唐氏综合征胎儿21号染色体上新微小RNA基因的鉴定和功能分析

Identification and functional analysis of chromosome 21-derived new microRNA gene in Down syndrome fetuses

目的:利用Solexa高通量深度测序技术鉴定21号染色体上新的微小RNA(miRNA)基因,为阐明21号染色体功能、探索唐氏综合征(DS)患者新的治疗靶点提供依据。方法:收集5例经染色体核型分析诊断为DS胎儿的脐带血和3例正常胎儿脐带血,分析染色体核型;提取胎儿脐带血单个核细胞总RNA,构建小RNA文库,采用Solexa高通量深度测序、计算机分析、Stem-loop RT-PCR法鉴定miRNA基因并分析其生物学功能。结果:血染色体核型分析,5例DS胎儿脐带血显示DS标准核型,3例正常胎儿脐带血显示正常核型;在DS胎儿脐带血中共发现21个新miRNA,其中仅1个候选miRNA来源于21号染色体,位于21号染色体的"DS关键区域",命名为"miR-nov21",后者在DS胎儿脐带血单个核细胞中表达水平高于正常胎儿。生物学功能分析,miR-nov21可调节211个靶基因共215个位点,与基因的表达、细胞分化的调节、胚胎形态的发生和心脏的发育有关。结论:21号染色体"DS关键区域"存在1个新的miRNA基因———miR-nov21。

Objective To identify the novel microRNA（miRNA） gene located at the chromosome 21 by Solexa high-throughput sequencing technology,and to provide basis for clarifying function of the chromosome 21 and investingating the new therapeutic target of Down syndrome（DS） patients.Methods The cord blood of 5 DS fetues verified by chromosome karyotype analysis and 3 normal fetuses were collected.Total RNA was extracted from mononuclear cells of cord blood in DS fetuses to construct the small RNA library.Then the miRNA gene was identified by Solexa high-throughput sequencing,computer analysis and Stem-loop RT-PCR,and its biological function was analyzed.Results The chromosome karyotype analysis showed that the karyetypes in cord blood of DS fetuses were standard DS karyotypes,and those in normal fetuses were normal karyotypes.A total of 21 novel miRNAs were identified in cord blood of DS fetuses.But only one candidate miRNA was located on the ＂DS critical region＂ of the chromosome 21,named as ＂miR-nov21＂,and its expression level in mononuclear cells of cord bood in DS fetuses was higher than that in normal fetuses.Biological funcation analysis revealed that miR-nov21 could regulate 211 conserved target genes containing 215 conserved sites,which were involved in gene expression,regulation of cell differentiation,embryonic morphogenesis,and heart development.Conclusion There is a novel miRNA gene in ＂DS critical region＂ of the chromosome 21.