MicroRNA作为原发性肝癌新标志物的应用前景*

董志珍，姚登福

(南通大学附属医院诊断学教研室，江苏南通226001)

MicroRNA作为原发性肝癌新标志物的应用前景*

董志珍，姚登福

(南通大学附属医院诊断学教研室，江苏南通226001)

原发性肝癌(primary liver cancer,PLC）发病率高，进展快，早期发现难，易产生多药耐药(MDR)和复发转移，治疗难度大，且预后差，仍是严重威胁人类健康的常见恶性肿瘤，亟待探索新的诊治方法[1-2]。体内广泛存在由20～25个核苷酸组成的非编码微小RNA(microRNAs，miRNAs)，在转录后水平调节基因表达，参与胚胎发育、细胞增殖与分化、凋亡等生命过程，在新血管生成、干细胞分化、浸润及转移等过程中发挥重要作用。循环血中稳定的miRNAs，不易被RNA酶消化，不受高或低pH、反复冻融和长期储存等影响，提示有可能成为新的肝癌标志物，近年倍受临床关注[3-4],在超过1 000多种miRNAs中，发现其中部分miRNAs有助于肝癌筛查或诊断、监测、治疗和预后评估。本文述评了肝癌miRNAs的诊断价值。

1 肝miRNAs经靶mRNA发挥作用

体内的miRNAs是一类内源性的具有调控功能的miRNAs，由较长的初级转录物(pri-miRNAs)经核酸酶(Drosha酶，Dicer酶)剪切加工而产生，随后组装进RNA诱导的沉默复合体(RNA-induced silencing complex,RISC)，以碱基互配方式识别靶mRNA，按互补程度不同指导沉默复合体降解或阻遏靶mRNA翻译。肝miRNAs参与多种调节途径包括发育、病毒防御、造血过程、器官形成、细胞增殖和凋亡、脂肪代谢等[5-6]。始于内胚层上皮细胞的肝细胞，在不同发育阶段miRNAs表达异同，在胚胎肝miRs-18a、miRs-92a、miRs-409-3p、miRs-451和miRs-483-3p高表达；成熟肝miRs-22、miRs-23b、miRs-99a、miRs-125b、miRs-192和let-7a、let-7b及let-7c高表达。

广泛存在非编码miRNAs，在转录后水平调节基因表达，参与胚胎发育、细胞增殖、细胞分化、细胞凋亡等维持机体正常生理功能。miRNAs可调控肝细胞再生如miRs-21、miRs-127和miRs-26a，在肝再生早期miR-21明显上调，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抑制剂p27和p57基因。在其它脏器很难检出的miR-122，是肝特异miRNA占肝miRNAs总量的70%，可作用于肝转录调节因子FoxA1和HNF4α，间接促进胚胎干细胞分化为成熟肝细胞。在肝细胞分化中，miRNAs可调节胆管形成，如miRs-30a、miRs-30c是胆管特异性miRNAs。经生物信息学分析，肝miRNAs涉及细胞增殖与分化，与靶mRNA共同作用，发挥生物学作用[7-8]。

2 肝miRNAs的促癌与抑癌功能

肝癌组织存在miRNA异常表达，以miRNA芯片技术对癌、癌周围组织及慢性肝炎组织,鉴定出差异表达miRNAs有30个,发现与分化相关的miRNAs有miR-92、miR-20和miR-18等,且表达水平与分化程度呈负相关[9-10]。miRNA异常表达促进肝癌的发生、发展,在癌组织有10种miRNAs表达明显上调,有16种miRNAs表达明显下调包括iR-199a-3p/miR-199b-3p等，可发生在肝细胞恶性转化过程中,即可见于慢性肝炎、肝硬化患者的肝组织[6-11]。肝miRNAs有多种上，可分为两类：致癌性miRNAs和抑癌性miRNAs。

2.1 致癌性miRNAs与功能

肝癌组织中明显上调、高表达的miRNAs，在肝癌发展过程中出现，其功能类似于癌基因的作用[12-22]（表1）。以实时定量PCR(qRT-PCR)法分析，发现在肝癌早期阶段miR-155、miR-221、miR-222和miR-21表达上调,其中miR-155异常表达促进癌细胞增殖[17]。在癌组织中miR-224、miR-34a和miR-362-5p表达显著上调,与肝癌发展密切相关。应用siRNA抑制肝癌细胞miR-362-5p表达，可显著降低细胞的增殖、克隆形成、侵袭和迁移及裸鼠肝癌生长和转移,通过靶基因CYLD激活NF-κB信号通路促进肝癌进展；肝癌组织和细胞株中miR-543表达升高,通过靶基因PAQR3促进肝癌细胞增殖及侵袭，发挥癌基因作用；miR-494通过直接靶向调控TET1（Ten eleven translocation 1)基因,使多种侵袭抑制miRNAs基因组DNA去甲基化，使基因沉默致肝癌血管浸润[23]。

肝癌组织miR-106a表达较癌旁组织明显升高,其启动子甲基化状态与其表达呈负相关,作用靶基因为TP53INP1和CDKN1A；与低表达miR-106a肝癌细胞株相比,高表达miR-106a细胞株更具侵袭性、更快细胞周期进程及更强的凋亡抵抗[24]；癌组织miR-520g表达上调,促进癌细胞侵袭、迁移和上皮间质转化(EMT),直接作用的靶点是Smad7。肝癌组织中miR-181a表达明显上调,TGF-β可促进miR-181a上调,诱导细胞发生EMT样变化[25]。癌组织miR-106b过度表达，与肿瘤分级显著相关，可通过Rho GTP酶、RhoA和RhoC来促进细胞迁移,促进转移。

表1 肝癌时上调的miRNAs及其生物学功能

2.2 抑癌性miRNAs与功能

肝癌组织另一类miRNA称为“抑癌性miRNA”,在正常肝组织中高表达、癌组织低表达（表2）如miR-122、miR-126和miR-375等[26-36]。肝癌组织miR-122、miR-125a/b、miR-26、miR-199和miR-375下调,在肝癌进展中发挥作用。肝癌组织miR-122下调致染色体不稳定性,而解除细胞周期G1期相关蛋白调控,间接地经细胞周期调节蛋白P53依赖途径发挥作用,使PPZA磷酸酶去磷酸化,激活Mdm-2致p53失活,抑制癌细胞增殖,促进凋亡，还可抑制血管内皮细胞分化,抑制血管生成。在非酒精性脂肪性肝病中miR-122基因沉默是个体早期事件，可作为评估患者发生癌变风险的理想指标。肝癌miRNA-21、miRNA-222和miRNA-145，经下游靶基因PTEN、p27和MAP3K发挥抑制作用[37]。

表2 肝癌时表达下调miRNAs及其生物学功能

肝癌组织及细胞株中miR-744明显下调,恢复其表达能降低癌细胞的增殖及引起细胞周期G1期阻滞,可通过靶向调控c-myc基因发挥抑制功能[38]。肝癌组织miR-148b表达显著低于对照肝组织,与肝癌血管浸润及TNM分期显著相关。多种肝癌细胞株如HepG2、MHCC97H、Hep3B及MHCC97L等miR-26b表达下降与肝癌分级显著相关,而恢复miR-26b表达能抑制肝癌细胞株侵袭及迁移力,伴随上皮标志物E-cadherin表达降低、间质标志物Vimentin表达增高及USP9X和Smad4受抑,miR-26b能经USP9X、Smad4和TGF-β信号通路抑制肝癌细胞EMT。众多miRNAs中,肝癌组织miR-125b表达下调,与细胞分化程度明显相关,可抑制肝癌细胞的EMT;主要通过Smad2和Smad4抑制EMT,改善肝癌细胞的化疗耐药等。肝癌miR-31可调节细胞周期蛋白(如HDAC2、CDK2)及EMT蛋白(如N-cadherin、E-cadherin和Vimentin等)表达，发挥抑癌功能[39]。

3 miRNAs表达与肝癌致病因素关系

肝癌发生与HBV及HCV感染、慢性酒精摄入、非酒精性脂肪性肝病及亚硝胺类物质、黄曲霉毒素及有害化学物质等诸多致病因素有关。以往不被重视的酒精性肝病、脂肪性肝病等慢性肝病发病率也急剧上升，成人酒精性脂肪性肝病患病率为4.5%，非酒精性脂肪性肝病(NAFLD)的患病率达15%，直追西方发达国家。如果不进行干预治疗，非酒精性脂肪性肝炎(NASH)也可发展为肝纤维化、肝硬化甚至肝癌[40]。综合有关肝癌的miRNAs表达的研究报道，很少见到一致性结果。探索的关键是发现肝癌特异miRNAs和肿瘤特异miRNAs。依据Columbia大学医学中心的报道，首先发现miRNAs表达与肝癌的致病因素相关（表3）。

表3 肝癌致病因素与特异miRNAs异常关系[41]

对癌症基因组图谱（TCGA）和肝癌数据库中9种实体瘤的临床、流行病学和miRNA表达谱资料综合比较分析，发现肝癌与癌周组织33种miRNAs差异表达(2倍以上)，其中绝多数(28 miRNAs)呈下调表达。在酒精性相关的79例肝癌组织中，发现有12种miRNAs改变明显，其中显著上调4种，显著下调8种；在HBV感染相关的79例肝癌组织中，其有7种miRNAs明显改变，其中miR-532，miR-93和miR-21呈明显上调表达，miR-424，miR-139， miR-24-1和miR-26b明显下调；在HCV相关的31例肝癌中，miR-93和miR-500a明显表达，而miR-424和miR-3607显著下调表达，提示有12种，7种和4种miRNAs分别对酒精性摄入、HBV和HCV感染具特异性并显著相关[41]。

4 miRNAs与肝癌诊断

PLC恶性程度高、病情进展快,发病隐匿,多数患者在诊断时即已出现肝内或肝外转移,失去了根治性手术治疗机会,且放、化疗效果也不理想。理想的肝癌标志物需要有较高的特异性,能将肝癌与肝硬化、肝炎、肝脏再生结节等区别开来：同时还需要较高的敏感性，早期诊断肝癌，且易检测、可重复、侵入少特点[42]。肝miRNA含量稳定,尽管血RNA酶会影响miRNA,但血miRNA表达稳定，可能是保护miRNA附加结构如膜性物质，包裹miRNA分子避免与RNA酶接触,或修饰miRNA以免于降解。肝癌患者血miR-122、miR-222和miR-223显著上调，miR-122可作为在HBV相关肝癌的潜在标志物；肝癌患者癌组织和血miR-21增高,改变比AFP早，可反映肝癌发生；以qRT-PCR法定量肝癌、慢性肝炎血miR-143和mi R-215表达水平,可作为肝癌诊断的潜在标志物[43]。

表4 人48配对肝癌与非癌组织中miRNAs的差异表达[41]

临床常用的肝癌标志物如外周血AFP、肝癌特异AFP(HS-AFP或AFP-L3)、异常凝血酶原、磷脂酰肌醇蛋白聚糖-3(GPC-3)[44]、肝癌特异γ-谷氨酰转移酶(HS-GGT)[45]、高尔基体蛋白酶-73、转化生长因子-β1、肝细胞生长因子、表皮生长因子受体和肿瘤特异生长因子等，它们单独或联合使用均有助于肝癌诊断，然而除HS-GGT和GPC-3外，良性肝病均有不同程度阳性率。肝癌、慢性肝病及健康人群血miRNAs如miR-106b、miR-10b及miR-181a均有表达，三者可鉴别良、恶性肝病，但单一miRNA与常规肝癌标志间尚未见优势，且不及Wnt3a的诊断与鉴别价值。miRNA筛选或诊断肝癌需大规模临床研究，以验证其诊断的特异价值。

5 miRNAs诊断肝癌的临床评价

肝miRNAs为重要的生物调节剂，在转录后水平上调控编码蛋白基因的表达。据估计约1/3人类基因直接或间接受其支配，并影响到肝细胞发生恶性转化的多种信号通路。有关肝癌组织及血miRNAs表达谱的研究，大多分析miRNAs异常与肝癌的临床病理学特征，并未涉及miRNAs表达与肝癌致病因素间的相互关系。在HBV相关肝癌和慢性肝病患者血中都见miR-155-5p、miR-24-3p、miR-490-3p、miR-210-3p和miR-335-5p表达，但miR-24-3p明显异常,且与肝癌患者血管浸润相关,可诊断和鉴别肝癌，ROC曲线下面积是0.636，AFP为0.627，两者联检可提高至0.834,可显著增加诊断准确度。另miR-125和miR-233可用于HBV阳性肝癌患者的早期诊断。纵观已报道miRNAs诊断肝癌的结果间差异很大。面临的最大挑战是miRNAs如何区别肝癌与其它实体肿瘤，尚未见肝癌特异miRNAs报道，临床大规模应用受限[14,16]。

令人高兴是新的肝癌特异miRNAs和肿瘤共有miRNAs被发现。在48对肝癌与癌周自身配对组织中miRNAs丰度的比较分析，发现miRNAs上调表达仅5种（miR-10b，miR-183，miR-182，miR-452和miR-21），下调表达居多为28种，有33种miRNAs可准确判别肝癌与非癌组织（表4）。

以上述33种miRNAs对癌与非癌的分类准确度较高，灵敏度为91.7%，特异性为100%，阳性预测值为100%，阴性预测值为92.3%，判别准确率95.8%和错判率为4.1%；以33种miRNAs用于302例非配对肝癌组织具同样效果，灵敏度为99.0%，特异性为97.9%，阳性预测值为99.7%，阴性预测值为94.0%，判别准确率为98.5%和错判率仅为1.5%。至于外周血中33种miRNAs的表达如何及其临床价值尚未见报道[41]。

6 展望

综上所述，肝癌相关miRNAs研究已取得较大进展,已为其诊断、治疗和预后等引入了新思路。因肝癌发生机制极其复杂，肝细胞恶性转化中涉及miRNA谱异常，除miRNA作用机制外，还需临床大规模研究包括不同病因、性别、年龄及进展阶段肝癌患者miRNAs动态表达，以筛选肝癌特异性miRNAs。miRNAs临床应用尚存亟待解决问题[46]：①内参miRNAs，以不同内参或非人源性miRNAs作为外参照，致各研究数据间兼容性和可比性差；②血miRNAs检测虽创伤小、可重复优势，需统一样本如血清或血浆；③许多因素可致结果偏倚，应流程标准化确保准确；④临床普及和提高对miRNAs的认识；⑤文献报道资料中miRNAs表达差异较大；⑥已筛选出识别肝癌与非癌的miRNAs谱，循环血中表达如何及其该成果转化亟待解决问题。但有理由相信，miRNAs应用在肝癌预防、临床诊断和治疗中具有前景[47,48]。

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（2016-7-30收稿）

R446.11

A

10.3969/j.issn.1000-2669.2016.05.002

*国家国际科技合作项目(2013DFA32150)，江苏省“六大人才高峰”项目(2014-YY-028)

姚登福，男，教授，博士生导师。E-mail:yaodf@ahnmc.com