不同病理类型的葡萄膜黑色素瘤中微小RNA差异表达谱分析

背景 目前研究证实微小RNA(miRNA)参与大多数人类肿瘤疾病的发生和发展,其作用类似于抑癌基因或癌基因.葡萄膜黑色素瘤(UM)是成人常见的眼部恶性肿瘤,其发生和转移机制仍未完全阐明.探讨UM组织中miRNA的差异表达情况有望为UM的靶向治疗提供依据. 目的 筛选不同病理类型的UM组织中特异性miRNA表达谱. 方法 收集于2013年3月至2015年10月在北京同仁医院手术局部切除并经常规组织病理学和免疫组织化学检测证实为梭型细胞型UM的标本4例和上皮细胞型UM标本4例,采用miRNA芯片分别检测2种UM组织中miRNA的表达,收集同期死于非肿瘤疾病的8个供体眼的正常葡萄膜组织作为对照,利用组间差异倍数筛选出差异≥2倍差异表达的miRNA;用在线软件预测差异表达miRNA的靶基因,采用生物信息学方法分析靶基因参与的信号功能通路.采用实时定量PCR法验证芯片检测结果.结果 收集的梭形细胞型和上皮细胞型UM标本经组织病理学检查均得到确诊,免疫组织化学检测梭形细胞型及上皮细胞型UM组织中HMB45、黑色素-A和S-100均呈阳性反应.与正常葡萄膜组织比较,在梭形细胞型UM组织中差异表达的miRNA有109个,其中29个上调,80个下调,上调的miRNA包括miR-146a-5p、miR-25-3p和miR-29b-1-5p,下调的miRNA包括miR-126-5 p、miR-183-5p和miR-96-5p;上皮细胞型UM中差异表达的miRNA有50个,其中23个上调,27个下调,上调的miRNA包括miR-155-5p、miR-210和miR-378 a-5p;下调的miRNA包括miR-199a-5p、miR-143-3p和miR-143-5p.在梭形细胞型和上皮细胞型UM组织中共同上调的miRNA为miR-132-3p、miR-21-5p、miR-34a-5p和miR-34b-5p,共同下调的miRNA为miR-125b-2-3p、miR-126-3p、miR-199a-3p和miR-214-3p.梭形细胞型和上皮细胞型UM组织中差异表达的miRNA所预测的靶基因分别参与癌症通路、丝裂原活化蛋白激酶(MAPK)信号通路、Wnt信号通路、细胞间黏附、胞吞作用、前列腺癌通路、结直肠癌通路和细胞黏附通路.结论 与正常葡萄膜组织相比,梭形细胞型UM和上皮细胞型UM组织中存在多种miRNA的差异表达,梭形细胞型UM和上皮细胞型UM组织之间也存在明显的miRNA差异表达,这些差异表达的miRNA可通过不同的信号转导通路参与调控UM的生物学行为.

Differential expression profile of microRNAs in different types of uveal melanoma

Background Researches showed that microRNA (miRNA) is involved in the pathogenesis and development of many tumors and plays a cancer-suppressing-gene like role or cancer-gene like action.Uveal melanoma (UM) is a common ocular malignant tumor in aduh,and the mechanism of UM pathogenesis and metastasis is still not elucidated.Understanding the differential expression of miRNAs in UM is expected to provide a basis for targeting treatment of UM.Objective This study was to screen and compare the expression profiles of miRNAs in epithelial type and spindle type of UM.Methods The use of specimens of UM and donor eyes was approved by Ethic Commission of Capital Medical University.The specimens of epithelial type (4 specimens) and spindle type (4 specimens) of UM confirmed by histopathology and immunochemistry were collected in Beijing Tongren Hospital from March 2013 to October 2015.The expression profile of miRNA was assayed by miRNA array.Normal uveal specimens were obtained from 8 donors as controls.The differentially expressing miRNAs were screened by intergroup differential folds of ≥2.The genes targeting differentially expressed miRNAs were predicted using multiples online software and the potential signal pathway was further analyzed by bioinformatics method.The microarray outcomes were validated by real-time quantitative PCR.Results Spindle cell type and epithelial cell type of UMs were verified by hematoxylin and eosin staining.Immunochemistry showed that HMB45,melanin-A and S-100 were positively expressed in the two types of UM.Compared with the normal uveal tissue,109 differentially expressed miRNAs,including 29 up-regulated and 80 down-regulated miRNAs were seen in the spindle cell type of UM,and in the epithelial cell type of UM,50 differentially expressed miRNAs were found,including 23 up-regulated and 27 down-regulated miRNAs.In spindle cell type of UM,the up-regulated miRNAs were miR-146a-5p,miR-25-3p and miR-29b-l-5p,and down-regulated ones were miR-126-5p,miR-183-5p and miR-96-5p.In epithelial cell type of UM,the up-regulated miRNAs were miR-155-5p,miR-210 and miR-378a-5p,and down-regulated ones were miR-199a-5p,miR-143-3p and miR-143-5p.In addition,the mutual up-regulated miRNA in both spindle cell type of UM and epithelial cell type of UM were miR-132-3p,miR-21-5p,miR-34a-5p and miR-34b-5p,and mutual down-regulated ones were miR-125b-2-3p,miR-126-3p,miR-199a-3p and miR-214-3p.Bioinformatics analysis showed that the targeting genes predicted by differentially expressed miRNAs participated in a number of biological pathways,including cancer-related pathway,mitogenactivated protein kinase (MAPK) pathway,Wnt signal pathway and intercellular adhesion,endocytosis,prostatic cancer,colorectal cancer pathways.Conclusions Many differentially expressed miRNAs exist among spindle cell type of UM,epithelial cell type of UM and normal uveal tissue.These miRNAs participate in or regulate the biological behaviour of UM via different signal pathways.