RESUMO
BACKGROUND: Hirschsprung's disease (HSCR) is a congenital disorder resulting from abnormal development of the enteric nervous system (ENS). Given the complexity of its pathogenesis, it is important to investigate the role of epigenetic inheritance in its development. As Circ-MTCL1 is abundant in brain tissue and colon tissue, whether it has a significant part in the development of ENS is worth exploring. This study clarifies its role in HSCR and identifies the specific molecular mechanisms involved. METHODS: Diseased and dilated segment colon tissues diagnosed as HSCR were collected for the assessment of gene expression levels using RT-PCR. EdU and CCK-8 assays were adopted to evaluate cell proliferation, and Transwell assay was adopted to assess cell migration. The interaction between Circ-MTCL1, miR-145-5p and SMAD3 was confirmed by dual luciferase reporter gene analysis, RT-PCR and Western blotting. RESULTS: Circ-MTCL1 was down-regulated in the aganglionic colon tissues. The decreased expression of Circ-MTCL1 associated with a reduction in cell migration and proliferation. Bioinformatics analysis and cellular experiments confirmed its role might have been associated with the inhibition of miR-145-5p. MiR-145-5p was up-regulated in HSCR diseased segment colon tissues, exhibiting a negative correlation with Circ-MTCL1. Overexpression of miR-145-5p reversed the inhibition of cell migration and proliferation associated with Circ-MTCL1 down-regulation. The expression of SMAD3 was inhibited by miR-145-5p. The overexpression of SMAD3 eliminated the miR-145-5p-associated inhibition of cell migration and proliferation. Overexpression of miR-145-5p reversed the inhibitory effects of Circ-MTCL1 down-regulation-associated inhibition of cell migration and proliferation, while suppressing SMAD3 expression. Conversely, overexpression of SMAD3 counteracted the miR-145-5p-associated inhibition of cell migration and proliferation. CONCLUSIONS: Circ-MTCL1 may function as a miR-145-5p sponge, regulating the expression of SMAD3 and influencing cell migration and proliferation, thus participating in the development of HSCR.
Assuntos
Doença de Hirschsprung , MicroRNAs , Humanos , Doença de Hirschsprung/genética , RNA Circular/genética , Proliferação de Células/genética , Movimento Celular/genética , MicroRNAs/genética , Proteína Smad3/genética , Proteínas Associadas aos MicrotúbulosRESUMO
BACKGROUND: Plasma 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 profiles of HSCR and elucidated the mechanism of the selected miR-199a-3p in the development of HSCR. METHODS: Plasma 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 confirmed 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 confirm the function of target gene, mammalian target of rapamycin (mTOR), in vitro. RESULTS: The 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 confirmed in tissue samples. mTOR can partially reverse the effect of miR-199a-3p on cell proliferation and migration function in vitro. CONCLUSIONS: miR-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.