【摘要】 目的先天性心脏病(CHD)是环境因素和遗传因素共同作用的结果。室间隔缺损(VSD)是CHD中最常见的一种。研究表明,microRNAs(miRNAs)与CHD的发生发展关系密切,但目前缺乏有关miRNAs与VSD的研究报道。本研究旨在通过血清差异miRNA的表达研究阐明miRNA与VSD发生发展的关系。方法采用miRNA表达谱芯片技术进行VSD患者血清miRNA表达差异分析,并对结果进行实时定量PCR(Real-time PCR)验证,进而对miRNA调控的靶基因进行生物信息学分析。结果通过miRNA芯片在VSD患者和对照血清样本中检测miRNA差异表达,筛选出36个表达差异的miRNA(P<0.05),其中21个miRNA在VSD患者表达显著下调,15个表达显著上调。经过Real-time PCR验证,证实8个miRNAs差异表达有意义。通过生物信息学预测出447个靶基因,经GO分析富集度分值(enrichment score value)最大的生物过程(biological process)为心脏右心室形态发生(cardiac right ventricle morphogenesis),对应的差异表达基因为NOTCH1、HAND1、ZFPM2和GATA3,调控这些基因的miRNA可能包括hsa-let-7e-5p、hsa-miR-222-3p和hsa-miR-433。结论通过微阵列技术分析和RT-PCR验证,筛选出8个差异表达miRNA,为后续实验提供了可供分析的miRNA。利用3个数据库及GO分析,发现hsa-let-7e-5p、hsa-miR-222-3p和hsa-miR-433的靶基因分别为NOTCH1、HAND1、ZFPM2和GATA3,为进一步在基因水平研究VSD的发病机制提供了新的突破口,为将来可能实现的基因诊断、基因干预治疗以及疾病的预防奠定了一定的实验基础。更多还原

【Abstract】 Objective: To characterize the expression of miRNAs that might be involved in the development or reflect the consequences of VSD. Methods: MiRNA microarray analysis and reverse transcription-polymerase chain reaction( RTPCR) were employed to determine the miRNA expression profile from 3 patients with VSD and 3 VSD-free controls. The expression of eight selected miRNAs( hsa-let-7e-5p,hsa-miR-155-5p,hsa-miR-222-3p,hsa-miR-379-5p,hsa-miR-409-3p,hsa-miR-433,hsa-miR-487 b and hsa-miR-498) were then independently validated by RT-PCR of plasma samples from20 VSD patients and 15 VSD-free controls. A total of 446 target genes were predicted by bioinformatic methods. Results:Thirty- six differentially expressed miRNAs were found in the patients with VSD and the VSD-free controls. Compared with VSD-free controls,expression of 15 miRNAs were up-regulated and 21 miRNAs were down-regulated in the VSD group.The results of the RT-PCR were consistent with those of the microarray analysis. Gene ontology analysis indicated that the top target genes were mainly related to cardiac right ventricle morphogenesis. Additionally,we predicted NOTCH1,HAND1,ZFPM2,and GATA3 as targets of hsa-let-7e-5p,hsa-miR-222-3p and hsa-miR-433. Conclusion: The study has reported for the first time three circulating miRNA profiles for patients with VSD and VSD-free controls and validated these results in a larger group of subjects. These findings may reveal important insights into the pathogenesis of VSD.更多还原