林德玲, 羅瑛 宋宜

1. 昆明理工大學(xué)醫(yī)學(xué)院, 昆明 650500;

2. 軍事醫(yī)學(xué)科學(xué)院放射與輻射醫(yī)學(xué)研究所, 北京 100850

基因轉(zhuǎn)錄后調(diào)控在DNA損傷反應(yīng)中的重要功能

林德玲1,2, 羅瑛1, 宋宜2

1. 昆明理工大學(xué)醫(yī)學(xué)院, 昆明 650500;

2. 軍事醫(yī)學(xué)科學(xué)院放射與輻射醫(yī)學(xué)研究所, 北京 100850

DNA損傷發(fā)生時(shí), 細(xì)胞會(huì)激活一系列復(fù)雜的信號(hào)網(wǎng)絡(luò)來調(diào)控細(xì)胞周期檢查, 完成DNA損傷修復(fù)或當(dāng)損傷超過修復(fù)能力時(shí)誘導(dǎo)凋亡, 這一信號(hào)網(wǎng)絡(luò)被稱為DNA損傷反應(yīng)(DNA damage response, DDR)。以往DDR信號(hào)網(wǎng)絡(luò)的研究主要集中于基因轉(zhuǎn)錄調(diào)控和蛋白共價(jià)修飾對(duì)功能分子的穩(wěn)定性和活性調(diào)控。近年來, mRNA穩(wěn)定性調(diào)控和 mRNA 翻譯調(diào)控等基因轉(zhuǎn)錄后調(diào)控機(jī)制在 DDR中的重要作用引起研究者越來越多的關(guān)注。研究證明：多種microRNAs和RNA結(jié)合蛋白(RNA-binding proteins, RBPs)在轉(zhuǎn)錄后水平調(diào)控諸多重要功能蛋白的表達(dá), 在 DDR信號(hào)網(wǎng)絡(luò)中起著不可或缺的作用。文章針對(duì) DDR反應(yīng)中轉(zhuǎn)錄后調(diào)控的研究進(jìn)展以及參與其中的microRNAs和RBPs進(jìn)行闡述和討論。

DNA損傷反應(yīng); microRNAs; RNA結(jié)合蛋白; 轉(zhuǎn)錄后調(diào)控

基因組穩(wěn)定使遺傳信息能夠忠實(shí)的傳遞給后代從而保證機(jī)體的生存。人體每個(gè)細(xì)胞每天都要面對(duì)數(shù)以萬計(jì)的 DNA損傷, 這些損傷可能來自內(nèi)源的DNA復(fù)制過程中的失誤, 也可能源于代謝副產(chǎn)品活性氧族引起的損傷[1]。與此同時(shí), 紫外照射(UV)、電離輻射(Ionizing radiation, IR)、化學(xué)物質(zhì)等許多胞外因素也會(huì)引起DNA損傷[2]。為保護(hù)基因組的完整性,真核細(xì)胞形成一套復(fù)雜的 DNA損傷反應(yīng)(DNA damage response, DDR)信號(hào)轉(zhuǎn)導(dǎo)系統(tǒng)。該信號(hào)網(wǎng)絡(luò)由眾多的功能蛋白組成, 按功能可分為損傷識(shí)別分子、信號(hào)傳導(dǎo)分子以及效應(yīng)分子, 通過對(duì)信息的收集、加工和傳遞最終決定細(xì)胞的命運(yùn), 即啟動(dòng)細(xì)胞周期阻滯(Cell cycle arrest)來完成對(duì)損傷DNA的修復(fù)或損傷超過修復(fù)能力時(shí)激活凋亡、自噬等細(xì)胞死亡途徑[3]。

DDR激活過程中, 細(xì)胞內(nèi)重要功能蛋白的表達(dá)水平被嚴(yán)格調(diào)控。研究證明, 除了mRNA轉(zhuǎn)錄和蛋白穩(wěn)定性、活性調(diào)控外, 對(duì)指導(dǎo)蛋白合成的 mRNA的穩(wěn)定性和翻譯活性調(diào)控等轉(zhuǎn)錄后調(diào)控機(jī)制也在DDR信號(hào)通路中發(fā)揮重要作用[4,5]。2005年美國(guó)NIH腫瘤所的 Cheadle等[6]通過基因組芯片(檢測(cè)胞內(nèi)mRNA豐度)和nuclear run-on芯片(檢測(cè)新生mRNA量)的比較研究證實(shí), 細(xì)胞激活過程中50%的mRNA豐度變化是由 mRNA穩(wěn)定性改變(而非基因轉(zhuǎn)錄改變)造成[6]。該數(shù)據(jù)直接說明：以往被忽視的基因轉(zhuǎn)錄后調(diào)控(Post-transcriptional regulation)同樣是真核基因表達(dá)調(diào)控的關(guān)鍵環(huán)節(jié)[6]。目前研究較明確的DDR轉(zhuǎn)錄后修飾機(jī)制包括：(1)選擇性的穩(wěn)定或降解特定mRNA; (2)對(duì)mRNA的翻譯活性進(jìn)行調(diào)控。這兩種機(jī)制都依賴于多種非編碼小RNA (microRNAs, miRNA)及 RNA 結(jié)合蛋白(RNA binding proteins, RBPs)的參與, 它們能直接結(jié)合mRNA, 調(diào)控其穩(wěn)定性及翻譯活性[7]。本文主要綜述了miRNA及RBPs介導(dǎo)的轉(zhuǎn)錄后修飾對(duì)DDR相關(guān)因子的調(diào)控。

1 miRNA介導(dǎo)的DDR調(diào)控

miRNA是一類內(nèi)源的非編碼小RNA(18～25 bp),通過與靶mRNA的3′端非翻譯區(qū)(3′UTR)結(jié)合來調(diào)節(jié)其穩(wěn)定性或翻譯活性, 進(jìn)而調(diào)控靶基因表達(dá)。細(xì)胞分化、細(xì)胞周期、凋亡等幾乎所有生物學(xué)過程中都存在miRNA的調(diào)控作用[8]。

研究證明, miRNA在DDR的起始和維持中都發(fā)揮著關(guān)鍵調(diào)控作用[9]。DDR起始于損傷識(shí)別分子對(duì)損傷DNA的識(shí)別。當(dāng)確認(rèn)DNA損傷后, 信號(hào)傳導(dǎo)器如ATM、ATR、DNA-PKcs等傳遞并放大原始損傷信號(hào)到下游的效應(yīng)器分子, 激活細(xì)胞周期檢查,進(jìn)而促進(jìn) DNA修復(fù)及凋亡途徑[10]。高通量生物信息學(xué)預(yù)測(cè)和實(shí)驗(yàn)已證實(shí)miRNA能調(diào)控DDR信號(hào)網(wǎng)絡(luò)中的多種關(guān)鍵因子。通過 MiRanda和 Targetscan這兩個(gè)獨(dú)立的目標(biāo)預(yù)測(cè)算法對(duì)142個(gè)DDR相關(guān)基因的3′UTR進(jìn)行檢測(cè), 發(fā)現(xiàn)超過一半的DNA損傷監(jiān)測(cè)和修復(fù)基因含有miRNA結(jié)合位點(diǎn)保守序列[9], 部分預(yù)測(cè)結(jié)果已被生物學(xué)實(shí)驗(yàn)驗(yàn)證。

1.1 miRNA對(duì)DDR識(shí)別分子的調(diào)控

DDR起始于 DNA損傷識(shí)別分子(如 H2AX、MDC1、53BP1等)對(duì)DNA損傷的識(shí)別。DNA損傷發(fā)生后, 損傷識(shí)別分子募集到損傷位點(diǎn), 而后將損傷信號(hào)放大, 傳遞到下游分子。組蛋白 H2AX的磷酸化即γH2AX的產(chǎn)生是DNA損傷信號(hào)級(jí)聯(lián)的早期事件, 能啟動(dòng)并維持DNA損傷位點(diǎn)對(duì)多種檢查點(diǎn)蛋白和修復(fù)蛋白的募集和激活[4]。Lal等[11]在分化晚期的血液細(xì)胞里發(fā)現(xiàn) miR-24能抑制 H2AX表達(dá)和DNA損傷修復(fù), 使細(xì)胞對(duì)γ射線及基因毒性藥物產(chǎn)生超敏反應(yīng)。此后又有研究發(fā)現(xiàn)在 U2OS細(xì)胞中, miR-138和 miR542-3p能抑制γH2AX聚集點(diǎn)(Foci)的形成[12], 其中miR-138的作用更為明顯。miR-138過表達(dá)抑制同源重組并且增加細(xì)胞對(duì)多種 DNA損傷劑的敏感性。值得注意的是, 在 U2OS細(xì)胞中沒有檢測(cè)到 miR-24, 提示 miRNA的功能和靶基因具有組織細(xì)胞特異性。

1.2 miRNA對(duì)DDR傳導(dǎo)器分子的調(diào)控

ATM(Ataxia-telangiectasia mutated)是磷脂酰肌醇-3-激酶相關(guān)激酶家族(PI3KK)的重要成員, 在DDR中催化多種底物分子的磷酸化來激活細(xì)胞周期檢查、損傷修復(fù)、凋亡等一系列信號(hào)途徑, 被譽(yù)為DNA雙鏈斷裂損傷反應(yīng)信號(hào)傳遞的“主導(dǎo)激酶”[13]。DNA損傷后, ATM 通過自身絲氨酸磷酸化而激活,其激酶活性主要被Wip1磷酸酶負(fù)調(diào)控[10]。最近, 有研究發(fā)現(xiàn)了新的調(diào)控機(jī)制, 即 miRNA通過靶向ATM的3′UTR負(fù)調(diào)控ATM, 越來越多的miRNA被發(fā)現(xiàn)通過靶向 ATM在 DDR中發(fā)揮作用, 如 miR-100、miR-101、miR-421、miR-18a、miR-181a/b等。上調(diào)miR-101、miR-100可增加多種腫瘤細(xì)胞的輻射敏感性; 過表達(dá)miR-421能抑制HeLa細(xì)胞的S期檢查點(diǎn)并增加細(xì)胞電離輻射敏感性; 乳腺癌細(xì)胞中過表達(dá)miR-18a、miR-181a/b導(dǎo)致DNA損傷修復(fù)能力下降[14～18]。

ATR(ATM-rad3-related)也屬于PI3KK激酶家族,在維持染色體完整和基因組穩(wěn)定性中發(fā)揮重要作用,它與 ATM的區(qū)別在于激活它們的基因損傷類型不同。ATM主要被DNA雙鏈斷裂激活, ATR主要由紫外線和 S期復(fù)制阻滯激活, 某些情況下這兩條信號(hào)途徑間存在交互調(diào)控和功能互補(bǔ)。研究發(fā)現(xiàn), 人腎癌細(xì)胞 IR處理后 miR-185表達(dá)降低, 而過表達(dá)miR-185能增加細(xì)胞 X射線敏感性, 進(jìn)一步研究發(fā)現(xiàn)此過程通過miR-185與ATR的3′UTR相互作用抑制 ATR 的表達(dá)來實(shí)現(xiàn)[19]。此外, 研究還發(fā)現(xiàn)miR-185、miR-300、miR-663高表達(dá)異常會(huì)導(dǎo)致DNA損傷并激活A(yù)TR/chk1 DDR通路[20]。

1.3 miRNA對(duì)DDR效應(yīng)器分子的調(diào)控

DDR中腫瘤抑制基因 BRCA1同其他腫瘤抑制因子、DNA損傷識(shí)別分子、信號(hào)傳導(dǎo)分子共同組成BRCA1相關(guān)基因組保護(hù)復(fù)合體, 調(diào)控細(xì)胞周期和損傷修復(fù)[21]。最近研究發(fā)現(xiàn), 卵巢癌中 miR-9可下調(diào)BRCA1表達(dá)并阻止DNA損傷修復(fù)過程[22]。另外在乳腺癌細(xì)胞中發(fā)現(xiàn)miR-182靶向BRCA1并抑制其表達(dá), 過表達(dá) miR-182影響同源重組修復(fù)并使細(xì)胞對(duì)IR敏感; 抑制miR-182能上調(diào)BRCA1表達(dá)并增加細(xì)胞對(duì)PARP1(Poly [ADP-ribose] polymerase 1)抑制劑的耐受性[23]。

腫瘤抑制因子p53是細(xì)胞DDR的核心調(diào)控分子之一。以往研究多集中于p53蛋白穩(wěn)定和活性調(diào)控;目前miRNA與 p53之間的關(guān)系開始被關(guān)注[24]。多種miRNA在DDR中作用于p53的3′UTR, 下調(diào)p53的表達(dá), 如miR-155、miR-125b、miR-504、miR-375等[25～28]。生物信息學(xué)分析結(jié)合實(shí)驗(yàn)研究發(fā)現(xiàn)：神經(jīng)細(xì)胞瘤和肺癌細(xì)胞中過表達(dá)的miR-125a抑制p53表達(dá)和細(xì)胞凋亡[29]。斑馬魚(Danio rerio)中缺失miR-125b會(huì)增加 p53依賴的凋亡。Hu等[28]發(fā)現(xiàn)miR-504在人腸癌細(xì)胞HCT116、肝癌細(xì)胞H460、乳腺癌細(xì)胞MCF-7中也都具有負(fù)調(diào)控p53的活性[28],并能促進(jìn)人大腸癌腫瘤在裸鼠體內(nèi)的增長(zhǎng)。另外,一些miRNA的表達(dá)受p53調(diào)控, 而這些miRNA所調(diào)控的基因又可與 p53發(fā)生相互作用形成反饋回環(huán)來調(diào)控DDR。Xiao等[30]發(fā)現(xiàn)DNA損傷激活的p53能夠轉(zhuǎn)錄激活miR-605, miR-605又靶向抑制mdm2表達(dá), mdm2又是p53的E3泛素連接酶, 這就間接上調(diào)p53水平, 保證了DNA損傷時(shí)p53的快速上調(diào)。

1.4 miRNA在DDR總體調(diào)控中的作用

DNA損傷發(fā)生后, 細(xì)胞啟動(dòng)一系列信號(hào)轉(zhuǎn)導(dǎo)系統(tǒng)來完成損傷修復(fù), 蛋白磷酸化是其中的重要事件。一旦DNA修復(fù)完成, 細(xì)胞需要關(guān)閉DNA損傷反應(yīng)途徑并恢復(fù)正常狀態(tài)[31]。Wip1是 ATM-p53 DNA損傷信號(hào)途徑的一個(gè)主要抑制因子, 它確保DDR的及時(shí)終止。DDR中很多關(guān)鍵蛋白都是Wip1的底物, 如 p53、H2AX、ATM、Chk2、p38MAPK等。研究發(fā)現(xiàn) miR-16和 miR-29等能靶向負(fù)調(diào)控Wip1, 在DNA損傷后這兩種miRNA被激活進(jìn)而通過與Wip1的mRNA相互作用抑制Wip1蛋白表達(dá)從而促進(jìn)DNA損傷信號(hào)的傳遞[32,33]。

除上述分子外, DDR途徑中的多種分子都受到miRNA的調(diào)節(jié), 如 PTEN、P21、RAD52等[34～36]。DDR調(diào)控的miRNA及作用于DDR的miRNA調(diào)控細(xì)胞對(duì)DNA損傷的敏感性, 并與腫瘤的產(chǎn)生及發(fā)展相關(guān)。眾所周知, DDR的缺陷及miRNA的廣泛抑制是人類多種腫瘤的標(biāo)志。因此, 進(jìn)一步發(fā)現(xiàn)并研究參與DDR信號(hào)途徑調(diào)控的miRNA將為人們提供更多信息, 如腫瘤細(xì)胞對(duì)基因毒性藥物的敏感性和耐受性等, 從而給腫瘤等DNA損傷相關(guān)疾病的治療提供新的策略。

2 RNA結(jié)合蛋白介導(dǎo)的DDR調(diào)控

轉(zhuǎn)錄生成的mRNA除了能被miRNA結(jié)合并調(diào)控外, 還能被多種蛋白結(jié)合調(diào)控。研究發(fā)現(xiàn)細(xì)胞內(nèi)的一些蛋白與 mRNA具有高度親和性, 通過與mRNA的結(jié)合而調(diào)控mRNA的穩(wěn)定性、翻譯活性等,這些蛋白被統(tǒng)稱為 RNA結(jié)合蛋白(RNA-binding proteins, RBPs)[37]。RBPs通過識(shí)別并結(jié)合mRNA上的順式調(diào)控元件能夠同時(shí)對(duì)一組mRNA進(jìn)行轉(zhuǎn)錄后水平的調(diào)控, 這類分布于mRNA上的順式元件被稱為“轉(zhuǎn)錄后調(diào)控密碼”。在DDR導(dǎo)致的細(xì)胞內(nèi)mRNA水平變化中, 有50%是mRNA自身的降解或穩(wěn)定性改變引起的, 還有50%是轉(zhuǎn)錄水平的改變。如Yaffe等[38]研究發(fā)現(xiàn), DDR中的重要功能蛋白14-3-3就是重要的轉(zhuǎn)錄后調(diào)控分子之一, 能結(jié)合并調(diào)節(jié)多種DDR效應(yīng)分子的mRNA的剪接和翻譯?，F(xiàn)已被鑒定的參與DDR反應(yīng)的RBPs分子包括HuR、TTP、TIAR等。

2.1 DDR中RBPs對(duì)靶mRNA穩(wěn)定性的調(diào)控

當(dāng)前, 在DDR中研究最多的“轉(zhuǎn)錄后調(diào)控密碼”是ARE基序(AU-richel e-ments, AREs)。AREs位于mRNA 3′UTR, AUUUA和UUAUUUAUU兩種形式的重復(fù)序列最為常見。人類抗原R(Human antigen R, HuR)、鋅指蛋白(Zinc finger protein Tristetraprolin, TTP)以及hnRNP A0 (Heterogeneous nuclear RNP A0)等RBPs分子都能夠識(shí)別并調(diào)控含AREs的mRNA分子的穩(wěn)定性。

細(xì)胞周期檢查激活分子p21作為p53的下游基因在DDR中激活, 多種RBPs可作用于p21 mRNA,調(diào)節(jié)其穩(wěn)定性。RBPs與 p21甚至各種RBPs之間形成復(fù)雜的正、負(fù)反饋調(diào)節(jié)環(huán)。一些RBPs可阻礙P53依賴的 p21表達(dá)上調(diào), 如 PCBP 家族、PCBP1、PCBP2、PCBP4及hnRNP K, 通過結(jié)合p21的 3′UTR富含CU區(qū)域下調(diào)其表達(dá)[39]。RBPs也可正調(diào)節(jié)p21 mRNA的穩(wěn)定性, 如HuR能通過自身RNA識(shí)別基序(RRM)與靶 mRNA 的 AREs結(jié)合, 促進(jìn) p21 mRNA穩(wěn)定性, 上調(diào)其表達(dá)[40]。然而HuR對(duì)p21的上調(diào)調(diào)控又可被另一種RBPs AUF1 (Heterogeneous nuclear ribonucleoprotein D)對(duì)p21的抑制所中和[40]。另一種周期調(diào)控分子cyclin D1也被HuR與AUF1這一對(duì)RBPs所調(diào)控。細(xì)胞發(fā)生UVC后, HuR-p21 mRNA 之間結(jié)合上升, AUF1-p21 mRNA作用減弱;相反, HuR-cyclin D1 mRNA 之間結(jié)合減少, AUF1-cyclin D1 mRNA復(fù)合物增多, 進(jìn)而p21表達(dá)上調(diào), cyclin D1被抑制, 出現(xiàn)G1/S阻滯[41]。HuR 和AUF1也可結(jié)合另一種p53靶基因14-3-3, 誘導(dǎo)細(xì)胞G2/M阻滯[41]。

在DDR反應(yīng)中, p38MAPK/MK2復(fù)合體與基因表達(dá)轉(zhuǎn)錄后調(diào)控過程高度相關(guān)。除了前面述及的HuR, MK2還能調(diào)節(jié)其他多種RBPs分子。Anderson等[42]發(fā)現(xiàn)在非應(yīng)激狀態(tài)時(shí), TTP通過與TNF mRNA的AREs結(jié)合負(fù)調(diào)控mRNA穩(wěn)定性, 因此包含AREs的mRNA在通常情況下不穩(wěn)定。當(dāng)受到DNA損傷如亞硝酸鹽處理后, 激活的MK2蛋白激酶催化TTP Ser-52和 Ser-178位磷酸化, 進(jìn)而被 14-3-3募集。TTP：14-3-3復(fù)合體的形成抑制TTP介導(dǎo)的mRNA降解[43]。除 TTP外, MK2還能直接磷酸化 hnRNP A0。Rousseau等[44]證明LPS處理激活的MK2能介導(dǎo) hnRNP A0 Ser-84位的磷酸化進(jìn)而穩(wěn)定其蛋白, hnRNP A0與TNFα mRNA的AREs結(jié)合進(jìn)而增加mRNA穩(wěn)定性。

2.2 DDR中RBPs對(duì)靶mRNA翻譯活性的調(diào)控

DDR是一個(gè)復(fù)雜的調(diào)控網(wǎng)絡(luò), 多種機(jī)制可以調(diào)控同一種分子, 一種分子又可參與多條途徑的修復(fù),多種復(fù)雜機(jī)制錯(cuò)綜復(fù)雜共同發(fā)揮作用。p53受激酶的調(diào)控, 又是miRNA的靶基因, 其mRNA的翻譯活性又受 RBPs Hzf與HuR的影響[45]。Gadd45可被IR、UV、氧化劑等誘導(dǎo), 在細(xì)胞周期阻滯、凋亡, p38MAPK與 JNK在激酶途徑中發(fā)揮重要作用。RBPs TIAR與上文提到的AUF1共同作用于Gadd45 mRNA 3′UTR, 不同的是TIAR作用于Gadd45的翻譯活性抑制[46]。此外, TIAR還能有效結(jié)合并抑制多個(gè)靶mRNA, 如IL8、TNF等的翻譯。通過芯片檢測(cè)從直腸癌細(xì)胞中IP獲得的TIAR-RNA復(fù)合物, 發(fā)現(xiàn)TIAR還存在28nt和32nt的兩個(gè)低親和性結(jié)合序列,該序列是一個(gè)位于mRNA 3′端的富含C的基序[47]。當(dāng)用UV處理細(xì)胞造成DNA損傷后, TIAR與這類3′富含C的mRNA之間的結(jié)合解離, 進(jìn)而導(dǎo)致這些基因表達(dá)上調(diào), 這些基因在DDR中的作用還需進(jìn)一步研究。

2.3 RBPs對(duì)DDR中miRNA的調(diào)控

miRNA和RBPs在DDR中發(fā)揮著關(guān)鍵的調(diào)控作用, 隨著miRNA研究的迅猛發(fā)展, RBPs在miRNA功能調(diào)控中的作用研究也逐漸引起關(guān)注[48,49]。RBPs結(jié)合蛋白常能夠通過破壞miRNA的生物合成、改變miRNA靶位點(diǎn)的二級(jí)結(jié)構(gòu)、影響miRNA加工的整體效率、選擇性的調(diào)控3′UTR等而改變miRNA的加工和活性。外部或內(nèi)部因素刺激引起的細(xì)胞反應(yīng)中, RBPs能夠動(dòng)態(tài)地控制miRNA介導(dǎo)的抑制作用的程度, 以維持正常的基因表達(dá)[48]。如為應(yīng)對(duì)細(xì)胞壓力, HuR可通過解除 miR-122對(duì) CAT1(Cationic amino acid transporter 1)mRNA 的抑制[50]。

因此, RBPs通過參與多種DDR因子的轉(zhuǎn)錄后修飾調(diào)控而在DDR中發(fā)揮重要功能, 并與腫瘤等DNA損傷相關(guān)疾病的發(fā)生有關(guān)(如HuR和TTP能與連接蛋白 cclaudin-1的 mRNA 3′UTR相互作用進(jìn)而穩(wěn)定其mRNA, 導(dǎo)致直腸癌細(xì)胞中l(wèi)audin-1異常高表達(dá))[51]。

3 結(jié)語與展望

近年來, DDR中轉(zhuǎn)錄后調(diào)控對(duì)基因表達(dá)的重要作用逐步引起研究者的重視, 這得益于 miRNA及RBPs參與的mRNA穩(wěn)定性及翻譯調(diào)控機(jī)制的發(fā)現(xiàn)。在IR引發(fā)的DNA損傷反應(yīng)中, 為防止DNA損傷錯(cuò)誤轉(zhuǎn)錄本的生成, 反應(yīng)早期會(huì)有RNA聚合酶II活性抑制和基因轉(zhuǎn)錄抑制[52]; 然而細(xì)胞又亟需多種DNA損傷反應(yīng)蛋白和修復(fù)蛋白以調(diào)控周期、修復(fù)損傷或誘導(dǎo)凋亡。因此, 轉(zhuǎn)錄后調(diào)控網(wǎng)絡(luò)(Post-transcription regulatory network)對(duì)mRNA的定位、穩(wěn)定性和翻譯活性進(jìn)行的調(diào)控在 IR反應(yīng)重要功能蛋白表達(dá)調(diào)控中的功能機(jī)制研究具有重要意義。除了 miRNA外,其他非編碼小RNA也通過多種方式參與DDR反應(yīng)。在對(duì)擬南芥 DNA雙鏈斷裂修復(fù)報(bào)告系統(tǒng)的研究中, Wei等[53]發(fā)現(xiàn)一種小分子RNA能特異性的從DNA雙鏈斷裂位點(diǎn)的鄰近序列產(chǎn)生并將其命名為diRNA(DSB-induced small RNA)。進(jìn)一步的研究證明, diRNA由DNA雙鏈斷裂位點(diǎn)處的RNA聚合酶NRPD1、NRPD2轉(zhuǎn)錄生成單鏈小 RNA, 隨后被RDPs加工成雙鏈并通過DCL2/3/4加工最后形成大小為 21個(gè)核苷酸的小 RNA, 并且在人類細(xì)胞中也廣泛存在。目前已證明其可與Argonatute2蛋白結(jié)合進(jìn)而募集多種組蛋白修飾酶類及 DNA修復(fù)復(fù)合體等到DNA雙鏈斷裂位點(diǎn)調(diào)控DSB修復(fù)。這一發(fā)現(xiàn)證明了 DDR反應(yīng)在轉(zhuǎn)錄調(diào)控機(jī)制之外還存在多種調(diào)控機(jī)制, 進(jìn)一步豐富和加深了人們對(duì)DDR的理解并為以后的藥物開發(fā)提供了更多的新靶點(diǎn)。

這一領(lǐng)域的挑戰(zhàn)在于對(duì) DDR中哪些基因受到轉(zhuǎn)錄后調(diào)控進(jìn)行確認(rèn), 并找到參與其調(diào)控的miRNA和RBPs。新的技術(shù)如全基因組范圍內(nèi)的RNAi掃描、下一代基因測(cè)序技術(shù)的運(yùn)用將促進(jìn)人們發(fā)現(xiàn)參與DDR的miRNA及RBPs, 并進(jìn)一步確認(rèn)和了解其功能機(jī)制[54]。綜上所述, 盡管許多疑問還未得到解答,但值得相信的是, 堅(jiān)持不懈的努力必將解決懸而未決的問題, 并產(chǎn)生新的診斷和治療策略來應(yīng)對(duì)人類DNA損傷缺陷引起的諸多疾病包括癌癥。

[1] Jackson SP, Bartek J. The DNA-damage response in human biology and disease. Nature, 2009, 461(7267): 1071-1078.

[2] Ciccia A, Elledge SJ. The DNA damage response: making it safe to play with knives. Mol Cell, 2010, 40(2): 179-204.

[3] Jackson SP. The DNA-damage response: new molecular insights and new approaches to cancer therapy. Biochem Soc Trans, 2009, 37(Pt 3): 483-494.

[4] Huen MS, Chen J. Assembly of checkpoint and repair machineries at DNA damage sites. Trends Biochem Sci, 2010, 35(2): 101-108.

[5] Polo SE, Jackson SP. Dynamics of DNA damage response proteins at DNA breaks: a focus on protein modifications. Genes Dev, 2011, 25(5): 409-433.

[6] Cheadle C, Fan J, Cho-Chung YS, Werner T, Ray J, Do L, Gorospe M, Becker KG. Stability regulation of mRNA and the control of gene expression. Ann N Y Acad Sci, 2005, 1058: 196-204.

[7] Boucas J, Riabinska A, Jokic M, Herter-Sprie GS, Chen S, H?pker K, Reinhardt HC. Posttranscriptional regulation of gene expression-adding another layer of complexity to the DNA damage response. Front Genet, 2012, 3: 159.

[8] Bhaskaran M, Mohan M. MicroRNAs: history, biogenesis, and their evolving role in animal development and disease. Vet Pathol, 2013, [Epub ahead of print].

[9] Wouters MD, van Gent DC, Hoeijmakers JH, Pothof J. MicroRNAs, the DNA damage response and cancer. Mutat Res, 2011, 717(1-2): 54-66.

[10] Stracker TH, Roig I, Knobel PA, Marjanovi? M. The ATM signaling network in development and disease. Front Genet, 2013, 4: 37.

[11] Lal A, Pan YF, Navarro F, Dykxhoorn DM, Moreau L, Meire E, Bentwich Z, Lieberman J, Chowdhury D. miR-24-mediated downregulation of H2AX suppresses DNA repair in terminally differentiated blood cells. Nat Struct Mol Biol, 2009, 16(5): 492-498.

[12] Wang YM, Huang JW, Li M, Cavenee WK, Mitchell PS, Zhou XF, Tewari M, Furnari FB, Taniguchi T. MicroRNA-138 modulates DNA damage response by repressing histone H2AX expression. Mol Cancer Res, 2011, 9(8): 1100-1111.

[13] Roos WP, Kaina B. DNA damage-induced cell death: from specific DNA lesions to the DNA damage response and apoptosis. Cancer Lett, 2013, 332(2): 237-248.

[14] Hu HL, Du LT, Nagabayashi G, Seeger RC, Gatti RA. ATM is down-regulated by N-Myc-regulated microRNA-421. Proc Natl Acad Sci USA, 2010, 107(4): 1506-1511.

[15] Ng WL, Yan D, Zhang XM, Mo YY, Wang Y. Overexpression of miR-100 is responsible for the low-expression of ATM in the human glioma cell line: M059J. DNARepair, 2010, 9(11): 1170-1175.

[16] Yan D, Ng WL, Zhang XM, Wang P, Zhang ZB, Mo YY, Mao H, Hao CH, Olson JJ, Curran WJ, Wang Y. Targeting DNA-PKcs and ATM with miR-101 sensitizes tumors to radiation. PloS ONE, 2010, 5(7): e11397.

[17] Song LB, Lin CY, Wu ZQ, Gong H, Zeng Y, Wu JH, Li MF, Li J. miR-18a impairs DNA damage response through downregulation of ataxia telangiectasia mutated (ATM) kinase. PloS ONE, 2011, 6(9): e25454.

[18] Bisso A, Faleschini M, Zampa F, Capaci V, De Santa J, Santarpia L, Piazza S, Cappelletti V, Daidone M, Agami R, Del Sal G. Oncogenic miR-181a/b affect the DNA damage response in aggressive breast cancer. Cell Cycle, 2013, 12(11): 1679-1687.

[19] Wang J, He J, Su F, Ding N, Hu W, Yao B, Wang W, Zhou G. Repression of ATR pathway by miR-185 enhances radiation-induced apoptosis and proliferation inhibition. Cell Death Disease, 2013, 4(6): e699.

[20] Chang L, Hu WT, Ye CY, Yao B, Song L, Wu X, Ding N, Wang JF, Zhou GM. miR-3928 activates ATR pathway by targeting dicer. RNA Biol, 2012, 9(10): 1247-1254.

[21] Rosen EM. BRCA1 in the DNA damage response and at telomeres. Front Genet, 2013, 4: 85.

[22] Li MQ, Song WT, Tang ZH, Lv SX, Lin L, Sun H, Li QS, Yang Y, Hong H, Chen XS. Nanoscaled poly(L-glutamic acid)/doxorubicin-amphiphile complex as pH-responsive drug delivery system for effective treatment of nonsmall cell lung cancer. ACS Appl Mater Interfaces, 2013, 5(5): 1781-1792.

[23] Moskwa P, Buffa FM, Pan YF, Panchakshari R, Gottipati P, Muschel RJ, Beech J, Kulshrestha R, Abdelmohsen K, Weinstock DM, Gorospe M, Harris AL, Helleday T, Chowdhury D. miR-182-mediated downregulation of BRCA1 impacts DNA repair and sensitivity to PARP inhibitors. Mol Cell, 2011, 41(2): 210-220.

[24] Krell J, Frampton AE, Colombo T, Gall TMH, De Giorgio A, Harding V, Stebbing J, Castellano L. The p53 miRNA interactome and its potential role in the cancer clinic. Epigenomics, 2013, 5(4): 417-428.

[25] Hunten S, Siemens H, Kaller M, Hermeking H. The p53/microRNA network in cancer: experimental and bioinformatics approaches. Adv Exp Med Biol, 2013, 774: 77-101.

[26] Wang J, Zong JY, Zhao D, Zhuo RX, Cheng SX. In situ formation of chitosan-cyclodextrin nanospheres for drug delivery. Colloids Surf B Biointerfaces, 2011, 87(1): 198-202.

[27] Wu N, Lin X, Zhao X, Zheng L, Xiao L, Liu J, Ge L, Cao S. MiR-125b acts as an oncogene in glioblastoma cells and inhibits cell apoptosis through p53 and p38MAPK-independent pathways. British J Cancer, 2013, 109(11): 2853-2863.

[28] Hu WW, Chan CS, Wu R, Zhang C, Sun Y, Song JS, Tang LH, Levine AJ, Feng ZH. Negative regulation of tumor suppressor p53 by microRNA miR-504. Mol Cell, 2010, 38(5): 689-699.

[29] Le MTN, Teh C, Shyh-Chang N, Xie HM, Zhou BY, Korzh V, Lodish HF, Lim B. MicroRNA-125b is a novel negative regulator of p53. Genes Dev, 2009, 23(7): 862-876.

[30] Xiao JN, Lin HX, Luo XB, Luo XY, Wang ZG. miR-605 joins p53 network to form a p53: miR-605: Mdm2 positive feedback loop in response to stress. EMBO J, 2011, 30(24): 5021.

[31] Macurek L, Benada J, Müllers E, Halim VA, Krej?iková K, Burdová K, Pechá?ková S, Hodny Z, Lindqvist A, Medema RH, Bartek J. Downregulation of Wip1 phosphatase modulates the cellular threshold of DNA damage signaling in mitosis. Cell Cycle, 2013, 12(2): 251-262.

[32] Zhang XN, Wan GH, Mlotshwa S, Vance V, Berger FG, Chen HX, Lu XB. Oncogenic Wip1 phosphatase is inhibited by miR-16 in the DNA damage signaling pathway. Cancer Res, 2010, 70(18): 7176-7186.

[33] Crescenzi E, Raia Z, Pacifico F, Mellone S, Moscato F, Palumbo G, Leonardi A. Down-regulation of wild-type p53-induced phosphatase 1 (Wip1) plays a critical role in regulating several p53-dependent functions in premature senescent tumor cells. J Biol Chem, 2013, 288(23): 16212-16224.

[34] Tan GY, Shi YL, Wu ZH. MicroRNA-22 promotes cell survival upon UV radiation by repressing PTEN. Biochem Biophys Res Commun, 2012, 417(1): 546-551.

[35] Yao YL, Wu XY, Wu JH, Gu T, Chen L, Gu JH, Liu Y, Zhang QH. Effects of MicroRNA-106 on Proliferation of Gastric Cancer Cell through Regulating p21 and E2F5. Asian Pac J Cancer Prev, 2013, 14(5): 2839-2843.

[36] Crosby ME, Kulshreshtha R, Ivan M, Glazer PM. MicroRNA regulation of DNA repair gene expression in hypoxic stress. Cancer Res, 2009, 69(3): 1221-1229.

[37] Khabar KS. Post-transcriptional control during chronic inflammation and cancer: a focus on AU-rich elements. Cell Mol Life Sci, 2010, 67(17): 2937-2955.

[38] Wilker EW, van Vugt MATM, Artim SA, Huang PH, Petersen CP, Reinhardt HC, Feng Y, Sharp PA, Sonenberg N, White FM, Yaffe MB. 14-3-3sigma controls mitotic translation to facilitate cytokinesis. Nature, 2007,446(7133): 329-332.

[39] Scoumanne A, Cho SJ, Zhang J, Chen XB. The cyclindependent kinase inhibitor p21 is regulated by RNA-binding protein PCBP4 via mRNA stability. Nucleic Acids Res, 2011, 39(1): 213-224.

[40] He Y, Zhang X, Zeng X, Huang Y, Wei JA, Han L, Li CX, Zhang GW. HuR-mediated posttranscriptional regulation of p21 is involved in the effect of Glycyrrhiza uralensis licorice aqueous extract on polyamine-depleted intestinal crypt cells proliferation. J Nutr Biochem, 2012, 23(10): 1285-1293.

[41] Barker A, Epis MR, Porter CJ, Hopkins BR, Wilce MC, Wilce JA, Giles KM, Leedman PJ. Sequence requirements for RNA binding by HuR and AUF1. J Biochem, 2012, 151(4): 423-437.

[42] Stoecklin G, Stubbs T, Kedersha N, Wax S, Rigby WFC, Blackwell TK, Anderson P. MK2-induced tristetraprolin: 14-3-3 complexes prevent stress granule association and ARE-mRNA decay. EMBO J, 2004, 23(6): 1313-1324.

[43] Kedersha N, Anderson P. Stress granules: sites of mRNA triage that regulate mRNA stability and translatability. Biochem Soc Trans, 2002, 30(Pt 6): 963-969.

[44] Rousseau S, Morrice N, Peggie M, Campbell DG, Gaestel M, Cohen P. Inhibition of SAPK2a/p38 prevents hnRNP A0 phosphorylation by MAPKAP-K2 and its interaction with cytokine mRNAs. EMBO J, 2002, 21(23): 6505-6514.

[45] Nakamura H, Kawagishi H, Watanabe A, Sugimoto K, Maruyama M, Sugimoto M. Cooperative role of the RNA-binding proteins Hzf and HuR in p53 activation. Mol Cell Biol, 2011, 31(10): 1997-2009.

[46] Lal A, Abdelmohsen K, Pullmann R, Kawai T, Galban S, Yang XL, Brewer G, Gorospe M. Posttranscriptional derepression of GADD45alpha by genotoxic stress. Mol Cell, 2006, 22(1): 117-128.

[47] Kim HS, Kuwano Y, Zhan M, Pullmann R Jr, Mazan-Mamczarz K, Li H, Kedersha N, Anderson P, Wilce MCJ, Gorospe M, Wilce JA. Elucidation of a C-rich signature motif in target mRNAs of RNA-binding protein TIAR. Mol Cell Biol, 2007, 27(19): 6806-6817.

[48] van Kouwenhove M, Kedde M, Agami R. MicroRNA regulation by RNA-binding proteins and its implications for cancer. Nat Rev Cancer, 2011, 11(9): 644-656.

[49] Ciafrè SA, Galardi S. MicroRNAs and RNA-binding proteins: a complex network of interactions and reciprocal regulations in cancer. RNA Biol, 2013, 10(6): 935-942.

[50] Bhattacharyya SN, Habermacher R, Martine U, Closs EI, Filipowicz W. Relief of microRNA-mediated translational repression in human cells subjected to stress. Cell, 2006, 125(6): 1111-1124.

[51] Sharma A, Bhat AA, Krishnan M, Singh AB, Dhawan P. Trichostatin-A modulates Claudin-1 mRNA stability through the modulation of Hu antigen R and tristetraprolin in colon cancer cells. Carcinogenesis, 2013, 34 (11): 2610-2621.

[52] Rybak A, Fuchs H, Smirnova L, Brandt C, Pohl EE, Nitsch R, Wulczyn FG. A feedback loop comprising lin-28 and let-7 controls pre-let-7 maturation during neural stemcell commitment. Nat Cell Biol, 2008, 10(8): 987-993.

[53] Wei W, Ba ZQ, Gao M, Wu Y, Ma YT, Amiard S, White CI, Rendtlew Danielsen JM, Yang YG, Qi YJ. A role for small RNAs in DNA double-strand break repair. Cell, 2012, 149(1): 101-112.

[54] Nie DM, Ouyang YD, Wang X, Zhou W, Hu CG, Yao JL. Genome-wide analysis of endosperm-specific genes in rice. Gene, 2013, 530(2): 236-247.

(責(zé)任編委: 朱衛(wèi)國(guó))

The post-transcriptional regulation of the DNA damage response

Deling Lin1,2, Ying Luo1, Yi Song2

1. Medical Faculty of Kunming University of Science and Technology, Kunming 650500, China;
2. Institute of Radiation Medicine, Academy of Military Medical Sciences, Beijing 100850, China

In response to DNA damage, a complex signaling network called DNA damage response (DDR) would be activated in cells, to arrest the cell cycle and initiate DNA repair. Previous studies were mainly focused on the transcriptional regulation of gene and covalent modification of protein . In recent years, mRNA stability and translation in DDR attracted more and more attention. Emerging evidence suggests that microRNAs and RNA-binding proteins (RBPs) play critical roles in protecting the heritable genome through participating in the regulation of the DNA damage response. In this review, we discuss the most recent findings regarding the post-transcriptional regulation of the DDR by microRNAs and RBPs.

DNA damage response; microRNAs; RNA-binding protein; post-transcriptional regulation

2013-10-10;

2014-01-08

國(guó)家自然科學(xué)基金項(xiàng)目(編號(hào)：31201014, 81372252)資助

林德玲, 碩士, 專業(yè)方向：遺傳學(xué)。E-mail: lindeling615@163.com

羅瑛, 博士, 教授, 研究方向：分子遺傳學(xué)。E-mail: luoyingabc@yahoo.com

宋宜, 博士, 副研究員, 研究方向：DNA損傷及修復(fù)。E-mail: songyibj@sina.com

10.3724/SP.J.1005.2014.0309

時(shí)間: 2014-2-24 17:44:01

URL: http://www.cnki.net/kcms/detail/11.1913.R.20140224.1744.001.html