Association between gut microbiota and Hirschsprung disease: a bidirectional two-sample Mendelian randomization study.

Background: Several studies have pointed to the critical role of gut microbiota (GM) and their metabolites in Hirschsprung disease (HSCR) pathogenesis. However, the detailed causal relationship between GM and HSCR remains unknown. Methods: In this study, we used two-sample Mendelian randomization (MR) analysis to investigate the causal relationship between GM and HSCR, based on the MiBioGen Consortium's genome-wide association study (GWAS) and the GWAS Catalog's HSCR data. Reverse MR analysis was performed subsequently, and the sensitivity analysis, Cochran's Q-test, MR pleiotropy residual sum, outlier (MR-PRESSO), and the MR-Egger intercept were used to analyze heterogeneity or horizontal pleiotropy. 16S rDNA sequencing and targeted mass spectrometry were developed for initial validation. Results: In the forward MR analysis, inverse-variance weighted (IVW) estimates suggested that Eggerthella (OR: 2.66, 95%CI: 1.23-5.74, p = 0.01) was a risk factor for HSCR, while Peptococcus (OR: 0.37, 95%CI: 0.18-0.73, p = 0.004), Ruminococcus2 (OR: 0.32, 95%CI: 0.11-0.91, p = 0.03), Clostridiaceae1 (OR: 0.22, 95%CI: 0.06-0.78, p = 0.02), Mollicutes RF9 (OR: 0.27, 95%CI: 0.09-0.8, p = 0.02), Ruminococcaceae (OR: 0.16, 95%CI: 0.04-0.66, p = 0.01), and Paraprevotella (OR: 0.45, 95%CI: 0.21-0.98, p = 0.04) were protective factors for HSCR, which had no heterogeneity or horizontal pleiotropy. However, reverse MR analysis showed that HSCR (OR: 1.02, 95%CI: 1-1.03, p = 0.049) is the risk factor for Eggerthella. Furthermore, some of the above microbiota and short-chain fatty acids (SCFAs) were altered in HSCR, showing a correlation. Conclusion: Our analysis established the relationship between specific GM and HSCR, identifying specific bacteria as protective or risk factors. Significant microbiota and SCFAs were altered in HSCR, underlining the importance of further study and providing new insights into the pathogenesis and treatment.

Identifying the potential transcriptional regulatory network in Hirschsprung disease by integrated analysis of microarray datasets.

Objective: Hirschsprung 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. Methods: Based 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. Results: We 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 (TP53 and TWIST1), 4 miRNAs (has-miR-107, has-miR-10b-5p, has-miR-659-3p, and has-miR-371a-5p), and 4 mRNAs (PIM3, CHUK, F2RL1, and CA1) 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). Conclusion: This 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.

Fecal microbiota transplantation enhances cell therapy in a rat model of hypoganglionosis by SCFA-induced MEK1/2 signaling pathway.

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. Transcriptome-wide 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 metabolism-induced MEK1/2 signaling.

Phenotypic and functional comparison of rat enteric neural crest-derived cells during fetal and early-postnatal stages.

In our previous study, we showed that with increasing time in culture, the growth characteristics of enteric neural crest-derived cells (ENCCs) change, and that the proliferation, migration and neural differentiation potential of these cells in vitro notably diminish. However, there are no studies on the developmental differences in these characteristics between fetal and early-postnatal stages in vitro or in vivo. In this study, we isolated fetal (embryonic day 14.5) and postnatal (postnatal day 2) ENCCs from the intestines of rats. Fetal ENCCs had greater maximum cross-sectional area of the neurospheres, stronger migration ability, and reduced apoptosis, compared with postnatal ENCCs. However, fetal and postnatal ENCCs had a similar differentiation ability. Fetal and postnatal ENCCs both survived after transplant into a rat model of Hirschsprung's disease. In these rats with Hirschsprung's disease, the number of ganglionic cells in the myenteric plexus was higher and the distal intestinal pressure change was greater in animals treated with fetal ENCCs compared with those treated with postnatal ENCCs. These findings suggest that, compared with postnatal ENCCs, fetal ENCCs exhibit higher survival and proliferation and migration abilities, and are therefore a more appropriate seed cell for the treatment of Hirschsprung's disease. This study was approved by the Animal Ethics Committee of the Second Affiliated Hospital of Xi'an Jiaotong University (approval No. 2016086) on March 3, 2016.

Involvement of the lncRNA AFAP1-AS1/microRNA-195/E2F3 axis in proliferation and migration of enteric neural crest stem cells of Hirschsprung's disease.

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.

NEAT1 Negatively Regulates Cell Proliferation and Migration of Neuroblastoma Cells by miR-183-5p/FOXP1 Via the ERK/AKT Pathway.

Neuroblastoma, a malignant tumor of the sympathetic nervous system, is an aggressive extracranial tumor in childhood. Long noncoding RNAs (lncRNAs) have been discovered to play a key role in the eukaryotic regulatory gene network and be involved in a wide variety of biological processes. We observed that the expression of lncRNA nuclear-enriched abundant transcript-1 (NEAT1) was significantly decreased in human neuroblastoma tissues and cell lines, compared with the normal. We observed cell proliferation, migration, and invasion with Cell Counting Kit-8 assay, colony formation assay, and Transwell assay to investigate the effects of NEAT1, miR-183-5p, or FOXP1 on neuroblastoma cells. And we also used StarBase and luciferase reporter gene assay to predict and confirm the interaction of NEAT1, miR-183-5p, and FOXP1 in neuroblastoma cells. First, overexpression of NEAT1 suppressed cell proliferation and played a key role in cell migration and invasion. In addition, NEAT1 was demonstrated to directly interact with miR-183-5p and exerted its antioncogenic role in neuroblastoma by negatively regulating miR-183-5p expression. miR-183-5p suppressed the expression of FOXP1 and regulated cell proliferation and migration by directly targeting FOXP1 mRNA 3'-untranslated region. Moreover, FOXP1 antagonized the effect of miR-183-5p on the phosphorylation of extracellular-regulated kinase/protein kinase B (ERK/AKT), while FOXP1 siRNA increased the reduced phosphorylation of ERK/AKT caused by miR-183-5p inhibitor in neuroblastoma cells. Taken together, these data showed that NEAT1 negatively regulated cell proliferation and migration of neuroblastoma by the miR-183-5p/FOXP1 axis via suppression of the ERK/AKT pathway. Our findings may provide a new target for the study of pathogenesis and treatment of neuroblastoma.

Inhibition of ROCK signaling pathway accelerates enteric neural crest cell-based therapy after transplantation in a rat hypoganglionic model.

BACKGROUND: Hirschsprung's disease (HSCR) is a congenital gastrointestinal disorder, characterized by enteric ganglia absence in part or entire of the colon, due to abnormal colonization and migration of enteric neural crest cells (ENCCs) during development. Currently, besides surgery which is the main therapy for HSCR, the potential of stem cell-based transplantation was investigated as an alternative option. Although promising, it has limitations, including poor survival, differentiation, and migration of the grafted cells. We hypothesized that modulation of extracellular factors during transplantation could promote ENCCs proliferation and migration, leading to increased transplantation efficiency. Considering that the RhoA/ROCK pathway is highly involved in cytoskeletal dynamics and neurite growth, our study explored the effect of inhibition of this pathway to improve the success of ENCCs transplantation. METHODS: Enteric neural crest cells were isolated from rat embryos and labeled with a GFP-tag. Cell viability, apoptosis, differentiation, and migration assays were performed with and without RhoA/ROCK inhibition. Labeled ENCCs were transplanted into the muscle layer of an induced hypoganglionic rat model followed by intraperitoneal injections of ROCK inhibitor. The transplanted segments were collected 3 weeks after for histological analysis. KEY RESULTS: Our results showed that inhibition of ROCK increased viable cell number, differentiation, and migration of ENCCs in vitro. Moreover, transplantation of labeled ENCCs into the hypoganglionic model showed enhanced distribution of grafted ENCCs, upon treatment with ROCK inhibitor. CONCLUSIONS AND INFERENCES: ROCK inhibitors influence ENCCs growth and migration in vitro and in vivo, and should be considered to improve the efficiency of ENCCs transplantation.

Correlation of spatio-temporal characteristics of intestinal inflammation with IL-17 in a rat model of hypoganglionosis.

Interleukin 17 expression is increased in children with Hirschsprung disease, which is characterized by intestinal inflammation. This study designed to exploit the characteristics of intestinal inflammation and examine the correlation of interleukin 17 in this process of hypoganglionosis model established by benzalkonium chloride treatment. Colon sections from female rats were treated with benzalkonium chloride to induce hypoganglionosis or with saline alone as a sham control. C-reactive protein and tumor necrosis factor-ɑ were used as markers of inflammation. Expression of C-reactive protein, tumor necrosis factor-ɑ, and interleukin 17 was assessed in colon tissue and blood serum on days 7, 14 and 21 after treatment. The correlation between C-reactive protein, tumor necrosis factor-ɑ, and interleukin 17 expression was estimated using the Spearman's rank-correlation coefficient. C-reactive protein, tumor necrosis factor-ɑ, and interleukin 17 were strongly expressed in submucosa and mucosa layers and serum from treated animals. The expression of C-reactive protein, tumor necrosis factor-ɑ, and interleukin 17 maintained the highest level at Day 21. Only C-reactive protein and tumor necrosis factor-ɑ expression was increased in control animals and only on day 7. Spearman's rank correlation coefficient was significant in C-reactive protein, tumor necrosis factor-ɑ, and interleukin 17 at Day 7, 14 and 21. Concomitant upregulation of C-reactive protein, tumor necrosis factor-ɑ, and interleukin 17 and significant positive correlations between C-reactive protein, tumor necrosis factor-ɑ, and interleukin 17 may imply that interleukin 17 is involved in spatio-temporal inflammation induced by benzalkonium chloride.