手足口病毒诱导肠上皮细胞差异表达miRNA的筛选及其在患儿粪便标本中的诊断效能分析  

Screening of differentially expressed miRNAs induced by hand-foot-mouth disease virus in intestinal epithelial cells and its diagnostic efficacy in feces specimens of children

作　　者：熊英[1] 唐晓磊 朱芮[1] XIONG Ying;TANG Xiaolei;ZHU Rui(Zhongnan Hospital of Wuhan University,Wuhan 430071,China;不详)

机构地区：[1]武汉大学中南医院,武汉430071 [2]皖南医学院第二附属医院

出　　处：《山东医药》2020年第35期1-5,共5页Shandong Medical Journal

基　　金：国家自然科学基金资助项目(81601806);湖北省自然科学基金资助项目(2017CFB701)。

摘　　要：目的筛选两种常见手足口病毒肠病毒71型(EV71)和柯萨奇病毒A16型(CA16)感染诱导人肠上皮细胞系(HIEC)差异表达的miRNA,并分析其在手足口病患儿粪便标本中的诊断价值。方法将EV71和CA16分别与HIEC共培养,感染复数为10,培养0、24、48、96 h后,行miRNA微阵列芯片分析,筛选差异表达miRNA。将HIEC分为观察组和对照组,观察组感染临床手足口病患儿分离病毒株46株,对照组不感染病毒株。培养72 h,采用实时荧光定量PCR法检测芯片筛选出的差异表达miRNA相对表达量,选择变化最大的前4种miRNA作为粪便miRNA联合检测法的靶标。选择手足口病患儿40例和健康体检儿童52例,比较粪便miRNA联合检测法、血清IgM抗体检测法、咽拭子病毒核酸检测法的诊断效能。结果miRNA微阵列芯片检测结果显示,HIEC感染EV71、CA16后miR-526a、miR-23b、miR-619-5p、miR-204-5p、miR-5585-3p和miR-146a表达明显升高且相对升高倍数均>2。观察组较对照组升高幅度最大的前4个miRNA分别为miR-23b、miR-619-5p、miR-204-5p、miR-5585-3p(P均<0.01)。粪便miRNA联合检测法对手足口病的阳性检出率为87.5%、灵敏度为90.91%、特异度为84.09%、准确性为92.30%,血清IgM抗体检测法对手足口病的阳性检出率为37.5%、灵敏度为63.64%、特异度为85.71%、准确性为70.60%,咽拭子病毒核酸检测法对手足口病的阳性检出率为90.0%、灵敏度为88.64%、特异度为92.86%、准确性为95.65%。结论EV71和CA16诱导HIEC表达明显升高的miRNA分别为miR-23b、miR-619-5p、miR-204-5p、miR-5585-3p,以其作为靶标进行粪便miRNA联合检测对手足口病的诊断价值较高。Objective To screen two common hand-foot-mouth disease(HFMD)viruses Enterovirus 71(EV71)and Coxsackie virus A16(CA16)-induced differentially expressed miRNAs in human intestinal epithelial cells(HIEC),and to analyze their diagnosis value for HFMD infection in children′s feces samples.Methods The two kinds of major viral pathogen(EV71 and CA16)were incubated and cultured with HIEC(MOI is 10 to 1).The total RNA was extracted from HIEC at 0,24,48 and 96 h.The miRNAs of six groups were detected by miRNA microarray chips and the differentially expressed miRNAs were analyzed for verification experiments.The real-time PCR(rt-PCR)was used to detect the differentially expressed miRNAs from infected HIEC by viral pathogen strains,and then we selected the top 4 miRNAs with the most changes as the targets of the fecal miRNA joint detection.We selected 40 children with HFMD and 52 children with healthy physical examination,and compared the diagnostic efficacy of fecal miRNA combined detection,serum IgM antibody detection and virus nucleic acid test of pharyngeal swab.Results The miRNAs microarray detection data showed the expression levels of miR-526a,miR-23b,miR-619-5p,miR-204-5p,miR-5585-3p and miR-146a increased significantly,with the increase multiple of more than 2.The first four miRNAs with the highest increase in the observation group compared with the control group were miR-5585-3p,miR-23b,miR-619-5p,and miR-204-5p(all P<0.01).The fecal miRNA combined detection method had a positive detection rate of 87.5%for HFMD,sensitivity of 90.91%,specificity of 84.09%,and accuracy of 92.3%;the serum IgM antibody detection had a positive detection rate of 37.5%,sensitivity of 63.64%,specificity of 85.71%,and accuracy of 70.6%;and the virus nucleic acid test of pharyngeal swab had a positive detection rate of 90%,sensitivity of 88.64%,specificity of 92.86%,and accuracy of 95.65%.Conclusion The miRNAs with significantly increased expression in HIEC induced by EV71 and CA16 are miR-5585-3p,miR-23b,miR-619-5p,and miR-204-5p,respect

关 键 词：手足口病 微小核糖核酸 粪便 血清IgM抗体检测法 咽拭子病毒核酸检测法 诊断 

分 类 号：R725.1[医药卫生—儿科]