張婁強(qiáng) 孫庚林

腫瘤干細(xì)胞miRNA及miRNA作為腫瘤標(biāo)志物的研究和臨床應(yīng)用進(jìn)展

張婁強(qiáng) 孫庚林△

腫瘤干細(xì)胞（CSCs）理論為腫瘤的研究開辟了一個(gè)新的方向，CSCs學(xué)說認(rèn)為腫瘤細(xì)胞具有異質(zhì)性，腫瘤中存在干細(xì)胞樣細(xì)胞，該群細(xì)胞是一種增殖失控、可形成腫瘤的細(xì)胞，只占腫瘤細(xì)胞很少部分，具有干細(xì)胞特性，是形成不同分化程度腫瘤細(xì)胞和腫瘤增長、復(fù)發(fā)及轉(zhuǎn)移的根源。微小RNA（miRNA）是廣泛存在的非編碼小RNA，調(diào)節(jié)著人類1∕3的基因，越來越多的證據(jù)顯示miRNA在腫瘤的發(fā)生發(fā)展中起著重要的作用，作為重要的轉(zhuǎn)錄后調(diào)控因子，廣泛參與腫瘤相關(guān)基因調(diào)控的生物程序，使不同類型的腫瘤表現(xiàn)出特異的miRNA表達(dá)譜。近年來，CSCs的miRNA研究日益成為熱點(diǎn)，已經(jīng)發(fā)現(xiàn)多種CSCs中存在特異性表達(dá)的miRNA，對(duì)CSCs的生物學(xué)行為有了更進(jìn)一步的認(rèn)識(shí)，發(fā)現(xiàn)腫瘤患者血漿中表達(dá)某些特異的miRNA，這些miRNA可以作為腫瘤的標(biāo)志物對(duì)患者的病情及預(yù)后進(jìn)行預(yù)測(cè)和判斷。本文就近來CSCs中miRNA研究進(jìn)展及miRNA作為腫瘤標(biāo)志物研究進(jìn)展進(jìn)行綜述。

腫瘤干細(xì)胞；微RNAs；腫瘤標(biāo)記,生物學(xué)；綜述

腫瘤干細(xì)胞（cancer stem cells,CSCs）被認(rèn)為是腫瘤組織中存在的極少量干細(xì)胞樣腫瘤細(xì)胞亞群，有無限的自我更新能力，在啟動(dòng)腫瘤形成和生長中起著決定性的作用，是腫瘤形成、生長、復(fù)發(fā)及耐藥性的根源[1-3]，CSCs的研究推動(dòng)了腫瘤學(xué)的發(fā)展。微小RNA（microRNA,miRNA）在CSCs中起到非常重要的調(diào)節(jié)作用，目前國內(nèi)外已有多項(xiàng)針對(duì)不同腫瘤CSCs miRNA的研究，成功篩選出在CSCs中與普通腫瘤細(xì)胞中表達(dá)差異的miRNA，對(duì)其中部分miRNA的生物學(xué)功能進(jìn)行了研究，發(fā)現(xiàn)某些特異性miRNA可用于腫瘤個(gè)體化治療，并可用于腫瘤的診斷及預(yù)后評(píng)價(jià)。本文對(duì)CSCs中miRNA研究和臨床應(yīng)用的最新進(jìn)展進(jìn)行綜述。

1 miRNA在CSCs中的表達(dá)及其功能

1.1 miRNA與乳腺癌干細(xì)胞（BCSCs） miRNA對(duì)BCSCs具有重要的調(diào)控作用，miR-200c-141、miR-200b-200a-429和miR-183-96-182在人BCSCs中的表達(dá)均下調(diào)[4-6]。B細(xì)胞特異的莫洛尼白血病病毒插入位點(diǎn)1基因（Bmi-1）屬于多梳基因家族成員，可與c-myc協(xié)同作用引起細(xì)胞轉(zhuǎn)化和腫瘤形成，miR-200c過表達(dá)可下調(diào)Bmi-1，從而抑制BCSCs增殖能力、抑制腫瘤的形成，并能抑制乳腺導(dǎo)管的形成[4]。在BCSCs 中Let-7下調(diào)，而且在動(dòng)物實(shí)驗(yàn)中發(fā)現(xiàn)，Let-7的過表達(dá)能夠下調(diào)癌基因H-Ras和高移動(dòng)族AT鉤2基因（HMGA2），從而抑制BCSCs的自我更新和分化能力。miR-30能夠通過調(diào)節(jié)泛素結(jié)合酶9（UBC9）和整聯(lián)蛋白β3（ITGB3）使BCSCs數(shù)量明顯減少。而且當(dāng)Let-7和miR-30同時(shí)表達(dá)時(shí)，BCSCs的自我更新和腫瘤細(xì)胞球的形成能力被更加完全地抑制[7]。在對(duì)BCSCs的研究中還發(fā)現(xiàn)：除了miR-200家族以外，miR-15b、miR-16、miR-103、miR-107、miR-145、miR-335和miR-128b均可影響B(tài)mi-1和調(diào)味12抑制因子同源物基因（Suz12）的表達(dá)，并影響作為DNA結(jié)合轉(zhuǎn)錄因子的鋅指E盒結(jié)合同源盒蛋白（Zeb）1、Zeb2及Kruppel樣因子4（Klf4）基因的表達(dá)，相反Zeb1和Zeb2能夠抑制以上miRNAs的表達(dá)[8]。

1.2 miRNA與腦腫瘤干細(xì)胞 目前miRNA對(duì)顱腦腫瘤CSCs的研究主要集中在抑癌方面，在神經(jīng)膠質(zhì)瘤干細(xì)胞的研究中發(fā)現(xiàn)，CD133+細(xì)胞與CD133-細(xì)胞在miRNA表達(dá)譜上存在差異，尤其miR-125b在CD133+細(xì)胞中被顯著下調(diào)，過表達(dá)miR-125b能夠抑制CD133+細(xì)胞的增殖，并能夠抑制內(nèi)源性核轉(zhuǎn)錄因子E2F2蛋白的水平[9]。miR-124和miR-137能夠誘導(dǎo)神經(jīng)干細(xì)胞及膠質(zhì)母細(xì)胞瘤干細(xì)胞的分化，并抑制腫瘤細(xì)胞增殖[10-12]，表明miR-124和miR-137對(duì)膠質(zhì)母細(xì)胞瘤具有治療作用。在對(duì)腦膠質(zhì)母細(xì)胞瘤的研究中還發(fā)現(xiàn)，miR-451在CD133+細(xì)胞中較CD133-細(xì)胞中表達(dá)明顯下調(diào)[13]，miR-451能抑制CD133+細(xì)胞自我更新形成細(xì)胞球，尤其與藥物甲磺酸伊馬替尼聯(lián)用后抑制作用更加明顯。

1.3 miRNA與胰腺癌腫瘤干細(xì)胞 在胰腺癌CSCs中Notch-1的表達(dá)可導(dǎo)致原癌miRNA（oncogenic miRNA）miR-21的表達(dá)升高，miR-200b、miR-200c、Let-7a、Let-7b和Let-7c的表達(dá)降低，而miR-200b的再次表達(dá)導(dǎo)致Zeb1和波形蛋白（vimentin）表達(dá)降低，并導(dǎo)致上皮性鈣黏附蛋白（E-cadherin）表達(dá)升高，Notch-1信號(hào)通路活化能夠使胰腺癌細(xì)胞產(chǎn)生上皮細(xì)胞間質(zhì)轉(zhuǎn)型（EMT），而miR-200b和CSCs的自我更新能力參與此過程的調(diào)節(jié)[14]。二甲雙胍可加強(qiáng)Let-7a、Let-7b、miR-26a、miR-101、miR-200b和miR-200c的表達(dá)，并降低胰腺癌CSCs相關(guān)基因的表達(dá)[15]。對(duì)吉西他濱具有抵抗性的胰腺癌細(xì)胞系MIA PaCa-2中包含有ALDH-positive腫瘤干細(xì)胞樣細(xì)胞，經(jīng)miR-205轉(zhuǎn)染后其干細(xì)胞標(biāo)志物Oct3∕4 和CD44及微管蛋白β3（TUBB3）表達(dá)均降低，從而恢復(fù)了對(duì)吉西他濱的敏感性[16]。

1.4 miRNA與前列腺癌腫瘤干細(xì)胞 miR-34a能夠抑制前列腺癌CSCs及前列腺癌的生長及轉(zhuǎn)移，CD44本身就是miR-34a調(diào)控的下游受體，在前列腺癌移植瘤和原代腫瘤中分離出的CD44+細(xì)胞中，miR-34a表達(dá)下調(diào)，在腫瘤細(xì)胞中增強(qiáng)表達(dá)miR-34a能夠抑制前列腺癌細(xì)胞的克隆及腫瘤的復(fù)發(fā)和轉(zhuǎn)移，在CD44-細(xì)胞中導(dǎo)入miR-34a反義序列能夠促進(jìn)腫瘤細(xì)胞的生長和轉(zhuǎn)移，動(dòng)物實(shí)驗(yàn)證實(shí)miR-34a對(duì)前列腺癌具有治療作用，可延長生存時(shí)間。以上提示miR-34a對(duì)前列腺癌CSCs具有重要的調(diào)控作用，可以作為治療前列腺癌的靶點(diǎn)[17]。

1.5 miRNA與肝癌腫瘤干細(xì)胞 miR-181家族成員在肝癌CSCs中表達(dá)上調(diào)，抑制miR-181可減少CSCs的細(xì)胞數(shù)量及其形成腫瘤的能力，外源性表達(dá)miR-181可增加CSCs細(xì)胞的數(shù)量，并發(fā)現(xiàn)miR-181能夠直接作用于肝臟細(xì)胞的尾型同源框轉(zhuǎn)錄因子2（CDX2）、GATA結(jié)合蛋白6（GATA6）以及Wnt∕β-catenin信號(hào)通路的抑制因子nemo樣激酶（NLK），提示miR-181可以作為肝癌治療的分子靶向目標(biāo)[18]。

1.6 miRNA與結(jié)腸直腸癌腫瘤干細(xì)胞 結(jié)腸癌細(xì)胞系SW1116的 CSCs中，CD133+∕CD44+與 CD133-∕CD44-細(xì)胞miRNA的表達(dá)存在差異，如miR-29a、miR-29b、miR-449b 和miR-4524等，miRNA在調(diào)節(jié)結(jié)腸癌干細(xì)胞的生物學(xué)特性中發(fā)揮重要的作用，通過調(diào)節(jié)結(jié)腸癌干細(xì)胞的信號(hào)通路、細(xì)胞骨架、膜蛋白的表達(dá)在腫瘤的復(fù)發(fā)和轉(zhuǎn)移中發(fā)揮其作用[19]。結(jié)腸直腸癌干細(xì)胞中miR-451下調(diào)導(dǎo)致環(huán)氧合酶（COX）-2的表達(dá)，從而激活對(duì)CSCs非常重要的Wnt信號(hào)通路，相反miR-451的表達(dá)恢復(fù)可引起多藥耐藥基因ABCB1的表達(dá)降低，從而提高結(jié)腸癌對(duì)伊立替康的敏感性，所以miR-451對(duì)結(jié)腸直腸癌的復(fù)發(fā)和耐藥性有關(guān)，可以作為結(jié)腸直腸癌患者對(duì)伊立替康化療反應(yīng)的標(biāo)志物[20]。還有研究發(fā)現(xiàn)，miR-21通過TGFβR2信號(hào)通路在結(jié)腸直腸癌干細(xì)胞中發(fā)揮重要的調(diào)節(jié)作用[21]。

1.7 miRNA與其他腫瘤干細(xì)胞 miR-21和miR-302在胃癌細(xì)胞系MKN-45的CSCs中表達(dá)升高，Let-7a表達(dá)降低，而在普通癌細(xì)胞中miR-372、miR-373和miR-520c-5p表達(dá)明顯高于CSCs[22]。

在卵巢癌干細(xì)胞中miR-214通過p53∕Nanog通路調(diào)節(jié)卵巢癌干細(xì)胞的生物學(xué)特性，miR-214表達(dá)加強(qiáng)可以提高其CSCs的數(shù)量、自我更新能力及Nanog表達(dá)水平，當(dāng)敲除miR-214時(shí)CSCs的數(shù)量、自我更新能力和Nanog的表達(dá)水平均降低，尤其在野生型p53基因的細(xì)胞系中還發(fā)現(xiàn)p53能夠直接被miR-214抑制，miR-214通過p53調(diào)節(jié)Nanog，揭示了miR-214在卵巢癌中的治療意義[23-24]。

頭頸部鱗癌中，ALDH1+∕CD44+細(xì)胞亞群具有腫瘤干細(xì)胞樣細(xì)胞的生物學(xué)特性，其miR-200c表達(dá)下調(diào)，Bmi-1表達(dá)上調(diào)，提高miR-200c的表達(dá)或敲除Bmi-1能夠?qū)LDH1+∕CD44+細(xì)胞亞群產(chǎn)生顯著的抑制作用，過表達(dá)miR-200c能夠下調(diào)Zeb1、鋅指轉(zhuǎn)錄因子Snail和神經(jīng)性鈣黏附蛋白（N-cadherin），上調(diào)E-cadherin，而且在ALDH1+∕CD44+細(xì)胞的移植瘤實(shí)驗(yàn)中發(fā)現(xiàn)，過度表達(dá)miR-200c或者敲除Bmi-1能夠有效地抑制腫瘤的肺轉(zhuǎn)移能力，提高實(shí)驗(yàn)動(dòng)物的生存率[25]。

2 miRNA作為腫瘤標(biāo)志物的研究

miRNA在腫瘤及CSCs中特異性表達(dá)的特征，使其有可能成為相應(yīng)腫瘤的生物學(xué)標(biāo)志物，用于對(duì)腫瘤進(jìn)行診斷并評(píng)價(jià)預(yù)后。腫瘤患者的血漿中可以檢測(cè)到某些特殊的miRNA，這些miRNA就是腫瘤細(xì)胞釋放入血液循環(huán)中的，最早驗(yàn)證血漿miRNA可以作為腫瘤生物標(biāo)志物的是對(duì)肺癌的研究，后來對(duì)肝癌患者血清miR-500檢測(cè)發(fā)現(xiàn)，術(shù)前患者miR-500呈高表達(dá)，術(shù)后恢復(fù)到正常水平，因此miR-500血清表達(dá)情況可以用于肝癌的診斷，同時(shí)還可以將miR-500血清表達(dá)水平用來判斷腫瘤復(fù)發(fā)及轉(zhuǎn)移情況[26]。乳腺癌患者低表達(dá)miR-200與其不良預(yù)后有關(guān)，提示miR-200可以作為乳腺癌的臨床標(biāo)志物，用來判斷患者預(yù)后[27]。腎透明細(xì)胞癌（RCC）患者血漿中的miR-221和miR-222的表達(dá)均高于非腫瘤患者，而腫瘤轉(zhuǎn)移患者血漿中的miR-221表達(dá)水平要高于未轉(zhuǎn)移患者，同時(shí)發(fā)現(xiàn)高表達(dá)miR-221的患者具有較低生存期，提示miR-221和miR-222可以作為RCC診斷和判斷預(yù)后的臨床標(biāo)志物[28]。

3 展望

miRNA對(duì)CSCs的生物學(xué)行為具有重要的調(diào)節(jié)作用，了解miRNA在CSCs中的調(diào)控機(jī)制必然會(huì)為腫瘤治療開辟新的思路，而且某些miRNA還可用來作為腫瘤的標(biāo)志物。明確CSCs中特異性表達(dá)的miRNA及其調(diào)控的靶基因、miRNA如何調(diào)控CSCs的生物學(xué)行為、腫瘤患者血漿中miRNA表達(dá)情況等將是今后研究的重點(diǎn)。

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(2014-01-08收稿 2014-04-17修回)

（本文編輯 李國琪）

Research and Clinical Application of miRNAs in Cancer Stem Cells and as Tumor Markers

ZHANG Louqiang,SUN Genglin△
Department of Stomatology,Tianjin Medical University General Hospital,Tianjin 300052,China△Reviser E-mail:lina823198@163.com

The theory of cancer stem cells(CSCs)provides a new perspective for the study of cancer.CSCs doctrine presumes that tumor cells are heterogeneity,and there are cancer stem-like cells in tumors,whose proliferation is uncontrolled.The cancer stem-like cells could form tumors from only few cells,who only account for a very small part of all the tumor cells.These cells with stem cells characteristics could form different extend of differentiation of tumor cells and work as source of tumor growth,recurrence and metastasis.miRNAs are widely-existed non-coding small RNAs,which regulate 1∕3 of all human genes and participate in a series of essential processes of life.More and more evidences indicate that miRNAs contribute significantly to cancer generation and development.They may exhibit oncogenic activity and act as tumor suppressors.As one of the post-transcriptional controlling factors,they regulate biological processes of tumor related genes extensively and specific miRNAs expression profiles were shown in different cancers.The research of miRNA about CSCs has become very hot.Some studies have confirmed that miRNAs play a very important role in a variety of CSCs,which act as a new target for tumor therapy.Researchers have found that some specific miRNAs express in plasma of cancer patients,which can be used as tumor markers to assist diagnosis and prognosis.This review focused on current progress of research in function of miRNAs in CSCs and its role as tumor markers.

neoplastic stem cells;microRNAs;tumor markers,biological;review

R730.4

A

10.3969∕j.issn.0253-9896.2014.10.025

天津醫(yī)科大學(xué)總醫(yī)院口腔科（郵編300052）△審校者 E-mail：lina823198@163.com