齊仁立 黃金秀 龍定彪 黃萍
(重慶市畜牧科學(xué)院 農(nóng)業(yè)部養(yǎng)豬科學(xué)重點實驗室 養(yǎng)豬科學(xué)重慶市市級重點實驗室,榮昌 402460)
MicroRNA與NF-κB調(diào)控的細(xì)胞凋亡
齊仁立 黃金秀 龍定彪 黃萍
(重慶市畜牧科學(xué)院農(nóng)業(yè)部養(yǎng)豬科學(xué)重點實驗室養(yǎng)豬科學(xué)重慶市市級重點實驗室,榮昌402460)
NF-κB是一種非常重要的核轉(zhuǎn)錄調(diào)控因子,激活后的NF-κB通過調(diào)節(jié)靶基因的轉(zhuǎn)錄表達(dá)參與細(xì)胞的各項生命活動,特別是炎癥、免疫和凋亡。NF-κB在不同類型的細(xì)胞凋亡中可能發(fā)揮不同的調(diào)控作用。而microRNA這類非編碼的RNA對于NF-κB的表達(dá)和功能又能夠產(chǎn)生系統(tǒng)性或者特異性的調(diào)節(jié)。綜述分析NF-κB對細(xì)胞凋亡的調(diào)控作用,并探討microRNA在此過程中的作用。
NF-κB;microRNA;細(xì)胞凋亡
早在1986年,Sen和他的同事們通過試驗在B淋巴細(xì)胞的核提取物中發(fā)現(xiàn)了一種能與免疫球蛋白κ輕鏈基因特異結(jié)合的蛋白因子,并將其命名為核因子κB(Nuclear factor Kappa B,NF-κB)[1]。NF-κB廣泛存在于多種細(xì)胞中,當(dāng)細(xì)胞受到外部刺激(藥物、輻射、缺氧等)或者細(xì)胞因子、病毒感染、脂多糖等刺激時,NF-κB被激活并與相應(yīng)的病毒、細(xì)胞因子、受體蛋白等所調(diào)節(jié)的基因增強(qiáng)子區(qū)結(jié)合,啟動基因轉(zhuǎn)錄[2-5]。隨著時間的推移,研究逐漸證明NF-κB是一種細(xì)胞核內(nèi)極為重要的轉(zhuǎn)錄調(diào)節(jié)因子,對于細(xì)胞的增殖和分化、衰老和死亡、黏附和遷移都具有重要的調(diào)節(jié)作用[3,6]。
NF-κB是由NF-κB/Rel蛋白家族成員NF-κB1(p50/前體為P105)、NF-κB2(p52/前體為P100)、Rel(p65)、RelB和C-Rel以同源或異源二聚體的形式組成。雖然有多個不同的二聚體組合,但p50和p65組成的二聚體最早被發(fā)現(xiàn),并且其分布和作用也最為廣泛,是通常所說的NF-κB。在細(xì)胞靜息狀態(tài)下,NF-κB以無活性狀態(tài)存在于細(xì)胞漿中,并與抑制因子IκBs(包括IκBα、IκBβ,IκBγ、IκBε、IκBζ、IκBS和Bcl-3等)一起組成了異源多聚體P50/P60/ IκBs。在這個多聚體中,IκBs能阻礙P50和P65的二聚體化,使NF-κB以無活性的形式存在。當(dāng)細(xì)胞受到腫瘤壞死因子α(Tumor necrosis factor α,TNFα)、白介素或者其他藥物的刺激時,IκBs的激酶IκK會被磷酸化激活,并作用于P50/P60/IκBs三聚體中的IκBs,使其從三聚體中脫離并被泛素化降解,暴露出P50亞基上的移位信號和P65亞基上的DNA結(jié)合位點,從而使P50/P65二聚體表現(xiàn)出NF-κB活性,并從細(xì)胞漿移位到細(xì)胞核中,然后與靶基因的κB基序結(jié)合,發(fā)揮轉(zhuǎn)錄調(diào)控作用。除了上述的典型激活方式,NF-κB的激活還存在其他非典型的方式[2](圖1)。在非經(jīng)典激活途徑中,P100和RelB的二聚體被激活,這種激活主要存在于B細(xì)胞和T細(xì)胞發(fā)育過程中。接收到特定的受體信號后(例如淋巴毒素B和B細(xì)胞活化因子)在NF-κB誘導(dǎo)激酶(NIK)的作用下,IκKa復(fù)合體被磷酸化進(jìn)而引起p100的磷酸化,經(jīng)蛋白激酶作用誘導(dǎo)產(chǎn)生p52-RelB異源二聚體,并使其進(jìn)入細(xì)胞核參與核轉(zhuǎn)錄活動。
圖1 NF-κB典型和非典型激活方式[2]
細(xì)胞凋亡是多細(xì)胞生物一種正常的生命現(xiàn)象,在受到外部或者內(nèi)部的刺激以后,細(xì)胞為了維護(hù)內(nèi)環(huán)境的穩(wěn)態(tài),出現(xiàn)由多種基因控制的細(xì)胞程序性的主動死亡。細(xì)胞凋亡對于維持細(xì)胞正常生長和代謝穩(wěn)定具有十分重要的意義,而細(xì)胞凋亡的異常與各種腫瘤的產(chǎn)生密切相關(guān)[7]。
活化的NF-κB與細(xì)胞凋亡緊密相關(guān),但是NF-κB對于細(xì)胞凋亡產(chǎn)生和發(fā)展的影響還存在很多謎團(tuán)。在不同的細(xì)胞中,不同的誘導(dǎo)刺激下,NF-κB似乎扮演了不同的角色。多數(shù)研究認(rèn)為NF-κB具有凋亡抑制作用[3,8-11],因為NF-κB 對于許多抗凋亡基因都有特異性調(diào)控作用,包括TRAF1、TRAF2、c-IAP1、c-IAP2、IEX-1L、Bcl-xL和Bfl-1/A1等[12-17]。典型的功能缺失試驗可以清楚地說明NF-κB抗凋亡作用。體外培養(yǎng)的胚胎成纖維細(xì)胞在剔除RelA時對于TNFα引起的凋亡更為敏感,而在個體試驗上,TNFα能夠促使RelA-/-小鼠出現(xiàn)肝細(xì)胞大量的凋亡,并最終導(dǎo)致小鼠的死亡[18,19]。
但是,另一方面,NF-κB的促凋亡作用也同樣明顯,Stefan等[20]報道了撤去培養(yǎng)基中的血清會造成293細(xì)胞的大量凋亡并伴隨著NF-κB的顯著激活。腫瘤抑制因子P53能夠改變細(xì)胞周期,誘發(fā)多種細(xì)胞凋亡,而NF-κB在其中起到了明顯的促凋亡作用。Ryan等[21]研究表明P53能夠以與TNFa不同的方式激活NF-κB,并且誘導(dǎo)細(xì)胞走向凋亡,而抑制或者減少NF-κB的活性,則會阻斷P53引起的凋亡。為了檢測NF-κB的促凋亡和抗凋亡作用,Kaltschmidt[22]和他的同事們通過遺傳學(xué)和藥理學(xué)方法阻止HeLa細(xì)胞中的NF-κB被激活,結(jié)果發(fā)現(xiàn)雖然細(xì)胞對于TNFα誘導(dǎo)的凋亡更為敏感,但是卻能夠完全阻止H2O2和pervanadate誘導(dǎo)的凋亡。他們認(rèn)為不同的凋亡誘導(dǎo)物引發(fā)的分子信號不同,決定了NF-κB在細(xì)胞凋亡產(chǎn)生和發(fā)展中的作用也不相同。
此外,似乎不同的NF-κB激活方式在細(xì)胞凋亡中也有不同的調(diào)控作用。與TNFα類似,F(xiàn)AS能夠激活死亡受體FASL誘發(fā)細(xì)胞凋亡。典型激活的NF-κB能直接與FAS基因啟動子結(jié)合,抑制NF-κB的典型激活能夠降低FAS表達(dá),而抑制非典型形式激活NF-κB則能夠增加FAS的表達(dá)[23]。
這些試驗結(jié)果提示,NF-κB在細(xì)胞凋亡中的調(diào)控作用十分復(fù)雜,受到激活方式、存在形式、細(xì)胞種類等多種因素的影響,在不同的細(xì)胞中可能分別或者同時具有抑制細(xì)胞凋亡和促進(jìn)細(xì)胞凋亡的作用。
MicroRNA是一種廣泛存在于動物、植物、細(xì)菌和病毒體內(nèi)的非編碼RNA(non-coding RNA),通過序列互補(bǔ)的方式引導(dǎo)沉默復(fù)合體降解靶mRNA或者抑制翻譯[24-26]。一個microRNA可以調(diào)控多個甚至影響數(shù)百個靶基因,而一個基因也可能同時受到多個microRNA的調(diào)節(jié)[27]。通過對靶基因的特異性/系統(tǒng)性調(diào)控,microRNA廣泛參與并調(diào)控了細(xì)胞增殖、分化、代謝、衰老和凋亡的各種轉(zhuǎn)錄激活和信號傳導(dǎo)[28]。反之,一些轉(zhuǎn)錄因子(如NF-κB)的激活和表達(dá)的改變又影響了microRNA的轉(zhuǎn)錄、加工和功能[29]。
目前已有一些研究探尋microRNA和NF-κB之間的相互作用關(guān)系,并且發(fā)現(xiàn)NF-κB的活化和功能的施展需要一些特定microRNA的參與[30,31](圖2)。此外,激活的NF-κB對于特定microRNA的調(diào)控機(jī)能也是必不可少的。最早被確認(rèn)參與調(diào)節(jié)NF-κB信號的是miR-146,它被認(rèn)為是Toll-like 受體介導(dǎo)的NF-κB激活的抑制者。Taganov等[32]研究發(fā)現(xiàn)miR-146是一個NF-κB依賴型基因,并能夠通過負(fù)反饋調(diào)節(jié)IL-1受體相關(guān)激酶1和TNFα受體相關(guān)蛋白6的表達(dá)參與到機(jī)體的免疫應(yīng)答反應(yīng)。Paik等[33]的試驗結(jié)果顯示,miR-146a在NKTL細(xì)胞系中的過表達(dá)能夠明顯抑制NF-κB的活性,阻滯細(xì)胞分化,誘導(dǎo)細(xì)胞凋亡。還有試驗發(fā)現(xiàn)miR-125家族能夠靶向NF-κB的抑制因子TNFα 誘導(dǎo)蛋白3(TNFAIP3)從而促進(jìn)NF-κB激活[34]。miR-21能夠通過PTEN/AKT通路促進(jìn)MCF-10A細(xì)胞中NF-κB信號,但是卻抑制了HEK293細(xì)胞中脂多糖LPS介導(dǎo)的NF-κB激活[35]。George等[30]分別于TNFα存在和不存在的情況下使用高通量library掃描NF-κB穩(wěn)轉(zhuǎn)報告細(xì)胞系中miRNA表達(dá)變化,并最終確認(rèn)miR-517a/c可以誘導(dǎo)細(xì)胞內(nèi)源性NF-κB的表達(dá),并促進(jìn)P65的核移位和IκB的降解。
在已經(jīng)確認(rèn)的參與NF-κB網(wǎng)絡(luò)的microRNA中,有許多都能夠直接參與細(xì)胞凋亡調(diào)控,如miR-21[36,37]、miR-29[38,39]和let-7[40]等。以miR-21為例,在許多實質(zhì)癌細(xì)胞中miR-21都有異常高表達(dá),它能夠靶向抑制細(xì)胞死亡因子PDCD4基因,并且影響細(xì)胞凋亡抑制基因BCL2,阻斷凋亡進(jìn)程,促進(jìn)癌細(xì)胞的生成[36]。miR-21還能夠通過降低靶基因PTEN的表達(dá),進(jìn)而影響AKT的磷酸化并導(dǎo)致NF-κB的激活[36]。反之,miR-21的轉(zhuǎn)錄也受到NF-κB的調(diào)控,P65亞基的缺失會抑制LPS誘導(dǎo)的miR-21表達(dá)[37],所以NF-κB的活性對于miR-21的調(diào)控功能是必不可少的。
圖2 參與NF-κB調(diào)控分子網(wǎng)絡(luò)的microRNA[31]
基于上述研究可以確認(rèn)一些microRNA能夠直接或者間接地參與NF-κB的表達(dá)和激活,或者是受到NF-κB的調(diào)控而影響下游靶基因,對細(xì)胞凋亡的啟動和發(fā)展起到重要的調(diào)節(jié)作用。但是這些不同的分子之間如何“交流”在很大程度上還充滿了懸念。此外,隨著高通量和新一代測序技術(shù)的迅速普及,將會有更多參與NF-κB分子網(wǎng)絡(luò)的microRNA被發(fā)現(xiàn)和確認(rèn)。
細(xì)胞凋亡有助于清除受損細(xì)胞,減少應(yīng)激損傷,維持機(jī)體穩(wěn)態(tài)。憑借其重要的調(diào)控作用和在分子網(wǎng)絡(luò)中的核心地位,NF-κB在細(xì)胞凋亡的產(chǎn)生和發(fā)展過程中扮演了重要的角色,它可能成為包括癌癥、心血管疾病、肥胖及代謝癥在內(nèi)的多種疾病診療的靶位點。未來的工作中,我們將更加清楚NF-κB在不同類型的細(xì)胞凋亡中的差異調(diào)控機(jī)制。生物體內(nèi)大量存在的microRNA對各種生命活動發(fā)揮著復(fù)雜而精巧的調(diào)控作用。迄今為止,只有極少數(shù)的microRNA被研究和確認(rèn)參與了NF-κB的激活和調(diào)控。但隨著新一代高通量測序和基因芯片技術(shù)的快速普及,將會有更多microRNA被發(fā)掘,有望在較短的時間內(nèi)了解更多影響和參與NF-κB分子調(diào)節(jié)的microRNA,并逐漸掌握它們之間的“對話機(jī)制”,一個完整系統(tǒng)的調(diào)控網(wǎng)絡(luò)圖譜將會逐漸清晰。
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(責(zé)任編輯 狄艷紅)
The Role of MicroRNA in the Cell Apoptosis Mediated by NF-κB
Qi Renli Huang Jinxiu Long Dingbiao Huang Ping
(Key Laboratory of Pig Industry Sciences,Ministry of Agriculture,Chongqing Key Laboratory of Pig Industry Sciences,Chongqing Academy of Animal Sciences,Rongchang402460)
As a key regulatory factor in cell, activated NF-κB induces the transcription of target genes involved in many cell activities,especially the inflammation, immunity and apoptosis. Previous studies proved that NF-κB plays various roles in different type of apoptosis. Moreover, expression and function of NF-κB could be systematically or specifically regulated by microRNA of non-encoding RNAS. The mediating effects of NF-κB on apoptosis were summarized, and the role of microRNA in the mediating process were further discussed .
NF-κB;microRNA;Apoptosis
10.13560/j.cnki.biotech.bull.1985.2015.05.005
2014-08-01
國家自然科學(xué)基金項目(31470117,31302055),重慶市農(nóng)發(fā)資金項目(13412),重慶市自然科學(xué)基金重點項目(2011BA1033)
齊仁立,男,博士,助理研究員,研究方向:脂肪細(xì)胞凋亡的分子機(jī)制;E-mail:qirenli1982@163.com
黃萍,女,高級畜牧師,研究方向:豬肉品質(zhì)的營養(yǎng)調(diào)控;E-mail:505254858@qq.com