早期胰腺癌患者血清外泌体差异miRNA筛选及hsa-let-7f-5p的诊断价值  

Screening of differential miRNAs in serum exosomes of early pancreatic cancer patients and the diagnostic value of hsa-let-7f-5p

作　　者：李源 任帅 曹营营 郭凯 杨梦 赵雅桐 张雅平 王中秋 Li Yuan;Ren Shuai;Cao Yingying;Guo Kai;Yang Meng;Zhao Yatong;Zhang Yaping;Wang Zhongqiu(Department of Radiology,Affiliated Hospital of Nanjing University of Chinese Medicine(Jiangsu Province Hospital of Chinese Medicine),Nanjing 21000,China)

机构地区：[1]南京中医药大学附属医院(江苏省中医院)放射科,南京210000

出　　处：《中华胰腺病杂志》2022年第6期432-438,共7页Chinese Journal of Pancreatology

基　　金：国家自然科学基金(82171925、82202135);"江苏省中医院优秀青年博士"资助项目;江苏省中医院高峰学术人才项目(y2021rc03);江苏省中医院创新发展基金面上项目(Y2021CX19)。

摘　　要：目的筛选早期胰腺癌患者血清外泌体差异表达的微小RNA(microRNA,miRNA),并评估外泌体hsa-let-7f-5p对早期胰腺癌的诊断价值。方法选取2019年1月至2020年1月间南京中医药大学附属医院行手术并经病理证实的19例早期胰腺癌患者(早期胰腺癌组)和16例慢性肿块型胰腺炎患者(胰腺炎组),收集两组患者的血清样本,同时选取19名健康志愿者的血清样本作为正常对照组。采用exoEasy Maxi Kit试剂盒分离血清外泌体,透射电子显微镜(TEM)下观察外泌体的结构特征,纳米颗粒追踪分析观察外泌体的粒径大小,蛋白质免疫印迹法检测外泌体表面特异性蛋白标志物CD63、CD81表达。利用miRNeasy Serum/Plasma Kit试剂盒抽提外泌体总RNA,对其进行质检后构建小RNA文库,参照小RNA数据库,对早期胰腺癌组、胰腺炎组及正常对照组差异表达的外泌体miRNA进行筛选。通过实时荧光定量PCR法验证候选外泌体miRNA的表达量,分析各组miRNA的表达量差异。应用基因本体(GO)分析和京都基因与基因组百科全书(KEGG)富集通路分析候选miRNA的靶基因及代谢通路在早期胰腺癌发生与发展中的作用。结果TEM可见外泌体特征性碟形双层囊膜结构,粒径主峰在150 nm左右,外泌体特异性蛋白CD63、CD81表达阳性,对比早期胰腺癌组与胰腺炎组和正常对照组miRNA表达的差异,筛选获得特异性的肿瘤标志物外泌体hsa-let-7f-5p,其在早期胰腺癌组中的表达显著高于胰腺炎组及正常对照组(P值均<0.05)。ROC曲线分析结果显示,外泌体hsa-let-7f-5p鉴别早期胰腺癌组和胰腺炎组的AUC值为0.843(95%CI0.640~1.000),灵敏度和特异度分别为100%和81.82%,鉴别早期胰腺癌组和正常对照组的AUC值为1.000(95%CI1.000~1.000),灵敏度和特异度均为100%,其诊断早期胰腺癌效能与CA19-9相当(P>0.05)。GO分析结果显示,外泌体hsa-let-7f-5p的靶基因主要参与补体激活凝集素途径,表达蛋白主要分�Objective To screen the serum exosomal microRNAs differentially expressed in early pancreatic cancer patients and evaluate the diagnostic value of exosomal hsa-let-7f-5p.Methods From January 2019 to January 2020,19 patients with early pancreatic cancer(early pancreatic cancer group)and 16 patients with chronic mass-forming pancreatitis(pancreatitis group)were selected from Affiliated Hospital of Nanjing University of Chinese Medicine who underwent surgery and were confirmed by pathology.Serum samples of the two groups of patients were collected.At the same time,serum samples of 19 healthy volunteers were selected as the normal control group.The exoEasy Maxi Kit was used to isolate serum exosomes.The structural characteristics of exosomes were observed by transmission electron microscopy(TEM).The particle size of exosomes was observd by nanoparticle tracking analysis.CD63 and CD81,the specific protein marker on the surface of exosomes,were identified by western blotting.The total RNA of exosomes was extracted by the miRNeasy Serum/Plasma Kit,and a small RNA library was constructed after quality inspection.With reference to the small RNA database,the differentially expressed exosomal microRNAs in early pancreatic cancer group,pancreatitis group and normal control group were filtered out.The miRNA candidates were validated by quantitative polymerase chain reaction(qPCR)and different expressions of them were analyzed.The role of target genes and metabolic pathways of candidate miRNAs in the occurrence and development of early pancreatic cancer were analyzed by gene ontology(GO)and Kyoto Encyclopeda of Genes and Genomes(KEGG)enrichment pathway.Results The isolated serum exosomes can be seen to have cup-like vesicle with the double lipid layer by TEM.The main peak of the particle size of target exosomes was about 150 nm.The expression of exosome specific protein markers CD63 and CD81 was positive.Comparing the expression of miRNAs among early pancreatic cancer group,pancreatitis group and normal control group,the speci

关 键 词：胰腺肿瘤 外泌体 微小RNA 糖类抗原19-9 胰腺炎 慢性 

分 类 号：R735.9[医药卫生—肿瘤]