高通量测序技术检测新生儿坏死性小肠结肠炎患儿微小RNA的差异表达  

Differential expression of microRNA in neonatal neerotizing enteroeolitis based on high-throughput sequencing

作　　者：杨丽[1] 周伟[1] 尧杰 袁伟明[1] 荣箫[1] 李雁彬[1] 唐娟[1] Yang Li Zhou Wei Yao Jie Yuan Weiming Rong Xiao Li Yanbin Tang Juan(Department of Neonatology, Guangzhou Women and Children＇s Medical Center, Guangzhou Medical University, Guangzhou 510120, China)

机构地区：[1]广州医科大学附属广州市妇女儿童医疗中心新生儿科,510120

出　　处：《中华围产医学杂志》2017年第1期31-37,共7页Chinese Journal of Perinatal Medicine

基　　金：广州市科技计划项目（201510010217）

摘　　要：目的探讨新生儿坏死性小肠结肠炎（necrotizing enterocolitis，NEC）患儿微小RNA（microRNA，miRNA）的差异表达情况及其意义。方法收集2014年10月至2015年11月在广州市妇女儿童医疗中心确诊为Bell分期Ⅱ期及Ⅱ期以上的NEC患儿25例，同期非NEC新生儿25例作为对照组。取外周血提取白细胞。随机选取对照组与NEC组各5例的标本，用Illumina二代高通量测序技术进行测序，筛选出有差异的miRNA，并进行靶基因预测及生物学功能分析。其余标本采用实时荧光定量一聚合酶链反应技术检测差异表达的miRNA的相对表达量，验证上述高通量测序的结果。应用DEGseq软件对2组数据的miRNA进行差异表达分析统计。P〈0．05为差异有统计学意义。以P〈0．01，g〈0．001，1Log2Ratiol≥1作为差异表达的筛选标准。采用MeV4．6软件对NEC组与对照组差异表达的miRNA进行聚类分析。结果NEC组与对照组有482个miRNA表达差异有统计学意义（P〈0．05），其中126个为已知显著差异表达的miRNA，其中上调58个，下调68个。表达上调的hsa—miR-223—5p、hsa—miR-183—3P和hsa—miR-2225p，以及下调的hsa—miR-23b5p、hsa—miR150—5p、hsa—miR-146a-3p和hsa—miR—1298—5p的验证结果与测序结果相符。生物信息学分析发现，在NEC患儿中有显著差异的miRNA的靶基因参与Toll样受体信号转导通路、丝裂原活化蛋白激酶通路、JAK—STAT等信号转导通路的调节。结论NEC患儿外周血白细胞miRNA表达存在显著差异，这些miRNA可能通过调节不同靶基因来调控相关信号通路参与NEC的发生和发展。Objective To analyze the differential expression of microRNA （miRNA） and its significance in patients with neonatal necrotizing enterocolitis （NEC）. Methods Twenty-five patients diagnosed with NEC with Bell stage ≥ Ⅱ , and 25 non-NEC patients as control group admitted to Guangzhou Women and Children＇s Medical Center between October 2014 and November 2015 were collected. White blood cells were extracted from the peripheral blood. Five samples were selected randomly each from NEC group and control group, and sequenced by second-generation Illumina high-throughput sequencing, screened for differentially expressed miRNA and analyzed for target genes prediction and biological function. The rest samples of the two groups were detected by real-time fluorescent quantitative polymerase chain reaction technology （RT-qPCR）, the results were used to validate the results of high-throughput sequencing. Differentially expressed miRNA in the two groups of data was analyzed using DEGseq software. P〈0.05 was considered statistieally significant. P〈0.01, q〈0.001 and ｜ Logs Ratio ｜ ≥ 1 were taken as criteria for screening the differential expression. The differential expressions of miRNA in NEC group and control group were analyzed by cluster analysis using MeV4.6 software. Results A total of 482 miRNAs were differentially expressed in the two groups, with significant difference （P〈0,05）. Among them, 126 were known miRNAs with significantly differential expression in the two groups, with 58 being up-regulated and 68 being down-regulated. The results of up-regulated miRNAs （hsa-miR-223-5p, -183-3p, -222-5p） and down-regulated miRNAs （hsamiR-23b-5p, -150-5p, -146a-3p, -1298-5p） were confirmed to be consistent with the results of sequencing. Bioinformatics analysis showed that the target genes with differential miRNA expression mainly involved Tolllike receptor signal transduction pathway, mitogen-activated protein kinase pathway, JAK-STAT and other signal transduction pathways. Conclusions T

关 键 词：小肠结肠炎 坏死性 微RNAS 高通量核苷酸序列分析 婴儿 新生 

分 类 号：R722.1[医药卫生—儿科]