BackgroundPlasma exosomal microRNAs have been suggested to be potential biomarkers of disease. However, the exosomal microRNAs in Hirschsprung's disease (HSCR) are still unclear. In this study, we analyzed the miRNA pro les of HSCR and elucidated the mechanism of the selected miR-199a-3p in the development of HSCR. MethodsPlasma exosomes were isolated, and exosomal miRNA high-throughput sequencing was performed to obtain differentially expressed miRNAs. CCK-8 and Transwell assay were used to determine the function of the most differentially expressed miRNA, which was con rmed in tissue specimen. Thereafter, target genes of the selected miRNAs were predicted by the databases. Gene Ontology (GO) analysis, Kyoto Encyclopedia of Genes Genomes (KEGG) analysis, and protein-protein interaction network (PPI) construction of possible target genes were used to perform enrichment analysis and interaction. Finally, the PCR, Western blot and recovery experiment were used to con rm the function of target gene, mammalian target of rapamycin (mTOR), in vitro. ResultsThe expression of miR-199a-3p was upregulated in plasma exosomes and diseased colonic tissues of patients with HSCR. In vitro, miR-199a-3p can inhibit cell proliferation and migration. Bioinformatic analysis suggested that mTOR might be a potential target of miR-199a-3p in HSCR. mTOR was discovered to be downregulated by miR-199a-3p in vitro. The negative connection between mTOR and miR-199a-3p was con rmed in tissue samples. mTOR can partially reverse the effect of miR-199a-3p on cell proliferation and migration function in vitro. ConclusionsmiR-199a-3p suppresses cell growth and motility, partially by targeting mTOR. Plasma exosomal miR-199a-3p, a diagnostic marker, is crucial for the development of HSCR.

Background METTL3, a mRNA m 6 A methyltransferase, has been implicated in various steps of mRNA metabolism, such as stabilization, splicing, nuclear transportation, translation, and degradation. However, whether METTL3 dysregulation is involved in Hirschsprung disease (HSCR) development remains unclear. In this study, we preliminarily elucidated the role of METTL3 in HSCR and sought to identify the associated molecular mechanism.Methods The gene expression levels of YAP and several methyltransferases, demethylases, and effectors were evaluated by RT-qPCR. Protein levels were evaluated by western blot and immunohistochemistry.Cell proliferation and migration were detected by CCK-8 and Transwell assays, respectively. The overall levels of m 6 A modi cation were determined by colorimetry.Results We found that m 6 A levels were reduced in stenotic intestinal tissue of patients with HSCR. When METTL3 was knocked down in SH-SY5Y and HEK-293T cells, the proliferative and migratory abilities of the cells were inhibited, m 6 A modi cation levels were reduced, and YAP expression was increased.Importantly, YAP and METTL3 expression displayed a negative correlation in both cell lines as well as in HSCR tissue.Conclusions Our results provide evidence for an interaction between METTL3 and YAP in HSCR, and further suggest that METTL3 is involved in the pathogenesis of HSCR by regulating neural crest cell proliferation and migration upstream of YAP.

Background The aim of this study was to identify hub genes, related transcription factors (TFs) and miRNAs from the miRNA–TF–gene interaction network in necrotizing enterocolitis (NEC). Methods Three expression data sets from GEO database that compared NEC with surgical negative controls were used to calculate differentially expressed miRNAs (DEMis) and genes (DEGs). A protein-protein interaction (PPI) network was constructed using DEGs and was used to determine hub genes. miRNAs related to hub genes were identified from the intersection between DEMis and predictions of hub gene-miRNA pairs using Starbase, TFs were predicted by hub genes, TF-miRNA pairs were predicted using miRNet. Finally, the miRNA–TF–hub gene interaction network was formed using these predicted pairs. Results A total of 14 DEMis and 123 DEGs were identified from the GEO datasets. One hundred and twenty DEGs were found in the PPI network. A pathogenic-associated interaction network was created by intersecting miRNAs, predicted TFs and hub genes. Article-published RNAs such as hsa-miR-7 or TLR4 were shown in this network, and novel RNAs and TFs (Hsa-miR-200a, GATA3, CXCL5) were shown in the network as important regulator. Conclusions This analysis displayed several important hub genes, TFs and miRNAs, some of which were not fully understood in previous studies of NEC. These results may play an important role in future studies on the etiology or treatment of NEC.