MicroRNA作為原發(fā)性肝癌新標志物的應用前景*

董志珍，姚登福

(南通大學附屬醫(yī)院診斷學教研室，江蘇南通226001)

MicroRNA作為原發(fā)性肝癌新標志物的應用前景*

董志珍，姚登福

(南通大學附屬醫(yī)院診斷學教研室，江蘇南通226001)

原發(fā)性肝癌(primary liver cancer,PLC）發(fā)病率高，進展快，早期發(fā)現(xiàn)難，易產(chǎn)生多藥耐藥(MDR)和復發(fā)轉移，治療難度大，且預后差，仍是嚴重威脅人類健康的常見惡性腫瘤，亟待探索新的診治方法[1-2]。體內廣泛存在由20～25個核苷酸組成的非編碼微小RNA(microRNAs，miRNAs)，在轉錄后水平調節(jié)基因表達，參與胚胎發(fā)育、細胞增殖與分化、凋亡等生命過程，在新血管生成、干細胞分化、浸潤及轉移等過程中發(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、反復凍融和長期儲存等影響，提示有可能成為新的肝癌標志物，近年倍受臨床關注[3-4],在超過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、治療和預后評估。本文述評了肝癌miRNAs的診斷價值。

1 肝miRNAs經(jīng)靶mRNA發(fā)揮作用

體內的miRNAs是一類內源性的具有調控功能的miRNAs，由較長的初級轉錄物(pri-miRNAs)經(jīng)核酸酶(Drosha酶，Dicer酶)剪切加工而產(chǎn)生，隨后組裝進RNA誘導的沉默復合體(RNA-induced silencing complex,RISC)，以堿基互配方式識別靶mRNA，按互補程度不同指導沉默復合體降解或阻遏靶mRNA翻譯。肝miRNAs參與多種調節(jié)途徑包括發(fā)育、病毒防御、造血過程、器官形成、細胞增殖和凋亡、脂肪代謝等[5-6]。始于內胚層上皮細胞的肝細胞，在不同發(fā)育階段miRNAs表達異同，在胚胎肝miRs-18a、miRs-92a、miRs-409-3p、miRs-451和miRs-483-3p高表達；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。

廣泛存在非編碼miRNAs，在轉錄后水平調節(jié)基因表達，參與胚胎發(fā)育、細胞增殖、細胞分化、細胞凋亡等維持機體正常生理功能。miRNAs可調控肝細胞再生如miRs-21、miRs-127和miRs-26a，在肝再生早期miR-21明顯上調，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%，可作用于肝轉錄調節(jié)因子FoxA1和HNF4α，間接促進胚胎干細胞分化為成熟肝細胞。在肝細胞分化中，miRNAs可調節(jié)膽管形成，如miRs-30a、miRs-30c是膽管特異性miRNAs。經(jīng)生物信息學分析，肝miRNAs涉及細胞增殖與分化，與靶mRNA共同作用，發(fā)揮生物學作用[7-8]。

2 肝miRNAs的促癌與抑癌功能

肝癌組織存在miRNA異常表達，以miRNA芯片技術對癌、癌周圍組織及慢性肝炎組織,鑒定出差異表達miRNAs有30個,發(fā)現(xiàn)與分化相關的miRNAs有miR-92、miR-20和miR-18等,且表達水平與分化程度呈負相關[9-10]。miRNA異常表達促進肝癌的發(fā)生、發(fā)展,在癌組織有10種miRNAs表達明顯上調,有16種miRNAs表達明顯下調包括iR-199a-3p/miR-199b-3p等，可發(fā)生在肝細胞惡性轉化過程中,即可見于慢性肝炎、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、高表達的miRNAs，在肝癌發(fā)展過程中出現(xiàn)，其功能類似于癌基因的作用[12-22]（表1）。以實時定量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表達上調,其中miR-155異常表達促進癌細胞增殖[17]。在癌組織中miR-224、miR-34a和miR-362-5p表達顯著上調,與肝癌發(fā)展密切相關。應用siRNA抑制肝癌細胞miR-362-5p表達，可顯著降低細胞的增殖、克隆形成、侵襲和遷移及裸鼠肝癌生長和轉移,通過靶基因CYLD激活NF-κB信號通路促進肝癌進展；肝癌組織和細胞株中miR-543表達升高,通過靶基因PAQR3促進肝癌細胞增殖及侵襲，發(fā)揮癌基因作用；miR-494通過直接靶向調控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[23]。

肝癌組織miR-106a表達較癌旁組織明顯升高,其啟動子甲基化狀態(tài)與其表達呈負相關,作用靶基因為TP53INP1和CDKN1A；與低表達miR-106a肝癌細胞株相比,高表達miR-106a細胞株更具侵襲性、更快細胞周期進程及更強的凋亡抵抗[24]；癌組織miR-520g表達上調,促進癌細胞侵襲、遷移和上皮間質轉化(EMT),直接作用的靶點是Smad7。肝癌組織中miR-181a表達明顯上調,TGF-β可促進miR-181a上調,誘導細胞發(fā)生EMT樣變化[25]。癌組織miR-106b過度表達，與腫瘤分級顯著相關，可通過Rho GTP酶、RhoA和RhoC來促進細胞遷移,促進轉移。

表1 肝癌時上調的miRNAs及其生物學功能

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、癌組織低表達（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,在肝癌進展中發(fā)揮作用。肝癌組織miR-122下調致染色體不穩(wěn)定性,而解除細胞周期G1期相關蛋白調控,間接地經(jīng)細胞周期調節(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,抑制癌細胞增殖,促進凋亡，還可抑制血管內皮細胞分化,抑制血管生成。在非酒精性脂肪性肝病中miR-122基因沉默是個體早期事件，可作為評估患者發(fā)生癌變風險的理想指標。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 肝癌時表達下調miRNAs及其生物學功能

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

3 miRNAs表達與肝癌致病因素關系

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、黃曲霉毒素及有害化學物質等諸多致病因素有關。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15%，直追西方發(fā)達國家。如果不進行干預治療，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]。綜合有關肝癌的miRNAs表達的研究報道，很少見到一致性結果。探索的關鍵是發(fā)現(xiàn)肝癌特異miRNAs和腫瘤特異miRNAs。依據(jù)Columbia大學醫(yī)學中心的報道，首先發(fā)現(xiàn)miRNAs表達與肝癌的致病因素相關（表3）。

表3 肝癌致病因素與特異miRNAs異常關系[41]

對癌癥基因組圖譜（TCGA）和肝癌數(shù)據(jù)庫中9種實體瘤的臨床、流行病學和miRNA表達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(2倍以上)，其中絕多數(shù)(28 miRNAs)呈下調表達。在酒精性相關的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4種，顯著下調8種；在HBV感染相關的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呈明顯上調表達，miR-424，miR-139， miR-24-1和miR-26b明顯下調；在HCV相關的31例肝癌中，miR-93和miR-500a明顯表達，而miR-424和miR-3607顯著下調表達，提示有12種，7種和4種miRNAs分別對酒精性攝入、HBV和HCV感染具特異性并顯著相關[41]。

4 miRNAs與肝癌診斷

PLC惡性程度高、病情進展快,發(fā)病隱匿,多數(shù)患者在診斷時即已出現(xiàn)肝內或肝外轉移,失去了根治性手術治療機會,且放、化療效果也不理想。理想的肝癌標志物需要有較高的特異性,能將肝癌與肝硬化、肝炎、肝臟再生結節(jié)等區(qū)別開來：同時還需要較高的敏感性，早期診斷肝癌，且易檢測、可重復、侵入少特點[42]。肝miRNA含量穩(wěn)定,盡管血RNA酶會影響miRNA,但血miRNA表達穩(wěn)定，可能是保護miRNA附加結構如膜性物質，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，miR-122可作為在HBV相關肝癌的潛在標志物；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表達水平,可作為肝癌診斷的潛在標志物[43]。

表4 人48配對肝癌與非癌組織中miRNAs的差異表達[41]

臨床常用的肝癌標志物如外周血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-谷氨酰轉移酶(HS-GGT)[45]、高爾基體蛋白酶-73、轉化生長因子-β1、肝細胞生長因子、表皮生長因子受體和腫瘤特異生長因子等，它們單獨或聯(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，三者可鑒別良、惡性肝病，但單一miRNA與常規(guī)肝癌標志間尚未見優(yōu)勢，且不及Wnt3a的診斷與鑒別價值。miRNA篩選或診斷肝癌需大規(guī)模臨床研究，以驗證其診斷的特異價值。

5 miRNAs診斷肝癌的臨床評價

肝miRNAs為重要的生物調節(jié)劑，在轉錄后水平上調控編碼蛋白基因的表達。據(jù)估計約1/3人類基因直接或間接受其支配，并影響到肝細胞發(fā)生惡性轉化的多種信號通路。有關肝癌組織及血miRNAs表達譜的研究，大多分析miRNAs異常與肝癌的臨床病理學特征，并未涉及miRNAs表達與肝癌致病因素間的相互關系。在HBV相關肝癌和慢性肝病患者血中都見miR-155-5p、miR-24-3p、miR-490-3p、miR-210-3p和miR-335-5p表達，但miR-24-3p明顯異常,且與肝癌患者血管浸潤相關,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,可顯著增加診斷準確度。另miR-125和miR-233可用于HBV陽性肝癌患者的早期診斷。縱觀已報道m(xù)iRNAs診斷肝癌的結果間差異很大。面臨的最大挑戰(zhàn)是miRNAs如何區(qū)別肝癌與其它實體腫瘤，尚未見肝癌特異miRNAs報道，臨床大規(guī)模應用受限[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對肝癌與癌周自身配對組織中miRNAs豐度的比較分析，發(fā)現(xiàn)miRNAs上調表達僅5種（miR-10b，miR-183，miR-182，miR-452和miR-21），下調表達居多為28種，有33種miRNAs可準確判別肝癌與非癌組織（表4）。

以上述33種miRNAs對癌與非癌的分類準確度較高，靈敏度為91.7%，特異性為100%，陽性預測值為100%，陰性預測值為92.3%，判別準確率95.8%和錯判率為4.1%；以33種miRNAs用于302例非配對肝癌組織具同樣效果，靈敏度為99.0%，特異性為97.9%，陽性預測值為99.7%，陰性預測值為94.0%，判別準確率為98.5%和錯判率僅為1.5%。至于外周血中33種miRNAs的表達如何及其臨床價值尚未見報道[41]。

6 展望

綜上所述，肝癌相關miRNAs研究已取得較大進展,已為其診斷、治療和預后等引入了新思路。因肝癌發(fā)生機制極其復雜，肝細胞惡性轉化中涉及miRNA譜異常，除miRNA作用機制外，還需臨床大規(guī)模研究包括不同病因、性別、年齡及進展階段肝癌患者miRNAs動態(tài)表達，以篩選肝癌特異性miRNAs。miRNAs臨床應用尚存亟待解決問題[46]：①內參miRNAs，以不同內參或非人源性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檢測雖創(chuàng)傷小、可重復優(yōu)勢，需統(tǒng)一樣本如血清或血漿；③許多因素可致結果偏倚，應流程標準化確保準確；④臨床普及和提高對miRNAs的認識；⑤文獻報道資料中miRNAs表達差異較大；⑥已篩選出識別肝癌與非癌的miRNAs譜，循環(huán)血中表達如何及其該成果轉化亟待解決問題。但有理由相信，miRNAs應用在肝癌預防、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

*國家國際科技合作項目(2013DFA32150)，江蘇省“六大人才高峰”項目(2014-YY-028)

姚登福，男，教授，博士生導師。E-mail:yaodf@ahnmc.com