非小细胞肺癌顺铂化疗耐药相关微RNA的筛选及鉴定

 目的　探讨肿瘤细胞微RNA（miRNA）对化疗药物敏感性的作用。方法　通过miRNA芯片技术检测顺铂（DDP）耐药细胞株A549／DDP与非耐药细胞株A549的miRNA表达的差异,利用荧光定量聚合酶链反应（PCR）技术验证相应miRNA的表达情况,通过在细胞株中抑制或过表达目标miRNA,研究其对细胞化疗药物敏感性的影响。结果　A549／DDP细胞对DDP的耐药为A549细胞的18倍。A549／DDP细胞与A549细胞存在51个表达水平差异在4倍以上的miRNA,其中24个表达上调,27个表达下调。PCR进一步证实miR-376c、miR-31、miR-29a、miR-221在A549／DDP细胞中显著上调,miR-196a、miR-20a、miR-20b、miR-17、miR-451在A549／DDP细胞中显著下调。在提高A549／DDP细胞中miR-17的表达后,细胞对DDP的敏感度增加了11.7 %,提高miR-451的表达或者抑制miR-29a的表达后,对DDP的敏感度分别下降了15.5 %、12.9 %,抑制miR-376c、miR-31、miR-221或过表达miR-196a、miR-20a、miR-20b均不影响A549／DDP细胞对DDP的敏感度。结论　非小细胞肺癌DDP耐药细胞与非耐药细胞的miRNA表达谱有差异,miRNA参与肺癌化疗耐药,miR-17具有逆转非小细胞肺癌DDP耐药的潜力。     

大肠癌相关microRNA研究进展

microRNA（miRNA）是一种大小约22个核苷酸的非编码单链小RNA分子，参与细胞增殖、分化、凋亡等多种重要细胞活动的调控。近来研究发现miRNA具有癌基因或抑癌基因样作用，参与多种恶性肿瘤的演进。是肿瘤发生、发展过程中重要分子。目前已发现多种miRNAs在大肠癌组织及大肠癌细胞系中异常表达，一部分在癌细胞中较正常细胞表达明显下降如miR-143、miR-145、let-7、miR-34a等，一部分表达则升高如miR-3l、miR-21等。体外试验中将miR-143、miR-145的前体导入大肠癌细胞中，可观察到癌细胞生长受到抑制，且呈剂量依赖性。miRNA表达谱与大肠癌的生物学行为和临床分期相关，如Ⅳ期大肠癌miR-31的表达水平明显较Ⅱ期升高。而大肠癌细胞系中miRNA表达谱与癌组织差别较大，由细胞系中得到miRNA表达谱可能不适于用来推断临床样本的相应表达谱。目前研究比较多的miRNAs如miR-143、miR-145、miR-34a、let-7a等，均有抑制细胞生长增殖的作用，它们在癌细胞中表达下降导致细胞过度生长增殖，可能参与大肠癌的发生。一些化疗药物能明显影响大肠癌细胞中miRNA的表达水平，如阿霉素可明显上调miR-34的表达水平，人们推测miRNA可能是一些化疗药物发挥抗肿瘤作用的重要分子。综上，阐明大肠癌相关miRNA的作用机制将可能丰富大肠癌的病因学及分子病理学理论，为大肠癌诊断治疗提供新策略和思路。     

miR-10a-5p基因甲基化对卵巢癌铂类耐药的影响

目的 卵巢癌是死亡率最高的妇科肿瘤,较强的化疗耐药性是其预后差的主要原因之一,为了阐明卵巢癌对铂类药物的耐药机制,本研究探讨miRNA基因的甲基化水平对卵巢癌铂类耐药的影响.方法 将卵巢癌组织分为敏感组和耐药组,每组各3例;采用基因芯片技术,对比分析了两组微RNA(microRNA,miRNA)的表达差异;采用实时荧光定量PCR,分别在6例敏感和3例耐药组织、铂类药物敏感(CoC1)和耐药的卵巢癌细胞系(CoC1/DDP),检测了候选miRNA的表达差异;应用Massarray技术,检测敏感组织(15例)与耐药组织(6例)中miRNA基因启动子的甲基化差异;应用生物信息学分析,鉴定目标miRNA的潜在靶基因.结果 以铂类药物敏感的卵巢癌组织样本为对照,利用基因芯片筛选,鉴定了6条在耐药组织样本中出现表达上调的miRNA(miR-493-3p、miR-10a-5 p、miR-16-2-3p、miR-1248、miR-451a、miR-628-3p)和6条表达下调的miRNA(miR-509-3p、miR-1197、miR-376a-3p、miR-1273a、miR-550a-3p、miR-19b-3p).组织验证发现,miR-509-3p、miR-493-3p、miR-10a-5p、miR-16-2-3p和miR-451a,与芯片结果一致;培养细胞研究发现,4条miRNA的表达调控方式与组织芯片结果一致,miR-10a-5p、miR-16-2-3p、miR-1248和miR-628-3p在耐药细胞系中高表达.进一步研究发现,与敏感肿瘤组织相比,耐药组织中miR-10a-5p基因启动子的甲基化水平出现显著降低,P=0.04.结合生物信息学预测HOXA1和USF2为miR-10a-5p与耐药相关的靶基因.结论 与敏感组相比,耐药组miR-10a-5p基因启动子甲基化水平显著降低,miR-10a-5p表达升高,通过抑制 HOXA1和USF2,抑制细胞凋亡,导致铂类化疗药物耐受.     

miR-21与消化系统肿瘤

microRNA（miRNA）是一类内源性非编码蛋白短链RNA,参与细胞增殖、分化、凋亡等多种重要活动的调控。近来研究发现miRNA具有癌基因或抑癌基因样作用,参与多种恶性肿瘤的演进,是肿瘤发生、发展过程中的重要分子,其中miR-21最受关注。miR-21在消化系统肿瘤如食管癌、胃癌、肝癌、胆管癌、胰腺癌、结直肠癌等的细胞或组织中表达上调,发挥着类似于癌基因的作用。miR-21通过对靶基因的调控,增强了肿瘤细胞的增殖、浸润及转移能力,并与消化系统肿瘤患者的无复发时间与总生存时间密切相关。目前体外研究证实,miR-21抑制物能够提高恶性肿瘤细胞对化疗药物的敏感性,使化疗药物的杀伤肿瘤效果更加明显,而miR-21类似物,可抵消一部分化疗药物的疗效,从而提示抑制miR-21的表达或阻碍miR-21与靶基因的相互作用在消化系统肿瘤的治疗中具有广阔的应用前景。本文就miR-21在消化系统肿瘤的调控机制、增殖与凋亡、侵袭、耐药与转移中的作用,及其临床价值作一综述。      

低表达miR-33a诱导胰腺癌细胞对吉西他滨的耐药

背景与目的：胰腺癌是一种恶性程度很高的肿瘤。由于其对一线化疗药物吉西他滨的耐受,往往导致预后较差。MicroRNA(miRNA,miR)是一类非编码小RNA,参与肿瘤的多种生物学功能。miR-33a作为代谢相关的miRNA被广泛研究,而与耐药之间关系的报道较少。该研究通过探讨miR-33a参与胰腺癌吉西他滨耐药及其作用解析,为胰腺癌化疗提供新的理论依据。方法：采用原位杂交方法检测胰腺癌组织中miR-33a的表达情况;采用实时荧光定量PCR(Real-time PCR)检测各胰腺癌细胞系中miR-33a的表达情况。利用SW1990和Miapaca-2胰腺癌亲本细胞株,构建吉西他滨耐药细胞株(SW1990^res,Miapaca-2^res)及miR-33a稳定表达细胞株(SW1990-miR-33a,Miapaca-2-miR-33a、SW1990^res-miR-33a和Miapaca-2^res-miR-33a);采用细胞毒性实验检测miR-33a的表达对胰腺癌细胞对吉西他滨敏感性的影响。结果：miR-33a在胰腺癌组织样本中普遍低表达。与HEK293T正常人胚肾细胞相比,其在各胰腺癌细胞系中均呈低表达。miR-33a过表达可以增加胰腺癌细胞对吉西他滨的药物敏感性,能有效逆转胰腺癌细胞对吉西他滨的耐药。结论：miR-33a在胰腺癌组织中低表达,导致胰腺癌患者对吉西他滨获得性耐药。增加miR-33a表达,从而增强了胰腺癌细胞对吉西他滨的药物敏感性,为开发新型胰腺癌分子靶向治疗药物,联合化疗提供新的理论依据。     

miR-214、miR-503在上皮性卵巢癌组织及血清外泌小体中表达的研究

目的 检测正常卵巢、化疗敏感上皮性卵巢癌(EOC)及化疗耐药EOC组织及血清外泌小体中p53相关miR-214、miR-503的表达差异,初探p53相关miRNA与EOC耐药的关系.方法 采用免疫荧光法检测正常卵巢、化疗敏感EOC及化疗耐药EOC组织中p53的表达;RT-PCR法检测血清外泌小体及上述3种组织中p53相关miR-214、miR-503表达的差异.结果 免疫荧光法发现,与正常组织相比,p53在EOC组织中荧光较弱,其中化疗耐药EOC组织与化疗敏感EOC组织间比较差异无统计学意义(P﹥0.05).与敏感组相比,耐药组血清外泌小体中miR-214上调3倍,miR-503下调3倍,与组织中变化一致.结论 p53相关miR-214及miR-503由外泌小体携带进入血液循环,并可能在EOC化疗耐药中发挥重要作用.     

结肠癌组织和癌旁组织miRNA表达谱研究

目的探讨结肠癌癌组织和癌旁组织中miRNA的表达差异,寻找潜在的结肠癌特异表达谱。方法miRNA表达芯片检测手术切除未发生转移的原位结肠癌组织和癌旁组织标本中miRNA表达水平,并采用Real-time PCR对差异表达的miRNA进行验证。结果miRNA表达芯片分析发现肿瘤细胞中24种miRNA表达下调,27种表达上调。miR-145、miR-143在癌组织中表达显著下调,miR-451在癌组织中表达显著上调,并被Real-time PCR方法进一步证实。结论结肠癌癌组织和癌旁组织中miRNA的表达存在差异,miR-145、miR-143在结肠癌组织中表达下调,miR-451表达上调,结肠癌组织具有特异miRNA表达谱,可作为结肠癌潜在诊断芯片进行开发。     

微小RNA与胶质瘤干细胞放化疗敏感性

微小RNA（miRNA）的表达与胶质瘤干细胞对放疗及化疗的敏感程度密切相关。许多研究发现,调节单一miRNA能起到提高胶质瘤干细胞的放化疗敏感性、增加细胞凋亡、多方面综合抑制胶质瘤干细胞生长的效果。胶质瘤的治疗策略有望以miRNA为靶点,通过调节miRNA在细胞中的表达,从而达到提高胶质瘤干细胞放化疗敏感性的目的。     

miR-21过表达与淋巴瘤诊治

微小RNA（microRNA,miRNA）是一类长20~25 nt的非编码RNA,广泛存在真核生物中。miRNA的表达呈现进化保守性、时序性及组织细胞特异性,并参与细胞生长、增殖、分化和凋亡等过程,miRNA的表达与多种肿瘤发病相关,具有肿瘤抑制因子及癌基因的作用。miR-21是在肿瘤中常见的过表达miRNA,在多种肿瘤组织中呈现高表达,其在淋巴瘤中也表达升高,并通过多种方式影响肿瘤抑癌基因及相关信号通路促进淋巴瘤的发生,影响肿瘤的进展及预后。使用转染反义寡核苷酸等分子生物学方法降低miR-21的表达可以抑制肿瘤细胞的生长等生物学的行为并提高肿瘤细胞对化疗的敏感性,深入研究miR-21在淋巴瘤中的作用有望为淋巴瘤的发病机制及治疗等方面提供新的思路。本文对miR-21在淋巴瘤中的表达情况,及其调控因素、作用机制、靶位点及与临床诊断治疗预后的关系进行综述。      

成骨肉瘤细胞SOSP-9607中miRNA的克隆与验证

背景与目的:microRNA(miRNA)是一类非编码蛋白,并参与转录后调节的单链小分子RNA(约20～25个碱基),对基因表达具有重要调控作用,与肿瘤的发生存在着密切的关系.本研究拟克隆成骨肉瘤细胞系SOSP-9607中miRNA,并对部分功能性基因进行验证.方法:以SOSP-9607细胞作为实验对象,分离提取细胞内小片段RNA(≤200 nt),多聚腺苷酸化和5'连接子连接后,通过RT-PCR进行反转录和扩增,克隆到pCR 4-TOPO载体上得到大约109 bp DNA片段,测序后经过生物信息学分析确定miRNA的表达情况.根据当前miRNA的研究现状,在克隆到的miRNA中选取部分功能性miRNA和新发现miRNA,合成相应探针后与从SOSP-9607细胞、HeLa细胞和成骨肉瘤组织中分离出的小片段RNA进行Northern印迹验证.结果:克隆测序后得到182个克隆体,经生物信息学分析发现有47个miRNA(25种),其中含有23种已知的miRNA和2种Nature杂志上预测的miRNA(miR-165和miR-166).选取的三条实体瘤相关的miRNA(miR-21、miR-20a、miR-17-5p)以及两条预测的miRNA进行Northern印迹验证,miR-21、miR-20a、miR-17-5p在SOSP-9607细胞和成骨肉瘤组织中均表达,预测的miR-165和miR-166在SOSP-9607细胞和成骨肉瘤组织中亦均表达,但miR-166在HeLa细胞中没有表达.结论:克隆了SOSP-9607细胞中的miRNA,并验证了部分功能性miRNA的表达,提示miRNA与肿瘤发生可能有密切的关系.     

Differential expression of miR200a-3p and miR21 in grade II–III and grade IV gliomas: Evidence that miR200a-3p is regulated by O6-methylguanine methyltransferase and promotes temozolomide responsiveness

Glioblastoma multiforme (GBM) is the most common primary brain tumor and is among the deadliest of human cancers. Dysregulation of microRNAs (miRNAs) expression is an important step in tumor progression as miRNAs can act as tumor suppressors or oncogenes and may affect cell sensitivity to chemotherapy. Whereas the oncogenic miR21 has been shown to be overexpressed in gliomas, the expression and function of the tumor-supressor miR200a in GBMs remains unknown. In this study, we show that miR21 is upregulated in grade IV (GBMs) vs. grade II–III (LGs) gliomas, confirming that miR21 expression level is correlated with tumor grade, and that it may be considered as a marker of tumor progression. Conversely, miR200a is demonstrated for the first time to be downregulated in GBMs compared with LGs, and overexpression of miR200a in GBM cells is shown to promote TMZ-sensitivity. Interestingly, miR200a but not miR21 expression level is significantly higher in TMZ-responsive vs. -unresponsive tumoral glial cells in primary culture. Furthermore, miR200a appears negatively correlated with the expression of the DNA repair enzyme O⁶-methylguanine methyltransferase (MGMT), and the inhibition of MGMT activity results in an increase of miR200a expression in GBM cells. Taken together, these data strongly suggest that miR200a is likely to act as a crucial antitumoral factor regarding glioma progression. Interplay between miR200a and MGMT should be considered as potential mechanism involved in therapeutic response.     

Plasma microRNAs are promising novel biomarkers for early detection of colorectal cancer

MicroRNA (miRNA) opens up a new field for molecular diagnosis of cancer. However, the role of circulating miRNAs in plasma/serum in cancer diagnosis is not clear. The aim of this study was to investigate whether plasma miRNAs can be used as biomarkers for the early detection of colorectal carcinoma (CRC). We measured the levels of 12 miRNAs (miR‐134, −146a, −17‐3p, −181d, −191, −221, −222, −223, −25, −29a, −320a and −92a) in plasma samples from patients with advanced colorectal neoplasia (carcinomas and advanced adenomas) and healthy controls using real‐time RT‐PCR. We found that plasma miR‐29a and miR‐92a have significant diagnostic value for advanced neoplasia. MiR‐29a yielded an AUC (the areas under the ROC curve) of 0.844 and miR‐92a yielded an AUC of 0.838 in discriminating CRC from controls. More importantly, these 2 miRNAs also could discriminate advanced adenomas from controls and yielded an AUC of 0.769 for miR‐29a and 0.749 for miR‐92a. Combined ROC analyses using these 2 miRNAs revealed an elevated AUC of 0.883 with 83.0% sensitivity and 84.7% specificity in discriminating CRC, and AUC of 0.773 with 73.0% sensitivity and 79.7% specificity in discriminating advanced adenomas. Collectively, these data suggest that plasma miR‐29a and miR‐92a have strong potential as novel noninvasive biomarkers for early detection of CRC.     

Regulation of onco and tumor suppressor MiRNAs by mTORC1 inhibitor PRP-1 in human chondrosarcoma

Metastatic chondrosarcoma of mesenchymal origin is the second most common bone malignancy and does not respond either to chemotherapy or radiation; therefore, the search for new therapies is relevant and urgent. This study aimed to reveal the comparative analysis of miRNAs and their targets in human JJ012 chondrosarcoma cell line between control and experimental samples, treated with mTORC1 inhibitor, cytostatic antiproliferative proline-rich polypeptide (PRP-1). Examination of tumor-specific microRNA expression profiles has revealed widespread deregulation of these molecules in diverse cancers. It was reported that microRNAs can function as novel biomarkers for disease diagnostics and therapy, as well as a novel class of oncogenes and tumor suppressor genes. mTORC 1 inhibitor PRP-1 caused significant upregulation of tumor suppressors, such as miR20a, miR125b, and miR192; and downregulation of onco miRNAs, miR509-3p, miR589, miR490-3p, miR 550 in human chondrosarcoma JJ012 cell line.     

MiRNA expression in urothelial carcinomas: Important roles of miR‐10a,miR‐222, miR‐125b,miR‐7 and miR‐452 for tumor stage and metastasis,and frequent homozygous losses of miR‐31

We analyzed 34 cases of urothelial carcinomas by miRNA, mRNA and genomic profiling. Unsupervised hierarchical clustering using expression information for 300 miRNAs produced 3 major clusters of tumors corresponding to Ta, T1 and T2‐T3 tumors, respectively. A subsequent SAM analysis identified 51 miRNAs that discriminated the 3 pathological subtypes. A score based on the expression levels of the 51 miRNAs, identified muscle invasive tumors with high precision and sensitivity. MiRNAs showing high expression in muscle invasive tumors included miR‐222 and miR‐125b and in Ta tumors miR‐10a. A miRNA signature for FGFR3 mutated cases was also identified with miR‐7 as an important member. MiR‐31, located in 9p21, was found to be homozygously deleted in 3 cases and miR‐452 and miR‐452* were shown to be over expressed in node positive tumors. In addition, these latter miRNAs were shown to be excellent prognostic markers for death by disease as outcome. The presented data shows that pathological subtypes of urothelial carcinoma show distinct miRNA gene expression signatures. © 2008 Wiley‐Liss, Inc.     

Plasma miRNAs as early biomarkers for detecting hepatocellular carcinoma

The early detection of hepatocellular carcinoma (HCC) presents a challenge because of the lack of specific biomarkers. Serum/plasma microRNAs (miRNAs) can discriminate HCC patients from controls. We aimed to identify and evaluate HCC‐associated plasma miRNAs originating from the liver as early biomarkers for detecting HCC. In this multicenter three‐phase study, we first performed screening using both plasma (HCC before and after liver transplantation or liver hepatectomy) and tissue samples (HCC, para‐carcinoma and cirrhotic tissues). Then, we evaluated the diagnostic potential of the miRNAs in two case–control studies (training and validation sets). Finally, we used two prospective cohorts to test the potential of the identified miRNAs for the early detection of HCC. During the screening phase, we identified ten miRNAs, eight of which (miR‐20a‐5p, miR‐25‐3p, miR‐30a‐5p, miR‐92a‐3p, miR‐132‐3p, miR‐185‐5p, miR‐320a and miR‐324‐3p) were significantly overexpressed in the HBV‐positive HCC patients compared with the HBV‐positive cancer‐free controls in both the training and validation sets, with a sensitivity of 0.866 and specificity of 0.646. Furthermore, we assessed the potential for early HCC detection of these eight newly identified miRNAs and three previously reported miRNAs (miR‐192‐5p, miR‐21‐5p and miR‐375) in two prospective cohorts. Our meta‐analysis revealed that four miRNAs (miR‐20a‐5p, miR‐320a, miR‐324‐3p and miR‐375) could be used as preclinical biomarkers (p_meta < 0.05) for HCC. The expression profile of the eight‐miRNA panel can be used to discriminate HCC patients from cancer‐free controls, and the four‐miRNA panel (alone or combined with AFP) could be a blood‐based early detection biomarker for HCC screening.     

Functional screening identifies a microRNA,miR‐491 that induces apoptosis by targeting Bcl‐XL in colorectal cancer cells

MicroRNAs (miRNAs) are a class of small noncoding RNAs that negatively regulate expression of target mRNA. They are involved in many biological processes, including cell proliferation, apoptosis and differentiation, and considered as new therapeutic targets for cancers. In our study, we performed a gain‐of‐function screen using 319 miRNAs to identify those affecting cell proliferation and death in human colorectal cancer cells (DLD‐1). We discovered a number of miRNAs that increased or decreased cell viability in DLD‐1. They included known oncogenic miRNAs such as miR‐372 and miR‐373, and tumor suppressive miRNAs such as miR‐124a, but also some for which this information was novel. Among them, miR‐491 markedly decreased cell viability by inducing apoptosis. We demonstrated that Bcl‐X_L was a direct target of miR‐491, and its silencing contributed to miR‐491‐induced apoptosis. Moreover, treatment of miR‐491 suppressed in vivo tumor growth of DLD‐1 in nude mice. Our study provides a new regulation of Bcl‐X_L by miR‐491 in colorectal cancer cells, and suggests a therapeutic potential of miRNAs for treating colorectal cancer by targeting Bcl‐X_L.     

Branched rolling circle amplification method for measuring serum circulating microRNA levels for early breast cancer detection

Serum circulating microRNAs (c‐miRNAs) are serving as useful biomarkers for cancer diagnosis. Here, we describe the development of a one‐step branched rolling circle amplification (BRCA) method to measure serum c‐miRNAs levels for early diagnosis of breast cancer. Four c‐miRNAs, c‐miRNA16 (c‐miR‐16), c‐miRNA21 (c‐miR‐21), c‐miRNA155 (c‐miR‐155), and c‐miRNA195 (c‐miR‐195) were isolated from the serum of 49 breast cancer patients (stages I‐IV) and 19 healthy controls, and analyzed using one‐step BRCA. The serum levels of c‐miR16, c‐miR21, c‐miR155, and c‐miR195 were higher (P < 0.0001) in stage I breast cancer patients than healthy controls. These levels were also higher in several breast cancer molecular subtypes (HER‐2 over‐expression, Luminal A, Luminal B, and triple negative breast cancer) than in healthy control subjects. The diagnostic accuracy of c‐miR16, c‐miR21, c‐miR155, and c‐miR195 for early diagnosis of breast cancer was confirmed by receiver operating characteristic (ROC) curve assay. These results show that the BRCA method can be used to measure serum c‐miRNAs levels, and that this method has high accuracy, sensitivity, and specificity. Moreover, both BRCA approach and quantitative real‐time PCR (qRT‐PCR) method show that the serum levels of c‐miR16, c‐miR21, c‐miR155, and c‐miR195 could be used as biomarkers to improve the early diagnosis of breast cancer, and distinguish different breast cancer molecular subtypes.     

miR‐210‐3p mediates metabolic adaptation and sustains DNA damage repair of resistant colon cancer cells to treatment with 5‐fluorouracil

Chemoresistance is the primary cause of chemotherapy failure. Compelling evidence shows that micro RNAs (miRNAs) contribute to reprogram cancer cells toward a resistant phenotype. We investigate the role of miRNAs in the response to acute treatment with 5‐FU in colon cancer‐resistant cells. We performed a global gene expression profile for the entire miRNA genome and found a change in the expression of four miRNAs following acute treatment with 5‐FU. Among them, we focused on miR‐210‐3p, previously described as a key regulator of DNA damage repair mechanisms and mitochondrial metabolism. We show that miR‐210‐3p downregulation enables resistant cells to counteract the toxic effect of the drug increasing the expression of RAD‐52 protein, responsible for DNA damage repair. Moreover, miR‐210‐3p downregulation enhances oxidative phosphorylation (OXPHOS), increasing the expression levels of succinate dehydrogenase subunits D, decreasing intracellular succinate levels and inhibiting HIF‐1α expression. Altogether, these adaptations lead to increased cells survival following drug exposure. These evidence suggest that miR‐210‐3p downregulation following 5‐FU sustains DNA damage repair and metabolic adaptation to counteract drug treatment.     

Hepatic miR‐126 is a potential plasma biomarker for detection of hepatitis B virus infected hepatocellular carcinoma

Controversies about the origin of circulating miRNAs have encouraged us to identify organ specific circulating miRNAs as disease biomarkers. To identify liver‐specific miRNAs for hepatocellular carcinoma (HCC), global expression profiling of miRNAs in liver tissue of HBV‐HCC and HBV‐control with no or mild fibrosis was evaluated. A total of 40 differentially expressed miRNAs were identified in HCC. Among ten highly altered miRNAs, six miRNAs were successfully validated in tissues, whereas only two miRNAs, miR‐126 and miR‐142‐3p showed increased expression in plasma of HBV‐HCC compared to HBV‐non‐HCC patients. Subsequently, ROC curve analysis revealed that neither miR‐126 nor miR‐142‐3p performed better than AFP in discriminating HCC from non‐HCC while combination of each with AFP showed significantly higher efficiency rather than AFP alone (AUC: 0.922, 0.908 vs. 0.88; sensitivity: 0.84, 0.86 vs. 0.82 and specificity: 0.92, 0.94 vs. 0.86 respectively). Interestingly, triple combination of markers (miR‐126 + miR‐142‐3p + AFP) showed no additive effect on efficiency (AUC: 0.925) over the dual combination. Again, the expression of only miR‐126 was noticed significantly higher in HBV‐HCC patients with low‐AFP [<250 ng/ml] compared to either non‐HCC or liver cirrhosis (AUC: 0.77, 0.64, respectively). Furthermore, no alteration in expression of mir‐126 in HCV‐HCC or non‐viral‐HCC revealed that miR‐126 + AFP might be specific to HBV‐HCC. To understand the physiological role of these two miRNAs in hepato‐carcinogenesis, target genes related to cancer pathways (APAF1, APC2, CDKN2A, IRS1, CRKL, LIFR, EGR2) were verified. Thus, combination of circulating miR‐126 + AFP is a promising noninvasive diagnostic biomarker for HBV‐HCC and may be useful in the management of HCC patients.