血浆外泌体miR-214-3p作为葡萄膜黑色素瘤诊断及预后评估生物标志物的研究

Evaluation of plasma exosomal miR-214-3p as a potential biomarker for diagnosis and prognosis of uveal melanoma

目的探讨微小RNA(miR)-214-3p在不同类型葡萄膜黑色素瘤(UM)患者血浆外泌体中的差异性表达,评估其是否可作为UM诊疗的新型分子生物标志物。方法收集2015年12月至2019年10月于北京同仁医院行眼球摘除术并确诊为UM的患者25例,其中原位梭形细胞型UM组和原位类上皮细胞型UM组各10例,转移型UM组5例(包括1例梭形细胞型UM患者和4例类上皮细胞型UM患者);同期收集10例健康对照者作为健康对照组,抽取所有受试者血液样本,提取血浆外泌体,透射电子显微镜下观察外泌体形态,分析外泌体粒径,Western blot法鉴定外泌体标记蛋白,采用实时荧光定量PCR法测定各组患者血浆外泌体miR-214-3p表达水平。采用差异性检验比较UM患者与健康对照者和不同类型UM患者间的血浆外泌体miR-214-3p的差异表达;通过受试者工作特征(ROC)曲线评价血浆外泌体miR-214-3p对UM的诊断及分型效能。结果透射电子显微镜下可见,所提取外泌体呈一侧凹陷的半球形结构,直径约100 nm。囊泡粒径大小为(82.0±2.7)nm。Western blot结果显示外泌体特异性标记蛋白TSG101条带均为阳性,阴性标记蛋白Calnexin均呈阴性。健康对照组、原位UM组和转移型UM组血浆外泌体miR-214-3p相对表达量分别为0.86(0.57,1.49)、0.24(0.10,0.67)和0.43(0.23,0.56),原位UM组血浆外泌体miR-214-3p相对表达量较健康对照组明显降低,差异有统计学意义(Z=2.62,P<0.01);ROC曲线对血浆外泌体中miR-214-3p的诊断效能分析显示,血浆外泌体miR-214-3p的AUC为0.795。转移型UM组血浆外泌体miR-214-3p相对表达量较健康对照组明显降低,差异有统计学意义(Z=2.08,P<0.05);原位梭形细胞型UM组和原位类上皮细胞型UM组血浆外泌体miR-214-3p相对表达量分别为0.11(0.07,0.64)和0.46(0.14,0.91),2个组间以及转移型UM组与原位类上皮型UM组间患者血浆外泌体miR-214-3p相对表达量比较差异均无统计学意义(均P>0.05)。结论血浆外泌体miR-214-3p在原位UM患者及转移型UM患者中均显著下降,循环miR-214-3p具有作为UM诊断生物标志物的潜力,但血浆外泌体miR-214-3p缺乏对UM分型的评估能力。

Objective To investigate the differential expression of microRNA(miR)-214-3p in plasma exosomes in different types of uveal melanoma(UM)and evaluate whether miR-214-3p is a potential molecular biomarker for the diagnosis and prognosis of UM.Methods Twenty-five UM in situ patients who received the enucleation of eyeball were enrolled at Beijing Tongren Hospital from December 2015 to October 2019,including 10 with epithelioid cell melanoma and 10 with spindle cell melanoma as well as 5 metastatic UM patients(1 with spindle cell-like melanoma and 4 with epithelioid cell-like melanoma)and 10 healthy subjects were enrolled during the same period.Blood sample was collected from all the subjects for the isolation of plasma exosomes.The morphology of exosomes was examined under the electron microscope.The exsomal marker proteins were identified by Western blot.The expression level of miR-214-3p in plasma exosomes was detected by real-time fluorescence quantitative PCR.The differential expression of miR-214-3p among different types of UM patients and healthy controls was compared.The diagnostic and classification performance of exosomal miR-214-3p was evaluated using receiver operating characteristic curve.Written informed consent was obtained from each subject prior to entering the study cohort.This study protocol was approved by an Ethics Committee of Capital Medical Univeristy(No.TRECKY2018-056).Results The isolated exosomes were hemispherical in shape with a concavity on one side.The diameter of the exosomes was about 100 nm and the particle diameter of vesicles from samples was(82.0±2.7)nm.TSG101 protein was detectable and Calnexin protein was not found in the exosomes.The relative expression levels of plasma exosomal miR-214-3p in healthy control group,in situ UM group and metastatic UM group were 0.86(0.57,1.49),0.24(0.10,0.67),and 0.43(0.23,0.56),respectively.The miR-214-3p relative expression level in plasma exosomes of in situ UM patients and metastatic UM patients was significantly lower than that of healthy controls,and the differences were statistically significant(Z=2.62,P<0.01;Z=2.08,P<0.05).The relative expression levels of exosomal miR-214-3p in spindle cell-like UM group and epithelioid cell-like UM group were 0.11(0.07,0.64)and 0.46(0.14,0.91),respectively,and no significant difference was found in the expression level of plasma exosomal miR-214-3p among different types of UM(all at P>0.05).The area under the curve of plasma exosomal miR-214-3p for UM diagnosis was 0.795.Conclusions Plasma exosomal miR-214-3p level is significantly reduced in both in situ UM patients and metastatic UM patients.Plasma exosomal miR-214-3p is a new potential diagnostic biomarker for UM,but the exosomal miR-214-3p appears to not be able to distinguish the types of UM.