Hirschsprung disease (HSCR), one of several neurocristopathies in children, is characterized by nerve loss in the large intestine and is mainly treated by surgery, which causes severe complications. Enteric neural crest-derived cell (ENCC) transplantation is a potential therapeutic strategy; however, so far with poor efficacy. Here, we assessed whether and how fecal microbiota transplantation (FMT) could improve ENCC transplantation in a rat model of hypoganglionosis; a condition similar to HSCR, with less intestinal innervation. We found that the hypoganglionosis intestinal microenvironment negatively influenced the ENCC functional phenotype in vitro and in vivo. Combining 16S rDNA sequencing and targeted mass spectrometry revealed microbial dysbiosis and reduced short-chain fatty acid (SCFA) production in the hypoganglionic gut. FMT increased the abundance of Bacteroides and Clostridium, SCFA production, and improved outcomes following ENCC transplantation. SCFAs alone stimulated ENCC proliferation, migration, and supported ENCC transplantation. Transcriptomewide mRNA sequencing identified MAPK signaling as the top differentially regulated pathway in response to SCFA exposure, and inhibition of MEK1/2 signaling abrogated the SCFA-mediated effects on ENCC. This study demonstrates that FMT improves cell therapy for hypoganglionosis via short-chain fatty acid metabolisminduced MEK1/2 signaling.
New Findings
What is the central question of this study?Long non‐coding RNAs (lncRNAs) are widely involved in the progression of Hirschsprung's disease (HSCR), but the role of actin filament associated protein 1 antisense RNA1 (AFAP1‐AS1), an lncRNA, in HSCR has not been explored before.
What is the main finding and its importance?Downregulation of AFAP1‐AS1 blocks enteric neural crest stem cell proliferation, differentiation, migration and invasion and promotes the occurrence of HSCR via the miR‐195/E2F3 axis, indicating thatAFAP1‐AS might be a potential biomarker for HSCR patients.
Abstract
Long non‐coding RNAs (lncRNAs) are involved in several human disorders. Nevertheless, it remains unclear whether they are implicated in the phenotypes of enteric neural crest stem cells (ENCSCs) in Hirschsprung's disease (HSCR). Therefore, we designed this study to explore the pathogenicity of AFAP1‐AS1 for HSCR. Microarray analysis and bioinformatic tools were used to screen out the differentially lncRNAs and microRNAs (miRNAs) in patients with HSCR. Small interference RNA transfection was applied to carry out functional experiments in ENCSCs. Cellular activities were detected by cell counting kit‐8, 5‐ethynyl‐2′‐deoxyuridine, Transwell assays and flow cytometry. Finally, rescue experiments were performed to examine the cofunction of AFAP1‐AS1 and miR‐195 and of miR‐195 and E2F transcription factor 3 (E2F3). AFAP1‐AS1 was reduced in HSCR patients. Meanwhile, knockdown of AFAP1‐AS1 reduced the cell migratory and proliferative capacities and facilitated cell apoptosis along with G0/G1 phase arrest. E2F3 was diminished when miR‐195 was upregulated, and AFAP1‐AS1 inhibition reduced its ability to bind to miR‐195. Altogether, AFAP1‐AS1 silencing acts as an endogenous RNA by interacting with miR‐195 to alter E2F3 expression, thus conferring repressive effects on ENCSC activity and promoting HSCR progression.
ObjectiveHirschsprung disease (HSCR) is one of the common neurocristopathies in children, which is associated with at least 20 genes and involves a complex regulatory mechanism. Transcriptional regulatory network (TRN) has been commonly reported in regulating gene expression and enteric nervous system development but remains to be investigated in HSCR. This study aimed to identify the potential TRN implicated in the pathogenesis and diagnosis of HSCR.MethodsBased on three microarray datasets from the Gene Expression Omnibus database, the multiMiR package was used to investigate the microRNA (miRNA)–target interactions, followed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. Then, we collected transcription factors (TFs) from the TransmiR database to construct the TF–miRNA–mRNA regulatory network and used cytoHubba to identify the key modules. Finally, the receiver operating characteristic (ROC) curve was determined and the integrated diagnostic models were established based on machine learning by the support vector machine method.ResultsWe identified 58 hub differentially expressed microRNAs (DEMis) and 16 differentially expressed mRNAs (DEMs). The robust target genes of DEMis and DEMs mainly enriched in several GO/KEGG terms, including neurogenesis, cell–substrate adhesion, PI3K–Akt, Ras/mitogen-activated protein kinase and Rho/ROCK signaling. Moreover, 2 TFs (TP53andTWIST1), 4 miRNAs (has-miR-107,has-miR-10b-5p,has-miR-659-3p, andhas-miR-371a-5p), and 4 mRNAs (PIM3,CHUK,F2RL1, andCA1) were identified to construct the TF–miRNA–mRNA regulatory network. ROC analysis revealed a strong diagnostic value of the key TRN regulons (all area under the curve values were more than 0.8).ConclusionThis study suggests a potential role of the TF–miRNA–mRNA network that can help enrich the connotation of HSCR pathogenesis and diagnosis and provide new horizons for treatment.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.