楊佳 李黎明 龍嘉 許翠 楊雪源 蔣明軍
黑素瘤相關(guān)微小RNA的研究進(jìn)展
楊佳 李黎明 龍嘉 許翠 楊雪源 蔣明軍
微小RNA參與細(xì)胞增殖、分化和凋亡是多種惡性腫瘤發(fā)生發(fā)展的重要調(diào)控因子。研究發(fā)現(xiàn),多種微小RNA在黑素瘤中表達(dá)異常,其中Let-7家族、微小RNA 137、微小RNA 200c和微小RNA 196a等發(fā)揮抑癌基因樣作用,而微小RNA 221/222、微小RNA 30b/30d、微小RNA 146a、微小RNA 214和微小RNA 182發(fā)揮癌基因樣作用。黑素瘤相關(guān)微小RNA的研究將豐富黑素瘤的分子生物學(xué)發(fā)病機(jī)制,為黑素瘤的早期診斷和治療提供新的思路。
黑色素瘤;微RNAs;細(xì)胞增殖;細(xì)胞分化;細(xì)胞凋亡
黑素瘤是一種高度惡性、易遠(yuǎn)處轉(zhuǎn)移的皮膚腫瘤,起源于黑素細(xì)胞,多發(fā)生于皮膚,亦可見于皮膚黏膜交界處、眼球脈絡(luò)膜等處,是皮膚癌中最具侵襲性的腫瘤。黑素瘤雖只占全部皮膚癌的4%,但因黑素瘤死亡的人數(shù)約占所有因皮膚癌死亡人數(shù)的75%[1]。研究顯示,多種微小 RNA(miRNA)在黑素瘤中表達(dá)異常,根據(jù)下游調(diào)控靶基因的不同,在黑素瘤中發(fā)揮癌基因或抑癌基因樣作用,參與黑素瘤發(fā)生、進(jìn)展等生物學(xué)進(jìn)程[2]。
研究顯示,miRNA編碼基因上游CpG島甲基化沉默相應(yīng) miRNA,如 miR-31、miR-18b、miR-375等。Asangani等[3]發(fā)現(xiàn),miR-31 由位于染色質(zhì)9p21.3的長約105 kB的miR-31宿主基因所編碼,該基因轉(zhuǎn)錄調(diào)節(jié)區(qū)域存在77個(gè)CpG島及豐富的H3K27Ac(組蛋白H3第27位賴氨酸乙?;?,而CpG島甲基化及多梳蛋白EZH2介導(dǎo)的H3K27me3(組蛋白第27位賴氨酸3甲基化)可沉默miR-31。黑素瘤細(xì)胞系及組織標(biāo)本中的miR-18b表達(dá)下降,基因序列分析顯示,miR-18b上游存在豐富的CpG島區(qū)域,經(jīng)甲基化轉(zhuǎn)移酶抑制劑處理的黑素瘤細(xì)胞系中,miR-18b上調(diào),提示DNA甲基化參與調(diào)控miR-18b 表達(dá)[4]。Mazar等[5]發(fā)現(xiàn),miR-375 編碼基因上游CpG島甲基化通過沉默miR-375促進(jìn)黑素瘤侵襲轉(zhuǎn)移進(jìn)程。此外,miR-29c通過下調(diào)DNA甲基轉(zhuǎn)移酶3A和3B影響基因組整體甲基化水平[6]。
2.1 let-7 家族:Schultz等[7]分析良性色素痣和原發(fā)性黑素瘤標(biāo)本中的157種miRNA表達(dá)譜,發(fā)現(xiàn)72種miRNA存在表達(dá)差異,其中5個(gè)let-7家族成員(let-7a、let-7b、let-7d、let-7e、let-7g)在原發(fā)性黑素瘤中表達(dá)下降,let-7b下降明顯。過表達(dá)let-7b可通過下調(diào)周期素D1、D3、A和細(xì)胞周期蛋白依賴性激酶4抑制細(xì)胞周期進(jìn)程,減少處于S期、增加處于G1的黑素瘤細(xì)胞數(shù)目,并抑制黑素瘤細(xì)胞的非貼壁依賴性生長。Müller等[8]研究指出,let-7 家族成員let-7a通過靶向調(diào)控整合素β3抑制黑素瘤侵襲,發(fā)揮抑癌基因樣作用。
作者單位:210042 南京,中國醫(yī)學(xué)科學(xué)院 北京協(xié)和醫(yī)學(xué)院 皮膚病研究所中心實(shí)驗(yàn)室
2.2 miR-137:miR-137 位于染色質(zhì) 1p22 區(qū)域,該區(qū)域與黑素瘤發(fā)病風(fēng)險(xiǎn)相關(guān)。Bemis等[9]發(fā)現(xiàn),miR-137與小眼球相關(guān)轉(zhuǎn)錄因子(MITF)3′UTR結(jié)合下調(diào)MITF,后者是黑素細(xì)胞發(fā)育、成熟、凋亡、黑素沉著的主要調(diào)節(jié)因子。上皮間充質(zhì)轉(zhuǎn)化參與腫瘤局部浸潤和遠(yuǎn)處轉(zhuǎn)移過程。E鈣黏蛋白能維持正常上皮細(xì)胞間連接的穩(wěn)定性,其黏附溶解是上皮間充質(zhì)轉(zhuǎn)化的起始步驟。Deng 等[10]研究指出,miR-137 通過靶向調(diào)控轉(zhuǎn)錄輔助抑制因子羧基末端結(jié)合蛋白上調(diào)E鈣黏蛋白,抑制上皮間充質(zhì)轉(zhuǎn)化介導(dǎo)的黑素瘤轉(zhuǎn)移、侵襲。多梳蛋白EZH2通過介導(dǎo)H3K27me3,抑制相關(guān)抑癌基因轉(zhuǎn)錄參與黑素瘤進(jìn)展,其表達(dá)水平與黑素瘤患者生存率相關(guān)。Luo等[11]研究認(rèn)為,過表達(dá)miR-137可抑制黑素瘤細(xì)胞的增殖、侵襲、遷移,其機(jī)制可能為miR-137下調(diào)MITF和癌基因c-Met、YB1、EZH2影響黑素瘤進(jìn)展過程中多種信號通路而發(fā)揮抑癌基因樣作用。
2.3 miR-200c:在轉(zhuǎn)移性黑素瘤較原發(fā)性黑素瘤中表達(dá)降低,且下降水平與瘤體厚度增加程度一致[12],其過表達(dá)可抑制黑素瘤細(xì)胞非貼壁依賴性生長,通過靶向調(diào)控E鈣黏蛋白轉(zhuǎn)錄抑制因子EZB2抑制上皮間充質(zhì)轉(zhuǎn)化介導(dǎo)的腫瘤浸潤侵襲[13],提示miRNA-200c在黑素瘤進(jìn)展、轉(zhuǎn)移中具有抑癌基因樣作用。
2.4 miR-196a:miR-196a靶向調(diào)控 HOX 轉(zhuǎn)錄因子超家族成員 HOX-C8[14]、HOX-B7[15]抑制黑素瘤侵襲進(jìn)展。Mueller等[14]指出,黑素瘤中 miR-196a 與HOX-C8表達(dá)水平負(fù)相關(guān),轉(zhuǎn)染miR-196a的黑素瘤細(xì)胞其侵襲能力較親代細(xì)胞下降約40%,并證實(shí)miR-196a可與HOX-C8 3′UTR起始區(qū)域結(jié)合下調(diào)HOX-C8引起下游相關(guān)基因表達(dá)紊亂發(fā)揮抑癌基因樣作用。Braig 等[15]發(fā)現(xiàn),miRNA-196a 表達(dá)缺失可通過上調(diào)HOX-B7,誘導(dǎo)堿性成纖維因子表達(dá),進(jìn)而活化原癌基因Ets-1,最終引起骨形成蛋白4上調(diào),促進(jìn)黑素瘤轉(zhuǎn)移。
2.5 其他黑素瘤相關(guān)的抑癌基因樣miRNA:Felli等[16]指出,miR-126/126*通過下調(diào)金屬蛋白酶9和基質(zhì)金屬蛋白酶7抑制肝素結(jié)合性表面生長因子前體蛋白水解,抑制黑素瘤細(xì)胞增殖、侵襲。Nyholm等[17]發(fā)現(xiàn),在黑素瘤細(xì)胞系中,轉(zhuǎn)染miR-125b可誘導(dǎo)細(xì)胞周期停滯于G0/G1期,并出現(xiàn)大量表達(dá)衰老標(biāo)記物如p21、p27、p53的細(xì)胞,提示miR-125b在黑素瘤中發(fā)揮抑癌基因樣作用。miR-18b在黑素瘤標(biāo)本及細(xì)胞系中表達(dá)降低,且miR-18b低水平表達(dá)的原發(fā)性黑素瘤患者總體生存率降低,過表達(dá)的miR-18b通過靶向調(diào)控抑癌基因鼠雙微基因2上調(diào)p53,抑制黑素瘤細(xì)胞增殖、侵襲及上皮間充質(zhì)轉(zhuǎn)化,誘導(dǎo)黑素瘤細(xì)胞凋亡[4]。
3.1 miR-221/222:Felicetti等[18]研究顯示,miR-221/222通過下調(diào)p27kipl/CDKN1B和c-kit受體促進(jìn) MITF 表達(dá),激活 Ref、C-myc,上調(diào)周期素 D1、D3和細(xì)胞周期蛋白依賴性激酶4,促進(jìn)黑素瘤進(jìn)展。Errico 等[19]發(fā)現(xiàn),轉(zhuǎn)錄調(diào)控因子 HOX-B7/PBX2 通過活化miR-221/222,下調(diào)c-Fos抑制腫瘤細(xì)胞凋亡,HOX-B7/PBX2拮抗劑HXR9通過阻斷上述通路誘導(dǎo)黑素瘤細(xì)胞凋亡,為黑素瘤的靶向治療提供了思路。
3.2 miR-30b/30d:Gaziel-Sovran 等[20]研究發(fā)現(xiàn),體外免疫抑制環(huán)境中過表達(dá)miR-30b/30d,可增強(qiáng)黑素瘤細(xì)胞的侵襲、轉(zhuǎn)移能力,其表達(dá)沉默具有相反的效應(yīng),過表達(dá)的miR-30b/30d通過下調(diào)GalNAc轉(zhuǎn)移酶GALNT7導(dǎo)致免疫抑制因子IL-10合成和分泌增加,抑制免疫細(xì)胞活化、募集,促進(jìn)黑素瘤轉(zhuǎn)移。
3.3 miR-146a:Forloni等[21]研究發(fā)現(xiàn),miR-146a 通過下調(diào)NUMB蛋白,激活Notch信號參與黑素瘤發(fā)生發(fā)展。黑素瘤中普遍存在BRAF、NRAS基因突變,其中BRAF V600E突變最為常見,miR-146a在BRAFV600E和NRASQ61K黑素瘤細(xì)胞系中表達(dá)水平較野生型中明顯增高,其機(jī)制可能為通過激活BRAF-MEK-細(xì)胞外調(diào)節(jié)蛋白激酶(ERK)信號通路活化MYC,促進(jìn)miR-146a轉(zhuǎn)錄表達(dá)。且前體miRNA-146a基因多態(tài)性(C>G rs2910164)影響黑素瘤進(jìn)展。因此,聯(lián)合應(yīng)用Notch抑制劑,即γ分泌酶抑制劑和BRAF抑制劑可能成為一種有效治療黑素瘤的方法。然而,有研究指出,由于γ分泌酶抑制劑存在較大的不良反應(yīng),需進(jìn)一步在黑素瘤細(xì)胞系和異種移植模型中進(jìn)行廣泛深入研究[22]。
3.4 miR-214:Penna 等[23]發(fā)現(xiàn),miR-214 在轉(zhuǎn)移性黑素瘤中表達(dá)上調(diào),其通過下調(diào)轉(zhuǎn)錄因子TFAP2C、TFAP2A、整合素α3及多種表面蛋白,如MET參與黑素瘤轉(zhuǎn)移過程。進(jìn)一步研究發(fā)現(xiàn)了一條由miR-214、miR-148b、激活蛋白2轉(zhuǎn)錄因子、活化真核細(xì)胞黏附分子構(gòu)成的調(diào)控黑素瘤細(xì)胞遠(yuǎn)處轉(zhuǎn)移的通路[24]。其中,miR-214具有核心作用,其通過在轉(zhuǎn)錄水平直接沉默TFAP2及轉(zhuǎn)錄后水平間接下調(diào)miR-148b兩條途徑上調(diào)活化真核細(xì)胞黏附分子,促進(jìn)黑素瘤細(xì)胞運(yùn)動(dòng)、侵襲、遠(yuǎn)處轉(zhuǎn)移。
3.5 miR-182:在黑素瘤中表達(dá)上調(diào)的miR-182是miR-183-96-182家族成員之一,位于染色質(zhì)7q31-34區(qū)域,兩側(cè)分別編碼癌基因c-MET和BRAF。Segura等[25]研究發(fā)現(xiàn),過表達(dá) miR-182通過直接下調(diào)MITF、FOXO3抑制凋亡、促進(jìn)黑素瘤細(xì)胞運(yùn)動(dòng)、轉(zhuǎn)移。Huynh等[26]在免疫低下小鼠脾臟內(nèi)注射A375黑素瘤細(xì)胞制作黑素瘤肝轉(zhuǎn)移小鼠模型,發(fā)現(xiàn)腹膜腔注射miR-182抑制劑的小鼠其肝臟轉(zhuǎn)移負(fù)荷較對照組明顯下降,提示miR-182抑制劑有望成為轉(zhuǎn)移性黑素瘤的治療方法。
Leidinger等[27]對 35例黑素瘤患者和 20例健康人外周血進(jìn)行高通量miRNA表達(dá)分析,發(fā)現(xiàn)21種表達(dá)下降,30種表達(dá)上升,其中16種表達(dá)明顯下降的miRNA,診斷黑素瘤準(zhǔn)確率高達(dá)97%,提示血清miRNA是一種有效的黑素瘤生物標(biāo)志物。Friedman等[28]基于統(tǒng)計(jì)學(xué)分析證實(shí),血清miRNA在判斷黑素瘤患者預(yù)后中的價(jià)值,指出5種血清miRNA,即miR-150、miR-15b、miR-199A-5p、miR-33a、miR-424與黑素瘤復(fù)發(fā)風(fēng)險(xiǎn)相關(guān)。Kanemaru等[29]研究發(fā)現(xiàn),黑素瘤患者血清miR-221水平較健康人明顯升高,Ⅰ~Ⅳ期黑素瘤患者血清miR-221水平較原位黑素瘤患者升高,且血清水平與瘤體厚度正相關(guān),隨訪顯示手術(shù)切除瘤體后血清miR-221水平下降,復(fù)發(fā)時(shí)其水平再度升高,提出miR-221可作為黑素瘤診斷、分期、判斷治療效果及預(yù)后的血清學(xué)標(biāo)志物。miR-21血清水平可反映腫瘤負(fù)荷,與已知黑素瘤預(yù)后指標(biāo)Breslow厚度和潰瘍深度相關(guān),且腫瘤組織內(nèi)miR-21表達(dá)水平與血清水平一致,提示血清miR-21可能是一種獨(dú)立的黑素瘤預(yù)后標(biāo)志物[30]。
miRNA在黑素瘤發(fā)生發(fā)展中具有強(qiáng)大的調(diào)控作用,且其種類及功能在不斷的發(fā)現(xiàn)之中。尋找與黑素瘤相關(guān)的miRNA,明確其靶基因及作用機(jī)制,并對其構(gòu)成的復(fù)雜調(diào)控網(wǎng)絡(luò)進(jìn)行分析,將有助于豐富黑素瘤的分子生物學(xué)發(fā)病機(jī)制,為將來基于miRNA的黑素瘤靶向治療開辟道路。
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Melanoma-related microRNAs
Yang Jia,Li Liming,Long Jia,Xu Cui,Yang Xueyuan,Jiang Mingjun.Central Laboratory,Institute of Dermatology,Chinese Academy of Medical Sciences and Peking Union Medical College,Nanjing 210042,China
MicroRNAs (miRNAs),as an important regulatory factor in development of multiple malignant tumors,take part in cell proliferation,differentiation and apoptosis.Studies have found aberrant expressions of various miRNAs in melanoma.Among these miRNAs,the Let-7 family,miRNA-137,miRNA-200c and miRNA-196a act as tumor suppressor genes,while miRNA-221/222,miRNA-30b/30d,miRNA-146a,miRNA-214 and miRNA-182 act as oncogenes.Studies on melanoma-related miRNAs will enrich knowledge about molecular pathogenesis of melanoma,and provide new ideas for early diagnosis and treatment of melanoma.
Melanoma;MicroRNAs;Cell proliferation;Cell differentiation;Apoptosis
s:YangXueyuan,Email:yangxueyuan@medmail.com.cn;JiangMingjun,Email:drmingjunjiang@163.com
2015-03-23)
國家自然科學(xué)基金(31470274);江蘇省臨床醫(yī)學(xué)科技專項(xiàng)(BL2012003)
Fund programs:National Natural Science Foundation of China(31470274);Jiangsu Provincial Special Programof Medical Science(BL2012003)
10.3760/cma.j.issn.1673-4173.2016.02.017
楊雪源,Email:yangxueyuan@medmail.com.cn;蔣明軍,Email:drmingjunjiang@163.com