降,而且無論在幼年還是老年大鼠的腦血管內(nèi)皮細胞中,miR-144的過表達均能顯著降低Nrf2的表達。經(jīng)進一步分析證明miR144可通過與Nrf2的3′-UTR結合,從而抑制Nrf2基因表達[43](圖1)。在此之前,也有學者在研究鐮刀型細胞貧血癥病人的血液細胞時,應用計算機靶區(qū)掃描分析發(fā)現(xiàn),人Nrf2基因的3′-UTR有2個潛在的miR-144結合區(qū)域,即nt265-271和nt370-377,miR-144能夠以Nrf2 mRNA為靶點,下調其表達,減少紅細胞對氧化應激的耐受性,導致貧血嚴重程度增加[44]。

抗氧化應激能力隨著年齡的增長逐漸減弱,在大鼠肝細胞中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,miR-146a能與Nrf2 mRNA結合,阻礙Nrf2轉錄后的翻譯,使其蛋白質含量下降,影響Nrf2-ARE通路的激活,導致下游抗氧化酶的表達下調,進而降低機體抗氧化應激能力[45]。

在正常細胞中,Nrf2對于癌癥化學預防和腫瘤抑制起到關鍵作用。但另一方面,在腫瘤形成和腫瘤保護中,Nrf2也起到主要作用。因此,miRNAs可以通過調控Nrf2的表達,發(fā)揮抑癌和致癌的雙重作用。

據(jù)報道,在乳腺癌細胞中過表達miR-153、miR-93、miR-28均能顯著降低Nrf2及下游基因的表達(圖1);同時,在乳腺上皮細胞中miR-153能夠減少凋亡、增加克隆形成。這些研究結果說明,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)膠質瘤細胞的研究中發(fā)現(xiàn),miR-153通過靶向Nrf2基因,抑制其蛋白質表達,從而促進凋亡、抑制細胞增殖,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)轉移的中晚期腫瘤的主要方案,但是藥物抵抗常導致化療效果差、預后不良。研究表明,Nrf2的過表達能夠導致癌細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細胞中,miR-340的表達顯著下調,Nrf2表達上調。生物信息學和熒光素酶報告基因實驗分析發(fā)現(xiàn)Nrf2是miR-340的直接靶基因。因此,上調miR-340,可以抑制Nrf2-依賴的抗氧化通路,從而增加HepG2細胞對順鉑的敏感性,成為靶向治療的依據(jù)。

在順鉑誘導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,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,從而下調Nrf2-ARE信號通路(表1)。不同細胞類型中調控Nrf2的miRNAs種類不同,miRNAs的表達是否與細胞類型有關、是否受Nrf2水平的影響還需進一步研究。Nrf2-ARE通路與腫瘤的發(fā)生發(fā)展密切相關,而miRNAs可以調控該通路,研究是否可以通過靶向miRNAs預防腫瘤形成或促進腫瘤凋亡具有重要的現(xiàn)實指導意義。

3.2 靶向Keap1的miRNAs

Nrf2活性受到胞漿中抑制性蛋白質Keap1的嚴格調控,多種miRNAs也可以通過作用Keap1來調控Nrf2-ARE信號通路。如miR-29可通過靶向Keap1來調節(jié)Nrf2-ARE信號通路(圖1)。研究表明,在高糖誘導的腎小管上皮細胞中,miR-29的表達下降,同時Keap1的表達增加,使得Nrf2泛素化增加,從而降低Nrf2的含量,引起腎小管上皮細胞的氧化損傷,導致腎功能紊亂[52]。但另一方面,也有研究指出Nrf2能夠調控miR-29家族的基因表達[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節(jié)以及具體調控機制還需進一步探討。

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

在缺血缺氧性心肌細胞中,過表達miR-200a能顯著增加Nrf2的核轉移以及下游抗氧化酶基因的表達;抑制miR-200a的表達顯示相反的效果,進一步的實驗證實miR-200a通過與Keap1的3′-UTR結合(圖1),下調Keap1蛋白的表達。由此說明miR-200a可通過抑制Keap1來增加Nrf2,激活Nrf2-ARE信號通路,保護心肌細胞免受低氧和ROS誘導的損傷,發(fā)揮細胞保護作用[56]。此外,miR-200a在肝星形細胞中也可以通過靶向Keap1,激活Nrf2-ARE通路,抵抗氧化應激,預防肝細胞纖維化病變[57]。

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

此外,有研究發(fā)現(xiàn)腦細胞中高度表達的miR-7在人神經(jīng)母細胞瘤細胞中也能夠與Keap1 mRNA的3′-UTR結合(圖1),抑制Keap1表達,進而導致Nrf2活性增加,促進其向核內(nèi)轉移,使下游靶基因HMOX1、GCLM等表達增加,減少ROS誘導的細胞死亡[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,上調Nrf2-ARE信號通路,參與調節(jié)機體的氧化還原反應(表1),但是否還有其他miRNAs調控Keap1仍需進一步研究。

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,調控Nrf2-ARE信號通路。

有文獻報道,miRNAs可以直接調控Cul3。研究發(fā)現(xiàn),低氧可以誘導miR-101的表達,進一步通過實驗證實miR-101能夠與Cul3 mRNA的3′-UTR結合,抑制Cul3蛋白表達,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miR-101能夠促進Nrf2向核內(nèi)轉移,促進HMOX1、血管內(nèi)皮生長因子的基因表達,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調控E3泛素化酶的研究尚少,關于其在泛素化酶系統(tǒng)中的作用尚需進一步研究。

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作用。

miR-155是與免疫調節(jié)最密切相關的一種miRNA,可以調控T細胞的成熟、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。相關研究發(fā)現(xiàn),在耗竭性CD4+T細胞中,miR-155表達增加;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 結合(圖1),導致HO-1表達降低[64],使機體免疫保護及免疫監(jiān)視等功能發(fā)生障礙,最終可能會導致自身免疫性疾病、慢性感染性疾病的發(fā)生。

然而,另有文獻報道,HO-1的表達是通過miR-155間接調控,而非直接調控。在人臍靜脈內(nèi)皮細胞中,腫瘤壞死因子-α(tumor necrosis factor-α,TNF-α)通過 NF-κB 通路誘導 miR-155 的表達,miR-155抑制轉錄因子BACH1的蛋白質表達,但對胞漿和核內(nèi)Nrf2的蛋白質表達無影響。BACH1是HMOX1基因的抑制因子,研究進一步發(fā)現(xiàn)HMOX1的mRNA和蛋白質表達增加。熒光素酶報告基因實驗顯示,miR-155能夠降低含有BACH1 mRNA 3′-UTR的報告基因活性,并且預測出4個結合位點。以上結果說明,miR-155通過靶向BACH1的表達,降低其活性,從而間接上調HO-1的表達[65],提高細胞的抗炎能力。

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結合,抑制基因表達。然而,有文獻報道在人非小細胞肺癌中,miR-1254可以通過其種子序列和非種子序列對HMOX1發(fā)揮雙重調控作用(圖1)。其一,miR-1254通過其種子序列直接與HMOX1 3′-UTR結合,抑制HO-1蛋白翻譯;其二,miR-1254通過其非種子序列與HMOX1的轉錄激活因子AP2A結合,抑制HMOX1基因轉錄。miR-1254通過以上兩種方式抑制HO-1表達,誘導細胞凋亡和細胞周期阻滯,從而抑制人非小細胞肺癌生長[23]。

HO-1參與調控炎癥反應、腫瘤發(fā)展,目前關于miRNAs調控HMOX1的報道較少,因此,還需進一步研究發(fā)現(xiàn)更多調控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,能夠調控細胞的氧化還原穩(wěn)態(tài),維持機體正常的生理功能。

研究發(fā)現(xiàn),在人臍靜脈血管內(nèi)皮細胞中過表達miR-433可下調GCLC、GCLM和GSH的水平,但對Nrf2的表達無影響。更進一步研究發(fā)現(xiàn)miR-433可以直接作用于GCLC、GCLM的mRNA 3′-UTR(圖1),影響其蛋白質表達,增加血管內(nèi)皮細胞的氧化損傷,而這種直接調控作用不依賴于Nrf2 的水平[66]。

除了miR-433外,目前尚未查到其他文獻報道直接調控GCL的相關miRNAs,因此,還需進一步探索以GCL為靶基因的miRNAs。

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

4 展望

關于miRNAs調控Nrf2-ARE信號通路中關鍵因子Nrf2、Keap1的研究較多,但是miRNAs調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,仍需要進一步研究miRNAs與Nrf2-ARE通路的調控關系,以便進一步了解該通路活化或失活的分子機制,為疾病預防提供新的思路。

表1 靶向調控Nrf2-ARE通路的miRNAsTable1 miRNAs targeting the regulation of Nrf2-ARE pathway

(接上表)

隨著miRNAs研究的不斷進展,將會發(fā)現(xiàn)更多與疾病發(fā)生發(fā)展有關的miRNAs,可以此為生物標志物,應用于臨床檢測;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。

參考文獻(References):

[1]Owusu-Ansah E,Banerjee U.Reactive oxygen species primeDrosophilahaematopoietic progenitors for differentiation[J].Nature,2009,461(7263):537-541.

[2]Roy K,Wu Y J,Meitzler J L,et al.NADPH oxidases and cancer[J].Clinical Science,2015,128(12):863-875.

[3]Reuter S,Gupta S C,Chaturvedi M M,et al.Oxidative stress,inflammation,and cancer:how are they linked?[J].Free Radical Biology and Medicine,2010,49(11):1603-1616.

[4]Li M,Marin-Muller C,Bharadwaj U,et al.MicroRNAs:control and loss of control in human physiology and disease[J].World Journal of Surgery,2009,33(4):667-684.

[5]Ludwig N,Becker M,Schumann T,et al.Bias in recent miRBase annotations potentially associated with RNA quality issues[J].Scientific Reports,2017,7(1):5162.

[6]Friedman R C,Farh K K,Burge C B,et al.Most mammalian mRNAs are conserved targets of microRNAs[J].Genome Research,2009,19(1):92-105.

[7]Espinosa-Diez C,Miguel V,Mennerich D,et al.Antioxidant responses and cellular adjustments to oxidative stress[J].Redox Biology,2015,6:183-197.

[8]Bartel D P.MicroRNAs:genomics,biogenesis,mechanism,and function[J].Cell,2004,116(2):281-297.

[9]McMahon M,Itoh K,Yamamoto M,et al.Keap1-dependent proteasomal degradation of transcription factor Nrf2 contributes to the negative regulation of antioxidant response element-driven gene expression[J].The Journal of Biological Chemistry,2003,278(24):21592-21600.

[10]Kobayashi A,Kang M I,Okawa H,et al.Oxidative stress sensor Keap1 functions as an adaptor for Cul3-based E3 ligase to regulate proteasomal degradation of Nrf2[J].Molecular and Cellular Biology,2004,24(16):7130-7139.

[11]Cullinan S B,Gordan J D,Jin J,et al.The Keap1-BTB protein is an adaptor that bridges Nrf2 to a Cul3-based E3 ligase:oxida tive stress sensing by a Cul3-Keap1 ligase[J].Molecular and Cellular Biology,2004,24(19):8477-8486.

[12]Rada P,Rojo A I,Chowdhry S,et al.SCF/β-TrCP promotes glycogen synthase kinase 3-dependent degradation of the Nrf2 transcription factor in a Keap1-independent manner[J].Molecular and Cellular Biology,2011,31(6):1121-1133.

[13]Qin S,Chen J,Tanigawa S,et al.Gene expression profiling and pathway network analysis of hepatic metabolic enzymes targeted by baicalein[J].Journal of Ethnopharmacology,2012,140(1):131-140.

[14]Qin S,Chen J,Tanigawa S,et al.Microarray and pathway analysis highlight Nrf2/ARE-mediated expression profiling by polyphenolicmyricetin[J].MolecularNutrition&FoodResearch,2013,57(3):435-446.

[15]Tanigawa S,Fujii M,Hou D.Action of Nrf2 and Keap1 in ARE-mediated NQO1 expression by quercetin[J].Free Radical Biology and Medicine,2007,42(11):1690-1703.

[16]Li L,Dong H,Song E,et al.Nrf2/ARE pathway activation,HO-1 and NQO1 induction by polychlorinated biphenyl quinone is associated with reactive oxygen species and PI3K/AKT signaling[J].Chemico-Biological Interactions,2014,209:56-67.

[17]Danilenko M,Studzinski G P.Keep harm at bay:oxidative phosphorylation induces Nrf2-driven antioxidant response via ERK5/MEF2/miR-23a signaling to Keap-1[J].EBioMedicine,2016,3:4-5.

[18]Shan J L,He H T,Li M X,et al.APE1 promotes antioxidant capacity by regulating Nrf-2 function through a redox-dependent mechanism[J].Free Radical Biology and Medicine,2015,78:11-22.

[19]Tertil M,Golda S,Skrzypek K,et al.Nrf2-heme oxygenase-1 axis in mucoepidermoid carcinoma of the lung:antitumoral effects associated with down-regulation of matrix metalloproteinases[J].Free Radical Biology and Medicine,2015,89:147-157.

[20]Cheng Z G,Zhang G D,Shi P Q,et al.Expression and antioxidation of Nrf2/ARE pathway in traumatic brain injury[J].Asian Pacific Journal of Tropical Medicine,2013,6(4):305-310.

[21]Saw C L L,Guo Y,Yang A Y,et al.The berry constituents quercetin,kaempferol,and pterostilbene synergistically attenuate reactive oxygen species:involvement of the Nrf2-ARE signaling pathway[J].Food and Chemical Toxicology,2014,72:303-311.

[22]Karnati H K,Panigrahi M K,Gutti R K,et al.MiRNAs:key players in neurodegenerative disorders and epilepsy[J].Journal of Alzheimer’s Disease,2015,48(3):563-580.

[23]Pu M,Li C,Qi X,et al.MiR-1254 suppresses HO-1 expression through seed region-dependent silencing and non-seed interaction with TFAP2A transcript to attenuate NSCLC growth[J].PLoS Genetics,2017,13(7):e1006896.

[24]Miao L,Yao H,Li C,et al.A dual inhibition:microRNA-552 suppresses both transcription and translation of cytochrome P450 2E1[J].Biochimica et Biophysica Acta-Gene Regulatory Mechanisms,2016,1859(4):650-662.

[25]Kim D H,Saetrom P,Sn?ve O,et al.MicroRNA-directed transcriptional gene silencing in mammalian cells[J].Proceedings of the National Academy of Sciences USA,2008,105(42):16230-16235.

[26]Prasad K N.Oxidative stress and pro-inflammatory cytokines may act as one of the signals for regulating microRNAs expression in Alzheimer’s disease[J].Mechanisms of Ageing and Development,2016,162:63-71.

[27]Lytle J R,Yario T A,Steitz J A.Target mRNAs are repressed as efficiently by microRNA-binding sites in the 5′UTR as in the 3′UTR[J].Proceedings of the National Academy of Sciences USA,2007,104(23):9667-9672.

[28]Lee I,Ajay S S,Yook J I,et al.New class of microRNA targets containing simultaneous 5′-UTR and 3′-UTR interaction sites[J].Genome Research,2009,19(7):1175-1183.

[29]Moretti F,Thermann R,Hentze M W.Mechanism of translational regulation by miR-2 from sites in the 5′untranslated region or the open reading frame[J].RNA,2010,16(12):2493-2502.

[30]Jin Y,Wang C,Liu X,et al.Molecular characterization of the microRNA-138-Fos-like antigen 1(FOSL1)regulatory module in squamous cell carcinoma[J].The Journal of Biological Chemistry,2011,286(46):40104-40109.

[31]Jopling C L,Schütz S,Sarnow P.Position-dependent function for a tandem microRNA miR-122-binding site located in the hepatitis C virus RNA genome[J].Cell Host&Microbe,2008,4(1):77-85.

[32]Orom U A,Nielsen F C,Lund A H.MicroRNA-10a binds the 5′UTR of ribosomal protein mRNAs and enhances their translation[J].Molecular Cell,2008,30(4):460-471.

[33]Tsai N P,Lin Y L,Wei L N.MicroRNA miR-346 targets the 5′-untranslated region of receptor-interacting protein 140(RIP140)mRNA and up-regulates its protein expression[J].Biochemical Journal,2009,424(3):411-418.

[34]Kurinna S,Werner S.Nrf2 and microRNAs:new but awaited relations[J].Biochemical Society Transactions,2015,43(4):595-601.

[35]Zhang C,Shu L,Kong A N.MicroRNAs:new players in cancer prevention targeting Nrf2,oxidative stress and inflammatory pathways[J].Current Pharmacology Reports,2015,1(1):21-30.

[36]Ayers D,Baron B,Hunter T.MiRNA influences in Nrf2 pathway interactions within cancer models[J].Journal of Nucleic Acids,2015,2015:143636.

[37]Ji Q,Gao J,Zheng Y,et al.Inhibition of microRNA-153 protects neurons against ischemia/reperfusion injury in an oxygen-glucose deprivation and reoxygenation cellular model by regulating Nrf2/HO-1 signaling[J].Journal of Biochemical and Molecular Toxicology,2017,31(7):e21905.

[38]Narasimhan M,Riar A K,Rathinam M L,et al.Hydrogen peroxide responsive miR153 targets Nrf2/ARE cytoprotection in paraquat induced dopaminergic neurotoxicity[J].Toxicology Letters,2014,228(3):179-191.

[39]Wang P,Liang X,Lu Y,et al.MicroRNA-93 downregulation ameliorates cerebral ischemic injury through the Nrf2/HO-1 defense pathway[J].Neurochemical Research,2016,41(10):2627-2635.

[40]Narasimhan M,Patel D,Vedpathak D,et al.Identification of novel microRNAs in post-transcriptional control of Nrf2 expression and redox homeostasis in neuronal,SH-SY5Y cells[J].PLoS One,2012,7(12):e51111.

[41]Ba Q,Cui C,Wen L,et al.Schisandrin B shows neuroprotective effect in 6-OHDA-induced Parkinson’s disease via inhibiting the negative modulation of miR-34a on Nrf2 pathway[J].Biomedicine&Pharmacotherapy,2015,75:165-172.

[42]Zhang X S,Ha S,Wang X L,et al.Tanshinone IIA protects dopaminergic neurons against 6-hydroxydopamine-induced neurotoxicity through miR-153/NF-E2-related factor 2/antioxidant response element signaling pathway[J].Neuroscience,2015,303:489-502.

[43]Csiszar A,Gautam T,Sosnowska D,et al.Caloric restriction confers persistent anti-oxidative,pro-angiogenic,and anti-inflammatory effects and promotes anti-aging miRNA expression profile in cerebromicrovascular endothelial cells of aged rats[J].American Journal of Physiology-Heart and Circulatory Physi ology,2014,307(3):H292-H306.

[44]Sangokoya C,Telen M J,Chi J T.MicroRNA miR-144 modulates oxidative stresstolerance and associates with anemia severity in sickle cell disease[J].Blood,2010,116(20):4338-4348.

[45]Smith E J,Shay K P,Thomas N O,et al.Age-related loss of hepatic Nrf2 protein homeostasis:potential role for heightened expression of miR-146a[J].Free Radical Biology and Medicine,2015,89:1184-1191.

[46]Wang B,Teng Y,Liu Q.MicroRNA-153 regulates Nrf2 expression and is associated with breast carcinogenesis[J].Clinical Laboratory,2016,62(1-2):39-47.

[47]Singh B,Ronghe A M,Chatterjee A,et al.MicroRNA-93 regulates Nrf2 expression and is associated with breast carcinogenesis[J].Carcinogenesis,2013,34(5):1165-1172.

[48]Yang M,Yao Y,Eades G,et al.MiR-28 regulates Nrf2 expression through a Keap1-independent mechanism[J].Breast Cancer Research and Treatment,2011,129(3):983-991.

[49]黃龍.Nrf2在miR-153調控神經(jīng)膠質瘤細胞增殖與凋亡中的作用[D].南京:南京大學(Huang Long.The Role of Nrf2 on miR-153 in the Regulation of Proliferation and Apoptosis of Glioblastoma Cells[D].Nanjing:Nanjing University),2013.

[50]Shi L,Chen Z G,Wu L L,et al.MiR-340 reverses cisplatin resistance of hepatocellular carcinoma cell lines by targeting Nrf2-dependent antioxidant pathway[J].Asian Pacific Journal of Cancer Prevention,2014,15(23):10439-10444.

[51]Liao W,Fu Z,Zou Y,et al.MicroRNA-140-5p attenuated oxidative stress in cisplatin induced acute kidney injury by activating Nrf2/ARE pathway through a Keap1-independent mechanism[J].Experimental Cell Research,2017,360(2):292-302.

[52]Zhou L,Xu D Y,Sha W G,et al.High glucose induces renal tubular epithelial injury via Sirt1/NF-kappaB/microR-29/Keap1 signal pathway[J].Journal of Translational Medicine,2015,13:352.

[53]Kurinna S,Schafer M,Ostano P,et al.A novel Nrf2-miR-29-desmocollin-2 axis regulates desmosome function in keratinocytes[J].Nature Communications,2014,5:5099.

[54]Cheng L B,Li K R,Yi N,et al.MiRNA-141 attenuates UV-induced oxidative stress via activating Keap1-Nrf2 signaling in human retinal pigment epithelium cells and retinal ganglion cells[J].Oncotarget,2017,8(8):13186-13194.

[55]Shi L,Wu L,Chen Z,et al.MiR-141 activates Nrf2-dependent antioxidant pathway via down-regulating the expression of Keap1 conferring the resistance of hepatocellular carcinoma cells to 5-fluorouracil[J].Cellular Physiology Biochemistry,2015,35(6):2333-2348.

[56]Sun X,Zuo H,Liu C,et al.Overexpression of miR-200a protects cardiomyocytes against hypoxia-induced apoptosis by modulating the kelch-like ECH-associated protein 1-nuclear factor erythroid 2-related factor 2 signaling axis[J].International Journal of Molecular Medicine,2016,38(4):1303-1311.

[57]Yang J J,Tao H,Hu W,et al.MicroRNA-200a controls Nrf2 activation by target Keap1 in hepatic stellate cell proliferation and fibrosis[J].Cellular Signalling,2014,26(11):2381-2389.

[58]Liu M,Hu C,Xu Q,et al.Methylseleninic acid activates Keap1/Nrf2 pathway via up-regulating miR-200a in human oesophageal squamous cell carcinoma cells[J].Bioscience Reports,2015,35(5):e00256.

[59]Eades G,Yang M,Yao Y,et al.MiR-200a regulates Nrf2 activation by targeting Keap1 mRNA in breast cancer cells[J].The Journal of Biological Chemistry,2011,286(47):40725-40733.

[60]Akdemir B,Nakajima Y,Inazawa J,et al.MiR-432 induces Nrf2 stabilization by directly targeting Keap1[J].Molecular Cancer Research,2017,15(11):1570-1578.

[61]Kabaria S,Choi D C,Chaudhuri A D,et al.MicroRNA-7 activates Nrf2 pathway by targeting Keap1 expression[J].Free Radical Biology and Medicine,2015,89:548-556.

[62]Kim J H,Lee K S,Lee D K,et al.Hypoxia-responsive microRNA-101 promotes angiogenesis via heme oxygenase-1/vascular endothelial growth factor axis by targeting cullin3[J].Antioxidants&Redox Signaling,2014,21(18):2469-2482.

[63]Yu D,Wang Y,Bi Y,et al.Salvianolic acid A ameliorates the integrity of blood-spinal cord barrier via miR-101/Cul3/Nrf2/HO-1 signaling pathway[J].Brain Research,2017,1657:279-287.

[64]張晉渝.MicroRNA-155在T細胞耗竭中的作用及其機制研究[D].重慶:第三軍醫(yī)大學(Zhang Jin-yu.The Characteristics and Roles of MicroRNA-155 in T Cell Exhaustion[D].Chongqing:Third Military Medical University),2013.

[65]Pulkkinen K H,Yla-Herttuala S,Levonen A L.Heme oxygenase 1 is induced by miR-155 via reduced BACH1 translation in endothelial cells[J].Free Radical Biology and Medicine,2011,51(11):2124-2131.

[66]Espinosa-Diez C,Fierro-Fernandez M,Sanchez-Gomez F,et al.Targeting of gamma-glutamyl-cysteine ligase by miR-433 reduces glutathione biosynthesis and promotes TGF-β-dependent fibrogenesis[J].Antioxidants&Redox Signaling,2015,23(14):1092-1105.

[67]Xue W,Bai X,Zhang L.RhTNFR:Fc increases Nrf2 expression via miR-27a mediation to protect myocardium against sepsis injury[J].Biochemical and Biophysical Research Communications,2015,464(3):855-861.

[68]Wang N,Zhang L,Lu Y,et al.Down-regulation of microRNA-142-5p attenuates oxygen-glucose deprivation and reoxygenation-induced neuron injury through up-regulating Nrf2/ARE signaling pathway[J].Biomedicine&Pharmacotherapy,2017,89:1187-1195.

[69]Yu M,Liu Y,Zhang B,et al.Inhibiting microRNA-144 abates oxidativestress and reducesapoptosis in heartsof streptozotocininduced diabetic mice[J].Cardiovascular Pathology,2015,24(6):375-381.

[70]Sun X,Liu D,Xue Y,et al.Enforced miR-144-3p expression as a non-invasive biomarker for the acute myeloid leukemia patients mainly by targeting Nrf2[J].Clinical Laboratory,2017,63(4):679-687.