薛迪新 陳積賢

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PDCD4基因在腫瘤中表達的調(diào)控及其功能的研究進展

薛迪新 陳積賢

PDCD4基因是一種新的抑癌基因，也是一種與細胞凋亡相關(guān)的基因。近年來研究表明在許多實體瘤中PDCD4基因的表達下降，甚至出現(xiàn)缺失。低表達的PDCD4基因促使腫瘤細胞增殖、侵襲和轉(zhuǎn)移。一般情況下，低表達的PDCD4提示腫瘤患者的不良預后，故PDCD4基因可能成為生物治療新的靶點。從分子水平闡述PDCD4基因的表達調(diào)控機制和功能，對恢復PDCD4基因的表達來治療腫瘤的意義重大，同時也可以從PDCD4基因的角度去闡述腫瘤的發(fā)生、侵襲和轉(zhuǎn)移的機制。本文主要是將近些年來PDCD4基因的功能與調(diào)控作一總結(jié)，并闡述PDCD4表達與腫瘤患者預后的關(guān)系。

1 PDCD4基因的發(fā)現(xiàn)及其編碼的蛋白結(jié)構(gòu)

1995 年，為了闡明細胞凋亡的分子機制，Shibahara K等[1]采用差異顯示法在各種凋亡細胞系中分離出cDNA克隆，在這些cDNA克隆中，MA-3 mRNA的表達被誘導。之后陸續(xù)克隆出雞PDCD4基因[2]和鼠的DUG基因[3]等，統(tǒng)稱為PDCD4。人類PDCD4基因定位于染色體10q24[4]，其表達的蛋白經(jīng)序列分析表明由469個氨基酸組成，包括N末端結(jié)構(gòu)域、C末端結(jié)構(gòu)域和2個保守的α螺旋MA-3結(jié)構(gòu)域[5]。N末端和C末端結(jié)構(gòu)域可能是PDCD4蛋白核定位信號區(qū)，但是關(guān)于PDCD4蛋白亞細胞定位的報道存在爭議。有文獻報道PDCD4蛋白定位在正常細胞的胞核和腫瘤細胞的胞質(zhì)中[6-7]，但也有文獻報道存在相反的分布[8]，這可能是由于PDCD4在細胞核與質(zhì)之間的轉(zhuǎn)運引起的[9]。MA-3結(jié)構(gòu)域通過與翻譯起始因子相互作用影響PDCD4的翻譯，包括eIF4GI、eIF4GII和eIF4A[10-11]。

2 PDCD4的功能

2.1 調(diào)節(jié)基因轉(zhuǎn)錄的功能 PDCD4蛋白能調(diào)節(jié)基因的轉(zhuǎn)錄從而發(fā)揮特定的生物學功能。用siRNA-Pdcd4轉(zhuǎn)染GEO細胞株，導致細胞u-PAR mRNA和蛋白的表達水平升高，促進腫瘤細胞的侵襲。進一步用染色質(zhì)免疫共沉淀技術(shù)（chromatin immunoprecipitation，ChIP）分析表明轉(zhuǎn)錄因子SP1/SP3與u-PAR基因的啟動子元件-152/-135和-380/-354相互作用，調(diào)節(jié)u-PAR基因的轉(zhuǎn)錄[12]。而在乳腺癌細胞株中，PDCD4表達能增加TIMP2基因的轉(zhuǎn)錄，抑制乳腺癌細胞的轉(zhuǎn)移[13]。在GEO細胞株中，敲除PDCD4能上調(diào)Snail蛋白來抑制E-cadherin的表達，刺激β-catenin/Tcf轉(zhuǎn)錄復合物依賴的轉(zhuǎn)錄，從而促進u-PAR和c-myc基因的轉(zhuǎn)錄，進一步用ChIP分析表明β-catenin/Tcf與u-PAR和c-myc基因的啟動子結(jié)合來激活基因的轉(zhuǎn)錄，u-PAR和c-myc基因與腫瘤細胞轉(zhuǎn)移相關(guān)，當敲除u-PAR和c-myc時，能夠抑制GEO-shPdcd4細胞的轉(zhuǎn)移[14]。此外，PDCD4蛋白通過直接抑制c-Jun的磷酸化，抑制轉(zhuǎn)錄因子AP-1的表達，從而抑制了細胞的惡性轉(zhuǎn)化[15-16]。轉(zhuǎn)錄因子AP-1可以誘導血管生成素Ang-2的轉(zhuǎn)錄[17]，用siRNA-Pdcd4轉(zhuǎn)染細胞株Bon-1和HCT116，PDCD4表達下降，而Ang-2 mRNA及其蛋白質(zhì)表達升高，進一步用血管生成分析法表明高表達Ang-2能刺激血管的形成[18]。PDCD4還能與轉(zhuǎn)錄因子Twist1相互作用，導致Twist1的靶基因YB-1表達下降，從而抑制腫瘤細胞的增殖[19]。

2.2 調(diào)節(jié)基因翻譯的功能 PDCD4蛋白不僅可以抑制帽子依賴的翻譯，還可以抑制內(nèi)核糖體進入位點（internal ribosome entry site，IRES）依賴的翻譯。真核生物翻譯起始因子eIF4A是依賴于ATP的RNA解旋酶，能展開5′mRNA的二級結(jié)構(gòu)，酵母雙雜交技術(shù)分析表明PDCD4蛋白能與翻譯起始因子eIF4A結(jié)合，抑制帽子依賴的翻譯，進而抑制AP-1的反式激活，從而抑制細胞的惡性轉(zhuǎn)化[11]。PDCD4表達抑制JNK的活性，抑制c-jun磷酸化，進而抑制真核生物翻譯起始因子eIF4E的表達和磷酸化，從而抑制帽子依賴的翻譯，而用H2O2處理肝癌細胞株MHCC97L后，eIF4E的表達和磷酸化水平升高，促進金屬蛋白酶MMP-2和MMP-9表達水平升高，從而促進肝癌細胞的轉(zhuǎn)移[20]。PDCD4能對DNA的損傷產(chǎn)生應答反應。PDCD4蛋白與eIF4A結(jié)合后，抑制eIF4A與p53 mRNA 5′非編碼區(qū)結(jié)合，從而抑制p53 mRNA的翻譯，用DNA損傷劑處理細胞后，PDCD4的表達下降，促進p53 mRNA的翻譯[21]，有利于DNA損傷的修復。XIAP和Bcl-xL mRNA的5′非編碼區(qū)包含IRES元件，PDCD4蛋白直接與XIAP和Bcl-xL mRNA的IRES元件結(jié)合，抑制48S起始復合物的形成，從而抑制XIAP和Bcl-xL mRNA的翻譯[22]。除此之外，PDCD4蛋白還可以直接調(diào)節(jié)基因的翻譯。c-myb編碼區(qū)包含PDCD4的反應元件，此反應元件存在于c-myb編碼區(qū)的XmaI到SalI之間，PDCD4蛋白的N端結(jié)構(gòu)域與c-myb mRNA中的PDCD4的反應區(qū)域直接結(jié)合抑制c-myb mRNA的翻譯[23]。

此外，PDCD4的表達還可以使腫瘤細胞對某些化學物質(zhì)或射線的敏感性發(fā)生變化。在正常生長的條件下，功能缺陷的PDCD4基因的雞淋巴瘤細胞株DT40的增殖和細胞周期并沒有受影響，但是在敲除PDCD4基因后，細胞對一些DNA損傷劑的敏感性增強，包括紫外線，依托泊甙和甲磺酸乙酯[24]。在胃癌細胞中敲除PDCD4基因后，癌細胞對腫瘤壞死因子相關(guān)凋亡誘導配體（TRAIL）的敏感性減弱[25]。PDCD4基因的功能見圖1。

圖1 PDCD4基因的功能

3 PDCD4基因表達的調(diào)控

3.1 轉(zhuǎn)錄水平的調(diào)控 在轉(zhuǎn)錄水平調(diào)節(jié)PDCD4基因的表達，主要是通過一些轉(zhuǎn)錄因子與PDCD4基因的調(diào)控區(qū)相互作用，或甲基化5′CpG島來實現(xiàn)的。轉(zhuǎn)錄因子v-Myb可以誘導雞細胞中PDCD4基因的表達[26]，而敲除c-Myb基因，PDCD4基因的表達明顯降低[27]。此外還有轉(zhuǎn)錄因子ZBP-89單獨或與SP家族成員相互作用后通過與PDCD4基因的啟動子結(jié)合促進PDCD4基因的轉(zhuǎn)錄[28]。在神經(jīng)膠質(zhì)瘤細胞株和組織中，PDCD4基因的5′CpG島的甲基化，抑制PDCD4 mRNA的轉(zhuǎn)錄，同時用DNA甲基化轉(zhuǎn)移酶抑制劑封閉5′CpG島的甲基化后，細胞中PDCD4基因的表達可以恢復[29]。

3.2 轉(zhuǎn)錄后水平的調(diào)控 最近發(fā)現(xiàn)一種約由20個核苷酸組成的非編碼的microRNA，它與靶基因轉(zhuǎn)錄的mRNA的3′非編碼區(qū)結(jié)合，通過抑制mRNA翻譯或直接降解mRNA來負性調(diào)節(jié)靶基因的功能[30-32]。許多實驗均表明PDCD4是miR-21的靶基因，miR-21在轉(zhuǎn)錄后水平負性調(diào)節(jié)PDCD4的表達，包括大腸癌[33-35]，胃癌[36-37]，胰腺癌[38-39]，惡性膠質(zhì)瘤[40]，膀胱癌[41]，子宮頸癌[42]等。此外，其他miRNA也同樣調(diào)節(jié)PDCD4基因的表達。用miR-183轉(zhuǎn)染人肝癌細胞株Huh7后，PDCD4蛋白的表達降低，用qRT-PCR技術(shù)檢測25對臨床活體肝癌及其正常肝臟組織中PDCD4 mRNA和miR-183的表達情況，結(jié)果顯示miR-183的表達與PDCD4 mRNA的表達存在明顯的負相關(guān)，進一步用生物信息學和熒光素酶報告基因檢測證實miR-183是與PDCD4 mRNA 3′非編碼區(qū)結(jié)合來負性調(diào)節(jié)PDCD4靶基因的表達[43]，表明PDCD4也是miR-183的靶基因。在大腸癌中，用上述同樣的實驗方法檢測，結(jié)果顯示microRNA-499-5p也可在轉(zhuǎn)錄后水平負性調(diào)節(jié)靶基因PDCD4的表達[44]。以上研究表明PDCD4基因的表達在轉(zhuǎn)錄后水平受到多種miRNA的調(diào)控。

3.3 其他信號轉(zhuǎn)導通路對PDCD4基因表達的調(diào)控多種信號轉(zhuǎn)導通路能共同調(diào)節(jié)基因PDCD4的表達。TGF-β信號轉(zhuǎn)導通路可誘導肝癌Huh7細胞株P(guān)DCD4的表達，用信號通路抑制物Smad7轉(zhuǎn)染細胞株后，可抑制TGF-β通路誘導PDCD4基因的表達[45]，但是在血管平滑肌細胞中，TGF-β通過上調(diào)miR-21的表達，負性調(diào)節(jié)PDCD4基因的表達[46]，此外用PKCδ和PKCεsiRNA轉(zhuǎn)染肝癌細胞，可增強TGF-β誘導的PDCD4蛋白的表達[47]。當腫瘤細胞處在炎癥的微環(huán)境下，能激活PI3K-mTOR信號途徑，增強蛋白酶體降解PDCD4蛋白的作用，從而使腫瘤細胞中PDCD4蛋白的表達下降[48]。用PI3K的抑制劑處理胃癌和卵巢癌細胞株，抑制PI3KAkt信號轉(zhuǎn)導通路，可使PDCD4蛋白的表達增加[25,49]。此外，激活FGF-2-S6K2信號途徑，也可以使PDCD4蛋白磷酸化而被降解[22]。還有一些信號轉(zhuǎn)導途徑通過調(diào)節(jié)miR-21的表達，從而間接調(diào)節(jié)PDCD4基因的表達。在TLR4信號途徑中，配體LPS（細菌內(nèi)毒素）結(jié)合Toll樣受體4（Toll-like receptor 4，TLR4）可激活銜接蛋白MyD88和NF-kappaB誘導miR-21的表達，可使PDCD4的表達降低[50]。在乳腺癌細胞株MCF-7中，透明質(zhì)烷（HA）能與受體CD44的結(jié)合激活PKCε,后者磷酸化干細胞標志物Nanog，磷酸化的Nanog從細胞質(zhì)進入細胞核，與RNase III DROSHA和RNA解旋酶p68結(jié)合，誘導miR-21的表達，從而負性調(diào)節(jié)PDCD4蛋白的表達[51]。在人類膠質(zhì)母細胞瘤U251細胞株中，熊果酸可抑制TGF-β信號轉(zhuǎn)導通路，進而抑制miR-21的表達，間接地促進了PDCD4蛋白的表達[52]。

此外，有一種稱為抗亞砷酸鹽蛋白-2（Ars2），它能夠沉默miRNA表達[53]，在人類膽管癌細胞和組織中，Ars2的缺失可引起miR-21的表達增加，上調(diào)的miR-21可抑制PDCD4蛋白的表達[54]。PDCD4表達的調(diào)節(jié)機制見圖2。

圖2 PDCD4基因表達的調(diào)控機制

4 PDCD4基因在臨床活體組織中的表達及預后判斷

在許多實體腫瘤中，PDCD4的表達與腫瘤的臨床預后密切相關(guān)。從正常、交界性到惡性卵巢癌組織中，PDCD4蛋白的表達逐漸下降，在卵巢癌患者中，PDCD4蛋白表達越低，患者生存率就越低，用免疫組化的方法檢測發(fā)現(xiàn)正常卵巢細胞中的PDCD4蛋白主要定位在細胞核，而卵巢癌細胞的PDCD4主要定位在細胞質(zhì)[55]，這可能與pAkt在細胞核和細胞質(zhì)中的表達有關(guān)[56]。從正常大腸黏膜、腺瘤到大腸癌的演變過程中，PDCD4蛋白的表達逐漸下降，在此過程中，細胞PDCD4蛋白核與質(zhì)表達的比率逐漸降低，pAkt與PDCD4蛋白從細胞核到細胞質(zhì)的轉(zhuǎn)移（即核與質(zhì)的比率）之間存在負相關(guān)，表明PDCD4的轉(zhuǎn)移受pAkt的調(diào)控。用Kaplan-Meier分析表明PDCD4蛋白表達越低，大腸癌患者的總生存時間越短，疾病相關(guān)存活率越低。PDCD4蛋白的缺失，提示大腸癌患者的不良預后[56]。在腎臟細胞癌中，PDCD4蛋白表達降低，與腎臟細胞癌的分級、分期和轉(zhuǎn)移有關(guān)，即PDCD4蛋白表達越低，腎癌的分期越晚，越容易轉(zhuǎn)移，患者的平均總生存時間越短，PDCD4蛋白的低表達提示腎臟細胞癌的不良預后[57]。Motoyama等[37]分析PDCD4 mRNA在105例胃癌中的表達與臨床病理特征的關(guān)系，結(jié)果顯示低表達的PDCD4 mRNA與胃癌的大小、浸潤深度、淋巴結(jié)轉(zhuǎn)移、血管轉(zhuǎn)移和臨床分期有關(guān)，表明低表達的PDCD4 mRNA提示胃癌的不良預后。Horiuchi等[58]分析326例大腸癌患者，在大腸癌Dukes′B和C期患者中，低表達的PDCD4 mRNA患者的總生存時間短，并且無瘤生存率低，在Dukes′D期的大腸癌患者中，低表達的PDCD4 mRNA患者的總生存時間短，但在Dukes′A期的患者中，低表達和高表達的PDCD4 mRNA患者之間的總生存時間和無瘤生存率均無差別。表明在Dukes′B、C和D期的患者中，低表達的PDCD4 mRNA提示大腸癌的不良預后。Gao等[29]研究表明在神經(jīng)膠質(zhì)瘤中，PDCD4基因5′CpG島的甲基化導致PDCD4表達的沉默，在84例神經(jīng)膠質(zhì)瘤患者中，PDCD4表達的缺失提示患者的不良預后。

一般情況下，低表達PDCD4提示腫瘤患者的不良預后，但是有一部分高表達的PDCD4的腫瘤患者的預后也很差，經(jīng)Powers等[59]的研究表明這與PDCD4蛋白N端被蛋白精氨酸甲基轉(zhuǎn)移酶（PRMT5）甲基化，影響了PDCD4蛋白的功能有關(guān)。故PDCD4蛋白的功能對腫瘤患者預后的判斷也是極其重要的。

5 結(jié)論

PDCD4表達的調(diào)節(jié)機制極其復雜，不僅在轉(zhuǎn)錄水平，轉(zhuǎn)錄后水平，而且一些信號轉(zhuǎn)導通路也可以調(diào)節(jié)PDCD4的表達。PDCD4的表達也可以通過調(diào)節(jié)其他基因的轉(zhuǎn)錄和翻譯，從而影響腫瘤細胞的增殖、侵襲和轉(zhuǎn)移，形成復雜的調(diào)節(jié)網(wǎng)絡(luò)。同時，PDCD4表達的缺失提示部分腫瘤患者的不良預后，因此，PDCD4可成為腫瘤治療新的靶點，恢復PDCD4的表達有望成為腫瘤治療新的策略。

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2012-07-19）

（本文編輯：胥昀）

瑞安市科技計劃項目（201102039）

325200 瑞安，溫州醫(yī)學院附屬第三醫(yī)院腫瘤外科

陳積賢，E-mail:CJX1957@sohu.com.