核黄素光化学处理下贮存末期血小板miRNA的表达谱分析  

Expression analysis of miRNA profiles in apheresis platelets at the end of storage under riboflavin and ultraviolet-Blight treatment

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

机构地区：[1]山东省血液中心,山东济南250014

出　　处：《中国输血杂志》2021年第9期961-966,共6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：目的分析核黄素光化学技术(VB2-PRT)处理下贮存d1和d5血小板微小RNA(miRNA)表达谱的变化,探讨贮存末期VB2-PRT处理miRNA参与调控血小板发生贮存损伤(PSL)的分子机制。方法留取无偿献血者单采血小板20人份(5 mL/份),混合摇匀,使用终浓度为50μmol/L VB2和6.24 J/mL紫外线(UV)B光照处理8 min后,均分为2等份,贮存于(22±2)℃恒温血小板振荡保存箱中,分别在d1和d5取样(5 mL),采用纳米球(DNB)测序技术对血小板miRNA组测序,采用DEGseq和MA-plot分析软件筛选2组(不同贮存期)差异表达的小RNA,当2组间的血小板miRNAs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:VB2-PRT处理后贮存d5与d1血小板相比,共有590个表达量差异明显的miRNAs,其中上调255个(如miR-99b、miR-7等),下调335个(如miR-451a、miR-19b等);已知272个miRNAs中,表达上调的112个,表达下调160个;新见miRNAs序列318条。d5与d1组差异表达miRNAs靶基因呈明显富集的GO条目(term)包括细胞组分、细胞器、细胞膜等细胞成分,涉及黏附、催化、分子转换、运输、转录因子和受体活性等分子功能,涉及细胞加工、代谢、生物调节、应激等生物过程。相应的KEGG富集前10的通路中主要是分泌、糖代谢、信号转导、膜转运、翻译、环境适应等信号通路。随机挑选的6个差异表达miRNAs所做荧光定量PCR结果与测序结果均一致。结论VB2-PRT处理下贮存d5血小板miRNA表达谱较贮存d1时发生明显变化,功能预测提示这些miRNAs可能参与VB2-PRT处理下血小板发生PSL的调控。Objective To investigate the changes of platelet microRNA(miRNA)expression profiles on d1 and d5 during storage with riboflavin and ultraviolet-B(UVB)light(VB2-PRT)treatment,and to explore the molecular mechanism of miRNAs involved in the regulation of platelet storage lesion(PSL)under VB2-PRT treatment.Methods 20 apheresis platelet concentrates(5 mL/sample)were collected from voluntary blood donors.After mixing and shaking,the samples was treated with riboflavin(final concentration 50μmol/L)and 6.24 J/mL UVB light for 8 min,then split into two aliquots and agitated stored at(22±2)℃.The concentrates were sampled(5 mL)on d1 and d5,respectively,and sequenced by DNA nanoball(DNB)sequencing technology.The differentially expressed miRNAs between the two groups(at different storage periods)were screened by DEGseq and MA-plot analysis software.The miRNAs,reached more than 2 times different expression between groups,were considered significant different(P<0.01).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 miRNA expression profile:compared with d1 platelets,there were 590 miRNAs with significantly different expression(P<0.01)in d5 group,including 255 up-regulated miRNAs(such as miR-99 b,miR-7)and 335 down regulated miRNAs(such as miR-451 a,miR-19 b).Among the 272 known miRNAs,112 were up-regulated and 160 were down regulated.There were 318 new miRNAs sequences.The enriched GO terms of target genes of differentially expressed miRNAs in d5 and d1 groups included cell components,organelles,cell membranes and other cellular structures,molecular functions such as adhesion,catalysis,molecular conversion,transportation,transcription factor and receptor activity,and biological processes such as cell

关 键 词：微小RNA 血小板 miRNA表达谱 核黄素光化学技术 血小板贮存损伤 纳米球测序 靶基因功能富集分析 靶基因信号通路富集分析 

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