贮存末期血小板miRNA 的表达谱分析  

Expression analysis of miRNA profiles in apheresis platelets at the end of storage

作　　者：谯铭铭 李伟 庄云龙[1] 叶辉[1] 刘群 盖厦 陈元锋[1] 申华[1] 蒋保云 王玉霞[1] QIAO Mingming;LI Wei;ZHUANG Yunlong;YE Hui;LIU Qun;GAI Xia;CHEN Yuanfeng;SHEN Hua;JIANG Baoyun;WANG Yuxia(Blood Center of Shandong Province,Jinan 250014,China;Heze Central Blood Station)

机构地区：[1]山东省血液中心,山东济南250014 [2]菏泽市中心血站

出　　处：《中国输血杂志》2021年第8期821-827,共7页Chinese Journal of Blood Transfusion

基　　金：山东省自然科学基金面上项目(ZR2017MH070);山东省医药卫生科技发展计划项目(2019WS540、2018WSB09004);中国输血协会威高科研基金资助项目(CSBT-WG-2017-03);山东省血液中心项目(201710)。

摘　　要：目的了解在体外常规贮存血小板微小RNA(miRNA)的表达谱变化,探讨miRNA参与调控血小板贮存损伤(PSL)的分子机制。方法留取来自无偿献血者的单采血小板20(人)份(5 mL/份),摇匀后等量混合,贮存于(22±2)℃恒温血小板振荡保存箱中,分别在d1和d5取样,采用纳米球(DNB)测序技术对血小板miRNA组测序;2组(不同贮存期)血小板相比miRNA表达量差异≥2倍(P<0.01)时,采用miRanda和TargetScan软件预测靶基因及基因本体论(GO)功能富集和京都基因与基因组百科全书(KEGG)信号通路富集分析。采用实时荧光定量PCR(qPCR)方法检测miRNA的表达以验证DNB测序结果。结果miRNA表达谱:贮存d5与d1血小板相比,共有315个表达量差异明显的miRNA,其中上调146个(如miR-146a、let-7b等),下调169个(如miR-30d、miR-142等);已知126个miRNA中,表达上调的43个,表达下调83个;新见miRNA序列189条。d5与d1组差异表达miRNA靶基因呈明显富集的GO条目(term)包括细胞组分、细胞器、细胞膜等细胞结构,黏附、催化、活性活性等分子功能,细胞加工、代谢、生物调节、应激等生物过程。相应的KEGG富集前10的通路中主要是信号转导、分泌、膜转运、氨基酸代谢、多糖代谢、蛋白质合成、环境适应等功能。随机挑选的6个差异表达miRNA所做qPCR结果与测序结果均一致。结论随着血小板体外贮存时间的延长,miRNA表达谱发生明显变化;功能预测提示这些miRNA可能参与体外贮存下血小板发生PSL的调控。Objective To investigate the changes of platelet microRNA(miRNA)expression profiles of storage in vitro,and to explore the molecular mechanism of miRNAs involved in the regulation of platelet storage lesion(PSL).Methods 20 platelet samples(5 mL/sample)were collected from apheresis platelet donors,fully mixed and stored in a shaker with(22±2)℃horizontal agitation,sampled on day 1 and day 5,and sequenced by DNA nanoball(DNB)sequencing technology.The miRNAs with more than 2 times expressions(P<0.01)were considered as significantly differences between d5 and d1 groups.The miRanda and TargetScan softwares were used to predict the target genes.Gene Ontology(GO)function enrichment analysis and Kyoto Encyclopedia of genes and genomes(KEGG)pathway enrichment analysis were performed on the target genes of significant differentially expressed miRNAs.The expression of miRNAs was verified by real-time fluorescence quantitative PCR(qRT-PCR).Results Compared with d1 group,315 miRNAs with significantly different expression(P<0.01)were screened in d5 group,including 146 up-regulated miRNAs(such as miR-146a,let-7b),and 169 down-regulated miRNAs(such as mir-30d,mir-142).Among 126 known miRNAs,43 were up-regulated and 83 were down-regulated.There are 189 new miRNA sequences.The enriched GO terms of target genes of differentially expressed miRNAs in d5 and d1 groups included cell components,organelles,cell membrane and other cell structures,molecular functions such as adhesion,catalysis and activity,and biological processes such as cell processing,metabolism,biological regulation and stress.The corresponding pathways in the top 10 of KEGG enrichment were mainly signal transduction,secretion,membrane transport,amino acid metabolism,polysaccharide metabolism,protein synthesis and environmental adaptation.The 6 randomly selected differentially expressed miRNAs verified by qRT-PCR were consistent with those of DNB sequencing.Conclusion The expression profiles of platelets miRNAs have changed significantly between the d1 and d5 of stor

关 键 词：微小RNA 血小板 miRNA表达谱 纳米球测序 功能分析 荧光定量PCR 小RNA 

分 类 号：R457.12[医药卫生—治疗学] R331.143[医药卫生—临床医学]