董志珍，姚登福

(南通大學(xué)附屬醫(yī)院診斷學(xué)教研室，江蘇南通226001)

MicroRNA作為原發(fā)性肝癌新標(biāo)志物的應(yīng)用前景*

董志珍，姚登福

(南通大學(xué)附屬醫(yī)院診斷學(xué)教研室，江蘇南通226001)

原發(fā)性肝癌(primary liver cancer,PLC）發(fā)病率高，進(jìn)展快，早期發(fā)現(xiàn)難，易產(chǎn)生多藥耐藥(MDR)和復(fù)發(fā)轉(zhuǎn)移，治療難度大，且預(yù)后差，仍是嚴(yán)重威脅人類健康的常見(jiàn)惡性腫瘤，亟待探索新的診治方法[1-2]。體內(nèi)廣泛存在由20～25個(gè)核苷酸組成的非編碼微小RNA(microRNAs，miRNAs)，在轉(zhuǎn)錄后水平調(diào)節(jié)基因表達(dá)，參與胚胎發(fā)育、細(xì)胞增殖與分化、凋亡等生命過(guò)程，在新血管生成、干細(xì)胞分化、浸潤(rùn)及轉(zhuǎn)移等過(guò)程中發(fā)揮重要作用。循環(huán)血中穩(wěn)定的miRNAs，不易被RNA酶消化，不受高或低pH、反復(fù)凍融和長(zhǎng)期儲(chǔ)存等影響，提示有可能成為新的肝癌標(biāo)志物，近年倍受臨床關(guān)注[3-4],在超過(guò)1 000多種miRNAs中，發(fā)現(xiàn)其中部分miRNAs有助于肝癌篩查或診斷、監(jiān)測(cè)、治療和預(yù)后評(píng)估。本文述評(píng)了肝癌miRNAs的診斷價(jià)值。

1 肝miRNAs經(jīng)靶mRNA發(fā)揮作用

體內(nèi)的miRNAs是一類內(nèi)源性的具有調(diào)控功能的miRNAs，由較長(zhǎng)的初級(jí)轉(zhuǎn)錄物(pri-miRNAs)經(jīng)核酸酶(Drosha酶，Dicer酶)剪切加工而產(chǎn)生，隨后組裝進(jìn)RNA誘導(dǎo)的沉默復(fù)合體(RNA-induced silencing complex,RISC)，以堿基互配方式識(shí)別靶mRNA，按互補(bǔ)程度不同指導(dǎo)沉默復(fù)合體降解或阻遏靶mRNA翻譯。肝miRNAs參與多種調(diào)節(jié)途徑包括發(fā)育、病毒防御、造血過(guò)程、器官形成、細(xì)胞增殖和凋亡、脂肪代謝等[5-6]。始于內(nèi)胚層上皮細(xì)胞的肝細(xì)胞，在不同發(fā)育階段miRNAs表達(dá)異同，在胚胎肝miRs-18a、miRs-92a、miRs-409-3p、miRs-451和miRs-483-3p高表達(dá)；成熟肝miRs-22、miRs-23b、miRs-99a、miRs-125b、miRs-192和let-7a、let-7b及l(fā)et-7c高表達(dá)。

廣泛存在非編碼miRNAs，在轉(zhuǎn)錄后水平調(diào)節(jié)基因表達(dá)，參與胚胎發(fā)育、細(xì)胞增殖、細(xì)胞分化、細(xì)胞凋亡等維持機(jī)體正常生理功能。miRNAs可調(diào)控肝細(xì)胞再生如miRs-21、miRs-127和miRs-26a，在肝再生早期miR-21明顯上調(diào)，其靶基因如Btg2、RhoB和Peli1。另如miR-34a靶向Met和Inhbb基因和miR-221靶向CDK抑制劑p27和p57基因。在其它臟器很難檢出的miR-122，是肝特異miRNA占肝miRNAs總量的70%，可作用于肝轉(zhuǎn)錄調(diào)節(jié)因子FoxA1和HNF4α，間接促進(jìn)胚胎干細(xì)胞分化為成熟肝細(xì)胞。在肝細(xì)胞分化中，miRNAs可調(diào)節(jié)膽管形成，如miRs-30a、miRs-30c是膽管特異性miRNAs。經(jīng)生物信息學(xué)分析，肝miRNAs涉及細(xì)胞增殖與分化，與靶mRNA共同作用，發(fā)揮生物學(xué)作用[7-8]。

2 肝miRNAs的促癌與抑癌功能

肝癌組織存在miRNA異常表達(dá)，以miRNA芯片技術(shù)對(duì)癌、癌周?chē)M織及慢性肝炎組織,鑒定出差異表達(dá)miRNAs有30個(gè),發(fā)現(xiàn)與分化相關(guān)的miRNAs有miR-92、miR-20和miR-18等,且表達(dá)水平與分化程度呈負(fù)相關(guān)[9-10]。miRNA異常表達(dá)促進(jìn)肝癌的發(fā)生、發(fā)展,在癌組織有10種miRNAs表達(dá)明顯上調(diào),有16種miRNAs表達(dá)明顯下調(diào)包括iR-199a-3p/miR-199b-3p等，可發(fā)生在肝細(xì)胞惡性轉(zhuǎn)化過(guò)程中,即可見(jiàn)于慢性肝炎、肝硬化患者的肝組織[6-11]。肝miRNAs有多種上，可分為兩類：致癌性miRNAs和抑癌性miRNAs。

2.1 致癌性miRNAs與功能

肝癌組織中明顯上調(diào)、高表達(dá)的miRNAs，在肝癌發(fā)展過(guò)程中出現(xiàn)，其功能類似于癌基因的作用[12-22]（表1）。以實(shí)時(shí)定量PCR(qRT-PCR)法分析，發(fā)現(xiàn)在肝癌早期階段miR-155、miR-221、miR-222和miR-21表達(dá)上調(diào),其中miR-155異常表達(dá)促進(jìn)癌細(xì)胞增殖[17]。在癌組織中miR-224、miR-34a和miR-362-5p表達(dá)顯著上調(diào),與肝癌發(fā)展密切相關(guān)。應(yīng)用siRNA抑制肝癌細(xì)胞miR-362-5p表達(dá)，可顯著降低細(xì)胞的增殖、克隆形成、侵襲和遷移及裸鼠肝癌生長(zhǎng)和轉(zhuǎn)移,通過(guò)靶基因CYLD激活NF-κB信號(hào)通路促進(jìn)肝癌進(jìn)展；肝癌組織和細(xì)胞株中miR-543表達(dá)升高,通過(guò)靶基因PAQR3促進(jìn)肝癌細(xì)胞增殖及侵襲，發(fā)揮癌基因作用；miR-494通過(guò)直接靶向調(diào)控TET1（Ten eleven translocation 1)基因,使多種侵襲抑制miRNAs基因組DNA去甲基化，使基因沉默致肝癌血管浸潤(rùn)[23]。

肝癌組織miR-106a表達(dá)較癌旁組織明顯升高,其啟動(dòng)子甲基化狀態(tài)與其表達(dá)呈負(fù)相關(guān),作用靶基因?yàn)門(mén)P53INP1和CDKN1A；與低表達(dá)miR-106a肝癌細(xì)胞株相比,高表達(dá)miR-106a細(xì)胞株更具侵襲性、更快細(xì)胞周期進(jìn)程及更強(qiáng)的凋亡抵抗[24]；癌組織miR-520g表達(dá)上調(diào),促進(jìn)癌細(xì)胞侵襲、遷移和上皮間質(zhì)轉(zhuǎn)化(EMT),直接作用的靶點(diǎn)是Smad7。肝癌組織中miR-181a表達(dá)明顯上調(diào),TGF-β可促進(jìn)miR-181a上調(diào),誘導(dǎo)細(xì)胞發(fā)生EMT樣變化[25]。癌組織miR-106b過(guò)度表達(dá)，與腫瘤分級(jí)顯著相關(guān)，可通過(guò)Rho GTP酶、RhoA和RhoC來(lái)促進(jìn)細(xì)胞遷移,促進(jìn)轉(zhuǎn)移。

表1 肝癌時(shí)上調(diào)的miRNAs及其生物學(xué)功能

2.2 抑癌性miRNAs與功能

肝癌組織另一類miRNA稱為“抑癌性miRNA”,在正常肝組織中高表達(dá)、癌組織低表達(dá)（表2）如miR-122、miR-126和miR-375等[26-36]。肝癌組織miR-122、miR-125a/b、miR-26、miR-199和miR-375下調(diào),在肝癌進(jìn)展中發(fā)揮作用。肝癌組織miR-122下調(diào)致染色體不穩(wěn)定性,而解除細(xì)胞周期G1期相關(guān)蛋白調(diào)控,間接地經(jīng)細(xì)胞周期調(diào)節(jié)蛋白P53依賴途徑發(fā)揮作用,使PPZA磷酸酶去磷酸化,激活Mdm-2致p53失活,抑制癌細(xì)胞增殖,促進(jìn)凋亡，還可抑制血管內(nèi)皮細(xì)胞分化,抑制血管生成。在非酒精性脂肪性肝病中miR-122基因沉默是個(gè)體早期事件，可作為評(píng)估患者發(fā)生癌變風(fēng)險(xiǎn)的理想指標(biāo)。肝癌miRNA-21、miRNA-222和miRNA-145，經(jīng)下游靶基因PTEN、p27和MAP3K發(fā)揮抑制作用[37]。

表2 肝癌時(shí)表達(dá)下調(diào)miRNAs及其生物學(xué)功能

肝癌組織及細(xì)胞株中miR-744明顯下調(diào),恢復(fù)其表達(dá)能降低癌細(xì)胞的增殖及引起細(xì)胞周期G1期阻滯,可通過(guò)靶向調(diào)控c-myc基因發(fā)揮抑制功能[38]。肝癌組織miR-148b表達(dá)顯著低于對(duì)照肝組織,與肝癌血管浸潤(rùn)及TNM分期顯著相關(guān)。多種肝癌細(xì)胞株如HepG2、MHCC97H、Hep3B及MHCC97L等miR-26b表達(dá)下降與肝癌分級(jí)顯著相關(guān),而恢復(fù)miR-26b表達(dá)能抑制肝癌細(xì)胞株侵襲及遷移力,伴隨上皮標(biāo)志物E-cadherin表達(dá)降低、間質(zhì)標(biāo)志物Vimentin表達(dá)增高及USP9X和Smad4受抑,miR-26b能經(jīng)USP9X、Smad4和TGF-β信號(hào)通路抑制肝癌細(xì)胞EMT。眾多miRNAs中,肝癌組織miR-125b表達(dá)下調(diào),與細(xì)胞分化程度明顯相關(guān),可抑制肝癌細(xì)胞的EMT;主要通過(guò)Smad2和Smad4抑制EMT,改善肝癌細(xì)胞的化療耐藥等。肝癌miR-31可調(diào)節(jié)細(xì)胞周期蛋白(如HDAC2、CDK2)及EMT蛋白(如N-cadherin、E-cadherin和Vimentin等)表達(dá)，發(fā)揮抑癌功能[39]。

3 miRNAs表達(dá)與肝癌致病因素關(guān)系

肝癌發(fā)生與HBV及HCV感染、慢性酒精攝入、非酒精性脂肪性肝病及亞硝胺類物質(zhì)、黃曲霉毒素及有害化學(xué)物質(zhì)等諸多致病因素有關(guān)。以往不被重視的酒精性肝病、脂肪性肝病等慢性肝病發(fā)病率也急劇上升，成人酒精性脂肪性肝病患病率為4.5%，非酒精性脂肪性肝病(NAFLD)的患病率達(dá)15%，直追西方發(fā)達(dá)國(guó)家。如果不進(jìn)行干預(yù)治療，非酒精性脂肪性肝炎(NASH)也可發(fā)展為肝纖維化、肝硬化甚至肝癌[40]。綜合有關(guān)肝癌的miRNAs表達(dá)的研究報(bào)道，很少見(jiàn)到一致性結(jié)果。探索的關(guān)鍵是發(fā)現(xiàn)肝癌特異miRNAs和腫瘤特異miRNAs。依據(jù)Columbia大學(xué)醫(yī)學(xué)中心的報(bào)道，首先發(fā)現(xiàn)miRNAs表達(dá)與肝癌的致病因素相關(guān)（表3）。

表3 肝癌致病因素與特異miRNAs異常關(guān)系[41]

對(duì)癌癥基因組圖譜（TCGA）和肝癌數(shù)據(jù)庫(kù)中9種實(shí)體瘤的臨床、流行病學(xué)和miRNA表達(dá)譜資料綜合比較分析，發(fā)現(xiàn)肝癌與癌周組織33種miRNAs差異表達(dá)(2倍以上)，其中絕多數(shù)(28 miRNAs)呈下調(diào)表達(dá)。在酒精性相關(guān)的79例肝癌組織中，發(fā)現(xiàn)有12種miRNAs改變明顯，其中顯著上調(diào)4種，顯著下調(diào)8種；在HBV感染相關(guān)的79例肝癌組織中，其有7種miRNAs明顯改變，其中miR-532，miR-93和miR-21呈明顯上調(diào)表達(dá)，miR-424，miR-139， miR-24-1和miR-26b明顯下調(diào)；在HCV相關(guān)的31例肝癌中，miR-93和miR-500a明顯表達(dá)，而miR-424和miR-3607顯著下調(diào)表達(dá)，提示有12種，7種和4種miRNAs分別對(duì)酒精性攝入、HBV和HCV感染具特異性并顯著相關(guān)[41]。

4 miRNAs與肝癌診斷

PLC惡性程度高、病情進(jìn)展快,發(fā)病隱匿,多數(shù)患者在診斷時(shí)即已出現(xiàn)肝內(nèi)或肝外轉(zhuǎn)移,失去了根治性手術(shù)治療機(jī)會(huì),且放、化療效果也不理想。理想的肝癌標(biāo)志物需要有較高的特異性,能將肝癌與肝硬化、肝炎、肝臟再生結(jié)節(jié)等區(qū)別開(kāi)來(lái)：同時(shí)還需要較高的敏感性，早期診斷肝癌，且易檢測(cè)、可重復(fù)、侵入少特點(diǎn)[42]。肝miRNA含量穩(wěn)定,盡管血RNA酶會(huì)影響miRNA,但血miRNA表達(dá)穩(wěn)定，可能是保護(hù)miRNA附加結(jié)構(gòu)如膜性物質(zhì)，包裹miRNA分子避免與RNA酶接觸,或修飾miRNA以免于降解。肝癌患者血miR-122、miR-222和miR-223顯著上調(diào)，miR-122可作為在HBV相關(guān)肝癌的潛在標(biāo)志物；肝癌患者癌組織和血miR-21增高,改變比AFP早，可反映肝癌發(fā)生；以qRT-PCR法定量肝癌、慢性肝炎血miR-143和mi R-215表達(dá)水平,可作為肝癌診斷的潛在標(biāo)志物[43]。

表4 人48配對(duì)肝癌與非癌組織中miRNAs的差異表達(dá)[41]

臨床常用的肝癌標(biāo)志物如外周血AFP、肝癌特異AFP(HS-AFP或AFP-L3)、異常凝血酶原、磷脂酰肌醇蛋白聚糖-3(GPC-3)[44]、肝癌特異γ-谷氨酰轉(zhuǎn)移酶(HS-GGT)[45]、高爾基體蛋白酶-73、轉(zhuǎn)化生長(zhǎng)因子-β1、肝細(xì)胞生長(zhǎng)因子、表皮生長(zhǎng)因子受體和腫瘤特異生長(zhǎng)因子等，它們單獨(dú)或聯(lián)合使用均有助于肝癌診斷，然而除HS-GGT和GPC-3外，良性肝病均有不同程度陽(yáng)性率。肝癌、慢性肝病及健康人群血miRNAs如miR-106b、miR-10b及miR-181a均有表達(dá)，三者可鑒別良、惡性肝病，但單一miRNA與常規(guī)肝癌標(biāo)志間尚未見(jiàn)優(yōu)勢(shì)，且不及Wnt3a的診斷與鑒別價(jià)值。miRNA篩選或診斷肝癌需大規(guī)模臨床研究，以驗(yàn)證其診斷的特異價(jià)值。

5 miRNAs診斷肝癌的臨床評(píng)價(jià)

肝miRNAs為重要的生物調(diào)節(jié)劑，在轉(zhuǎn)錄后水平上調(diào)控編碼蛋白基因的表達(dá)。據(jù)估計(jì)約1/3人類基因直接或間接受其支配，并影響到肝細(xì)胞發(fā)生惡性轉(zhuǎn)化的多種信號(hào)通路。有關(guān)肝癌組織及血miRNAs表達(dá)譜的研究，大多分析miRNAs異常與肝癌的臨床病理學(xué)特征，并未涉及miRNAs表達(dá)與肝癌致病因素間的相互關(guān)系。在HBV相關(guān)肝癌和慢性肝病患者血中都見(jiàn)miR-155-5p、miR-24-3p、miR-490-3p、miR-210-3p和miR-335-5p表達(dá)，但miR-24-3p明顯異常,且與肝癌患者血管浸潤(rùn)相關(guān),可診斷和鑒別肝癌，ROC曲線下面積是0.636，AFP為0.627，兩者聯(lián)檢可提高至0.834,可顯著增加診斷準(zhǔn)確度。另miR-125和miR-233可用于HBV陽(yáng)性肝癌患者的早期診斷?？v觀已報(bào)道m(xù)iRNAs診斷肝癌的結(jié)果間差異很大。面臨的最大挑戰(zhàn)是miRNAs如何區(qū)別肝癌與其它實(shí)體腫瘤，尚未見(jiàn)肝癌特異miRNAs報(bào)道，臨床大規(guī)模應(yīng)用受限[14,16]。

令人高興是新的肝癌特異miRNAs和腫瘤共有miRNAs被發(fā)現(xiàn)。在48對(duì)肝癌與癌周自身配對(duì)組織中miRNAs豐度的比較分析，發(fā)現(xiàn)miRNAs上調(diào)表達(dá)僅5種（miR-10b，miR-183，miR-182，miR-452和miR-21），下調(diào)表達(dá)居多為28種，有33種miRNAs可準(zhǔn)確判別肝癌與非癌組織（表4）。

以上述33種miRNAs對(duì)癌與非癌的分類準(zhǔn)確度較高，靈敏度為91.7%，特異性為100%，陽(yáng)性預(yù)測(cè)值為100%，陰性預(yù)測(cè)值為92.3%，判別準(zhǔn)確率95.8%和錯(cuò)判率為4.1%；以33種miRNAs用于302例非配對(duì)肝癌組織具同樣效果，靈敏度為99.0%，特異性為97.9%，陽(yáng)性預(yù)測(cè)值為99.7%，陰性預(yù)測(cè)值為94.0%，判別準(zhǔn)確率為98.5%和錯(cuò)判率僅為1.5%。至于外周血中33種miRNAs的表達(dá)如何及其臨床價(jià)值尚未見(jiàn)報(bào)道[41]。

6 展望

綜上所述，肝癌相關(guān)miRNAs研究已取得較大進(jìn)展,已為其診斷、治療和預(yù)后等引入了新思路。因肝癌發(fā)生機(jī)制極其復(fù)雜，肝細(xì)胞惡性轉(zhuǎn)化中涉及miRNA譜異常，除miRNA作用機(jī)制外，還需臨床大規(guī)模研究包括不同病因、性別、年齡及進(jìn)展階段肝癌患者miRNAs動(dòng)態(tài)表達(dá)，以篩選肝癌特異性miRNAs。miRNAs臨床應(yīng)用尚存亟待解決問(wèn)題[46]：①內(nèi)參miRNAs，以不同內(nèi)參或非人源性miRNAs作為外參照，致各研究數(shù)據(jù)間兼容性和可比性差；②血miRNAs檢測(cè)雖創(chuàng)傷小、可重復(fù)優(yōu)勢(shì)，需統(tǒng)一樣本如血清或血漿；③許多因素可致結(jié)果偏倚，應(yīng)流程標(biāo)準(zhǔn)化確保準(zhǔn)確；④臨床普及和提高對(duì)miRNAs的認(rèn)識(shí)；⑤文獻(xiàn)報(bào)道資料中miRNAs表達(dá)差異較大；⑥已篩選出識(shí)別肝癌與非癌的miRNAs譜，循環(huán)血中表達(dá)如何及其該成果轉(zhuǎn)化亟待解決問(wèn)題。但有理由相信，miRNAs應(yīng)用在肝癌預(yù)防、臨床診斷和治療中具有前景[47,48]。

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（2016-7-30收稿）

R446.11

A

10.3969/j.issn.1000-2669.2016.05.002

*國(guó)家國(guó)際科技合作項(xiàng)目(2013DFA32150)，江蘇省“六大人才高峰”項(xiàng)目(2014-YY-028)

姚登福，男，教授，博士生導(dǎo)師。E-mail:yaodf@ahnmc.com