室间隔缺损胎儿心室肌组织微小RNAs时序性表达差异研究

目的采用微小RNAs（miRNAs）表达谱芯片分析不同孕龄（孕早期与孕中期）室间隔缺损（VSD）胎儿心室肌组织中时序性表达差异的miRNAs。方法连续性纳入2009年7～12月南京医科大学附属南京妇幼保健院因病理因素流产经解剖证实为VSD且不合并其他畸形的胎儿为VSD组。以同期因生理性难免流产,并经解剖证实无心脏畸形和其他器官畸形的胎儿为对照组,依据孕龄与VSD组1：1匹配。根据孕龄,将VSD组和对照组分别分为孕早期亚组和孕中期亚组。采用Agilent Human2.0 miRNAs表达谱芯片观察胎儿心室肌组织miRNAs表达变化,芯片数据采用生物信息学方法进行分析,包括差异miRNAs筛选,预测miRNAs靶基因Gene Ontology分析,靶基因信号通路分析,并采用实时PCR法验证芯片结果。结果 VSD组和对照组各纳入6例,两组孕早期亚组和孕中期亚组均各3例。①通过差异miRNAs筛选,发现孕早期VSD亚组与孕中期VSD亚组间有33个时序性表达差异的miRNAs。19个miRNAs在孕早期VSD亚组表达上调,在孕中期VSD亚组表达下调;14个miRNAs在孕早期VSD亚组表达下调,在孕中期VSD亚组表达上调。②生物信息学预测到2761个靶基因,大部分miRNAs的靶基因中含有与心脏发育直接相关的关键基因（TBX5、GATA4、TBX1和NKX2-5等）。③靶基因GeneOntology分析表明其中与细胞进程、代谢过程和生物调控相关的靶基因分别占整个靶基因数量的23.5%、18.3%和17.7%。④靶基因信号通路分析发现,WNT信号通路中的靶基因在孕早期VSD亚组与孕中期VSD亚组中存在时序性差异。⑤随机挑选孕早期VSD亚组与孕中期VSD亚组时序性表达差异的4个miRNAs（hsa-miR-19a、hsa-let-7e、hsa-miR-134和hsa-miR-206）进行验证,定量PCR结果显示,孕早期VSD亚组分别上调3.2（hsa-miR-19a）和4.1倍（hsa-let-7e）,下调5.3（hsa-miR-134）和4.4倍（hsa-miR-206）;孕中期VSD亚组分别下调4.8（hsa-miR-19a）和3.4倍（hsa-let-7e）,上调4.5（hsa-miR-134）和3.9倍（hsa-miR-206）。结论时序性表达差异的miRNAs在胎儿VSD畸形的发生中可能起着重要作用,但本研究样本量较小,需要进一步扩大样本量进行验证。这些差异表达miRNAs的预测靶基因与细胞发育、分化和代谢密切相关,含有与心脏发育直接相关的关键基因,部分靶基因为WNT信号通路中的关键因子,提示VSD的发生发展是机体在miRNAs的参与下,在多个层面上使基因表达失控的共同结果。

Differential time-specific expression of miRNAs in ventricular myocardium of fetus with ventricular septal defect

Objective MiRNAs microarray was employed to determine the differential time-specific expression of miRNAs in ventricular myocardium of fetus with ventricular septal defect（VSD） in the different gestational ages（the first trimester and the second trimester）.Methods The VSD group was recruited from the fetuses with VSD aborted for pathological factors,and was divided into VSD subgroups：subgroup 1 for the first trimester and subgroup 2 for the second trimester according to the gestational ages.The control group was chosen from the fetuses of inevitable abortion without cardiac malformations,and was divided into the control subgroups in the same way.The fetal ventricular myocardiums were acquired and RNAs were extracted and processed.The Agilent Human 2.0 microarrays containing 723 known miRNAs were applied to get the data about miRNAs expression changes,the data were analyzed by bioinformatics methods：differential miRNAs expression analysis,target gene ontology analysis and pathway-express analysis.Differential expressions of miRNAs microarray were certificated by real-time PCR.Results VSD group and control group had 6 fetuses,respectively,with 3 fetuses in the first trimester and 3 in the second trimester,respectively.①There were 33 differential time-specific expression of miRNAs in the 2 VSD subgroups.Expressions of 19 miRNAs were up-regulated in subgroup 1 and down-regulated in subgroup 2.Expressions of 14 miRNAs were up-regulated in subgroup 1 and down-regulated in subgroup 2.②A total of 2 761 target genes were predicted by bioinformatic methods.The most of target genes were directly associated with the key genes of heart development（such as TBX5,GATA4,TBX1,NKX2-5,etc.）.③The gene ontology analysis indicated that target genes were mainly related to the cellular process,metabolic process and biological regulation,and accounted for 23.5 %,18.3%,17.7% of the total number of target genes,respectively.④The pathway-express analysis indicated that the WNT signaling pathway played an important role in both VSD subgroups.⑤Four miRNAs（hsa-miR-19a,hsa-let-7e,hsa-miR-134 and hsa-miR-206） were randomly selected and verified by real-time PCR,the results of quantitative PCR were basically consistent with the microarray results.Conclusions The differential time-specific expression of miRNAs may play an important role in the occurrence and development of fetal VSD malformations,but it needs confirmation by study with larger sample size.These predicted target genes were mainly related to cell growth,differentiation and metabolism;partially were directly associated with the key genes of heart development;partially were key factors of the WNT signaling pathway.The occurrence and development of fetal VSD malformations were the joint results of imbalance gene expression which functioned at different levels under the participation of miRNAs.