Identification Of Differentially Expressed MiRNAs In Anencephaly And Control And Target MRNA Prediction

Neural tube defects (NTDs) are a group of severe congenital malformations characterized by a failure of neural tube closure during early embryonic development. NTDs are complex birth defects with a multi-factorial pattern of inheritance, requiring both genetic and environmental factors to contribute to their etiology.NTDs are caused by the failure of the neural tube to close. Therefore, factors that impact the development of the neural tube are likely to be involved in the pathogenesis of NTDs. MicroRNAs (miRNAs) are small, single-stranded, and non-coding RNA molecules that possess about 22 nucleotides. The function of miRNAs in mammalian cells is to repress the translation or cleavage of target gene messenger RNA (mRNA) by base-pairing with the 3'-untranslated regions (UTRs) of target mRNAs.A number of miRNA profiling studies have shown that the expression of miRNAs changes during neural stem cell differentiation and during the morphological development of the mammalian brain. These researches suggest that miRNAs may be important players in these processes. However, neither the expression nor the role of miRNAs in NTDs has been characterized. Thus, it is necessary to unravel the expression and function of the miRNAs in NTDs. Our study may provide a further understand the etiology of NTDs.Objective:To investigate the expression of miRNAs in tissues from fetuses with the most severe NTDs, anencephaly and normal control, and discuss the roles of miRNAs in the pathogenesis of NTDs. To predict the target genes of miRNAs which the expression change was confirmed and analysis the function of target genes.Methods:The profiling of miRNAs from area cerebrovasculosa of fetuses with anencephaly and normal brains was performed using an LNA mercuryTM microarray. Real-time RT-PCR analysis was carried out using an NCodeTM miRNA First-Strand cDNA Synthesis and a qRT-PCR kit. Relative expression was calculated using the AACT method and normalizing to the expression of U6 snRNA.The target gene sets for miRNAs were determined using miRGen v3. The human protein interaction data set was obtained from the Human Protein Reference Database (HPRD).Using these data, we constructed a protein interaction network of the predicted miRNA target genes. GeneCodis 2.0 was used to search for biological features of the interacting proteins involved in the network.Results:1. Compared to healthy human fetal brain tissue, tissue from fetuses with anencephaly has a specific miRNA expression profile. In anencephaly, there were 97 downregulated miRNAs and 116 upregulated miRNAs. These miRNAs include two downregulated miRPlus and nine upregulated miRPlus.2. The microarray findings were extended using real-time qRT-PCR for nine miRNAs. Of these nine miRNAs validated, miR-126, miR-198, and miR-451 were upregulated, whereas miR-9, miR-212, miR-124, miR-138, and miR-103/107 were downregulated in the tissues from fetuses with anencephaly.3. Bioinformatic analysis shows 881 potential target genes that are regulated by the validated miRNAs. Of these potential genes,79 genes are involved in a protein interaction network.4. There are six co-occurrence annotations (transcription, signal transduction, cell cycle, cell growth, cell death, and cell-cell signaling) within the GOSlim Process. There are seven co-occurrence annotations (protein binding, transcription factor activity, receptor activity, Nucleotide binding, DNA binding, receptor binding, and transferase activity) within the GOSlim Function found by Genecodis2. And there are thirty-five co-occurrence annotations (such as focal adhesion,actin cytoskeleton and Jak-STAT signaling pathway et al.) within the KEGG pathway found by Genecodis2.Conclusion:This study confirms that anencephaly has a specific miRNA expression profile compared to gestational age-matched normal fetal brain tissue. The results show that different miRNAs are deregulated in NTDs, suggesting the involvement of these miRNAs in the pathogenesis of NTDs. We also provide a target gene map of miRNAs that are predicted to contribute to future investigations of the regulatory mechanisms of miRNA in human anencephaly.