基于表达谱芯片和下一代测序技术筛选先天性心脏病胎儿心肌组织差异表达的miRNA

目的 联合采用表达谱芯片和下一代测序技术同时高通量筛选先天性心脏病胎儿心肌组织表达差异的miRNA.方法 实验组为孕中期先天性畸形胎儿,对照组为同胎龄无心脏畸形的难免流产的胎儿,取胎儿心室心肌组织,联合采用Agilent Human 2.0 microRNAs表达谱芯片和SOLiD下一代测序技术同时观察心肌组织microRNA的表达变化,数据采用生物信息学方法进行分析,并用实时PCR方法验证芯片结果.结果 通过差异miRNA筛选,发现先天性心脏畸形组在表达谱芯片和下一代测序中共同差异的24个miRNA,生物信息学预测到1 606个靶基因,靶基因Gene Ontology分析表明其中与细胞进程、代谢过程、生物调控相关的靶基因为主,Pathway显著性分析表明,部分靶基因为生物信号通路中的关键因子;随机挑选共同表达差异的4个miRNA进行验证,结果表明定量PCR检测结果与芯片与下一代测序共同筛选结果基本相符.结论 这些在先天性心脏病中异常表达的miRNA为研究先天性心脏病分子水平上的发病机制提供了重要的线索,将有可能为心脏相关疾病的诊断和治疗提供新的靶点和研发新的药物.

Differential Expression of miRNA in Ventricular Myocardium of Fetus with Congenital Heart Disease by Using Next-generation Sequen- cing and Microarray Analysis

Objective MiRNA microarray and next-generation sequencing were employed to determine the differential expression of miRNA in ventricular myocardium of fetus with congenital heart disease （CHD） . Methods The fetuses with CHD in the second trimester were included in the experimental group. The controls were chosen from the fetuses of inevitable abortion without cardiac malformations. The fetal ventricular myocardia were acquired and RNA was extracted and processed. The Agilent Human 2.0 microarrays and SOLID next-generation sequencing were applied to get the data about miRNA expression changes, and the data were analyzed by bioinformatics meth- ods. Differential expression of miRNA was certificated by real-time PCR. Results There were 24 miRNAs with differential expression. 1606 target genes were predicted by bioinformatics methods. The Gene Ontology analysis indicated that target genes were mainly related to the cellular process, metabolic process and biological regulation. The pathway-express analysis indicated that some signaling pathways played important role in the occurrence of CHD. Four miRNAs were ran-domly selected and verified by real-time PCR, and the results of quantitative PCR were basically consistent with those detected by MiRNA microarray and next-generation sequencing. Conclusion The mRNAs with differential expression in CHD provides important clues to study the pathogenesis of the occurrence and development of fetal CHD, which will be possible to supply new targets and develop new drugs for diagnosing and treating CHD.