目的 检测唐氏综合征(DS)患儿与健康儿童外周血单个核细胞miRNA表达的差异,探索DS患儿21号3染色体扰乱2倍体基因表达和免疫缺陷的形成机制.方法 收集6例标准核型DS患儿(DS组)和6例健康儿童(non-DS组)外周血标本,分别提取各组样本外周血单个核细胞总RNA等量混合,构建DS组和non-DS组small RNA文库,采用Illumina测序技术对两组样本外周血单个核细胞miRNA的表达水平进行差异分析,同时对差异表达miRNA调控靶基因进行生物信息学功能分析,并用Stem-loop qRT-PCR对Illumina测序结果 和差异表达miRNA的调控靶基因进行验证.结果两样本总共表达miRNA有911种,其中只有114种miRNA显著性差异表达(差异倍数>2,P<0.001),其中有11种在DS组中低表达,103种高表达,包括编码于21号染色体的5个miRNA(miR-99a、let-7c、miR-125b、miR-155、miR-802).生物学功能分析显示高丰度差异表达miRNA调控靶基因与造血和淋巴器官的发育、胸腺的发育、T/B细胞的分化和活化有关.结论 DS患儿21号3染色体对2倍体miRNA表达丰度的影响较小,21号染色体编码miRNA也未均呈现1.5倍以上的升高,高丰度差异表达的miRNA可能与DS患儿免疫系统的发育缺陷以及DS患儿循环T、B淋巴数量的异常有关.%Objective To detect the differentially expressed miRNA in peripheral blood mononuclear cells be-tween Down syndrome ( DS ) and non -DS children , and thus to uncover the molecular mechanisms in DS children . Methods The peripheral blood of 6 DS children with standard DS karyotypes (DS group) and 6 non-DS children (non-DS group) were collected.Total RNA was extracted from peripheral blood mononuclear cells .Two small RNA libraries were constructed using pooled RNA from DS and non -DS children, respectively.Illumina deep sequencing technology was conducted to evaluate the miRNA expression .The target genes of differentially expressed miRNAs were annotated by Gene Ontology using DAVID .Furthermore, Stem-loop qRT-PCR was performed to verify the results of Illumina deep sequencing and some target genes of differentially expressed miRNAs .Results A total of 911 miRNAs were identified and most of them were expressed at the similar levels in two groups .Of these identified miRNAs , 114 miRNAs were iden-tified to be differentially expressed with a fold changes >2.0 and P-value <0.001.Among these differentially expressed miRNAs, 11 miRNAs were down-regulated and 103 miRNAs were up-regulated in DS children including the five miR-NAs (miR-99a, let-7c, miR-125b, miR-155 and miR-802) encoded by chromosome 21.Function annotation of target genes indicated that a set of highly abundantly and significantly differentially expressed miRNAs involved in hemato -poietic or lymphoid organ development , thymus development , T/B cell differentiation and activation .Conclusion The chromosome 21 contributes to the abundantly expressed miRNAs is small , and not all Hsa 21-derived miRNAs are over-expressed with ratios significantly ≥1.5 in DS peripheral blood mononuclear cells .The abundantly differentially expressed miRNAs might be associated with the immune deficiency and the abnormally number of circulatory T /B lymphocytes in DS children.
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