Multiple 'omics'-analysis reveals the role of prostaglandin E2 in Hirschsprung's disease.

The etiology and pathogenesis of Hirschsprung's disease (HSCR) remain largely unknown. We examined colon tissues from three independent populations with a combined analysis of metabolomics, transcriptomics and proteomics to understand HSCR pathogenesis, according to which mouse model was used to examine prostaglandin E2 (PGE2) induced clinical presentation of HSCR. SH-SY5Y and SK-N-BE(2) cell lines were studied for PGE2 inhibited cell migration through EP2. Our integrated multiple 'omics'-analysis suggests that the levels of PGE2, the expression of the gene encoding PGE2 receptor (EP2), and PGE2 synthesis enzyme genes (PTGS1 and PTGES) increased in HSCR colon tissues, together with a decreased synthesis of PGE2-related byproducts. In vivo, the pregnant mice treated with PGE2 gave birth to offspring with the decrease of ganglion cells in their colon and gut function. In in vitro study, when EP2 was blocked, the PGE2-inhibited cell migration was recovered. Our study identified a novel pathway highlighting the link between expression of PTGS1 and PTGES, levels of PGE2, expression of PTGER2, and neural crest cell migration in HSCR, providing a novel strategy for future diagnosis and prevention of HSCR.

MPGES-1 derived PGE2 inhibits cell migration by regulating ARP2/3 in the pathogenesis of Hirschsprung disease.

BACKGROUND: We previously studied the metabolomics, transcriptomics and proteomics of intestinal tissue of Hirschsprung disease (HSCR) patients; the results suggested that the expression of prostaglandin E2(PGE2), prostaglandin E receptor 2(PTGER2) and microsomal prostaglandin E synthase-1 (mPGES-1) notably increased in HSCR colon tissues. We already verified the differential expression of PGE2/EP2 in HSCR patients; therefore we investigate how mPGES-1 derived PGE2 affects the migration and the potential mechanism in cells, revealing the role of mPGES-1 derived PGE2 in the pathogenesis of Hirschsprung disease. METHODS: SH-SY5Y and SK-N-BE2 cell lines were obtained from American Type Culture Collection (ATCC, USA). Prostaglandin E2 and its synthetase inhibitors were purchased from Med Chem Express (MCE, USA). Migration assays were performed with transwell and scratch assays. Cell proliferation was confirmed by CCK8 method. Flow cytometer was used to detect the cell cycle and cell apoptosis. The expressions of mRNA and protein of EP2, ARP2/3 were determined by qRT-PCR and western blot respectively. Immunofluorescence and confocal laser scanning microscopy were used to observe the morphology and function of cytoskeleton. RESULTS: MPGES-1 derived PGE2 decreased the relative expression of EP2 and ARP2/3 and caused damage to cytoskeleton. As to cell functions, PGE2 inhibited cell migration while having no effects on the proliferation, cell cycle and apoptosis. By adding mPGES-1 inhibitor MK886 the abnormal expression and damaged cell function were reversed. CONCLUSIONS: MPGES-1 derived PGE2 inhibits the cell migration by regulating ARP2/3 complex via prostaglandin E2 receptor. Potential mechanisms are the damage of cytoskeleton and related proteins leading to failure of cell polarize and migration. Here we thoroughly inquire the role mPGES-1 derived PGE2 plays in cell migration which might provide a new thinking in the investigation interrelated to the pathogenesis of HSCR.

Circular RNA CCDC66 targets DCX to regulate cell proliferation and migration by sponging miR-488-3p in Hirschsprung's disease.

It has been suggested that circular RNAs play critical roles in natural growth and disease development. Nevertheless, whether the circular RNAs were related in Hirschsprung's disease (HSCR) remains unknown. Thus, we discovered the cir-CCDC66 was downregulated in HSCR compared with the normal gut tissues. The cir-CCDC66 reduction might inhibit cells' proliferation and migration in vitro. Then, we found that DCX transcript was putative cir-CCDC66 competing endogenous RNA. Furthermore, the function of cir-CCDC66 as a sponge for miR-488-3p to regulate DCX RNA expression was demonstrated by immunoprecipitation and luciferase reporter assays. In conclusion, this is the first report revealing that cir-CCDC66 modulates DCX expression through sponging miR-488-3p and thus participates in the onset of HSCR.

Long non-coding RNA FAL1 functions as a ceRNA to antagonize the effect of miR-637 on the down-regulation of AKT1 in Hirschsprung's disease.

OBJECTIVES: Emerged evidence demonstrates that long non-coding RNAs (lncRNAs) may play quintessential regulatory roles in the cellular processes, tumourigenesis and the development of disease. Though focally amplified lncRNA on chromosome 1 (FAL1) has been identified to have crucial functions in many diseases, its biological mechanism in the development of Hirschsprung's disease (HSCR) still remains unknown. MATERIALS AND METHODS: The expression levels of FAL1 in HSCR aganglionic tissues and matched normal specimens were detected by quantitative real-time PCR (qRT-PCR). Cell proliferation and migration were detected by Cell Counting Kit-8 (CCK-8) assay, Ethynyl-deoxyuridine (EdU) assay and transwell assay relatively. Cell cycle and apoptosis were assessed using flow cytometer analysis. Moreover, the novel targets of FAL1 were confirmed with the help of bioinformatics analysis and dual-luciferase reporter assay. Western blot assay as well as RNA immunoprecipitation (RIP) assay was conducted to investigate the potential mechanism. RESULTS: FAL1 expression was markedly down-regulated in HSCR aganglionic tissues and decreased FAL1 expression was associated with the diagnosis of HSCR. Cell functional analyses indicated that FAL1 overexpressing notably promoted cell proliferation and migration, while down-regulation of FAL1 suppressed cell proliferation and migration. Additionally, Flow cytometry assay demonstrated that knockdown of FAL1 induced markedly cell cycle stalled in the G0/G1 phase. Furthermore, FAL1 could positively regulate AKT1 expression by competitively binding to miR-637. CONCLUSIONS: These results illuminated that FAL1 may work as a ceRNA to modulate AKT1 expression via competitively binding to miR-637 in HSCR, suggesting that it may be clinically valuable as a biomarker of HSCR.

Down-regulation of circ-PRKCI inhibits cell migration and proliferation in Hirschsprung disease by suppressing the expression of miR-1324 target PLCB1.

Circular RNAs (circRNAs) are a novel class of noncoding RNAs (ncRNAs), which have been shown to participate in intracellular RNA regulatory networks and play vital roles in many pathological processes. Recently, circular RNA_PRKCI (circ-PRKCI) has been reported to regulate cell proliferation, migration and invasion in several human cancers. Hirschsprung disease (HSCR) is a well-known congenital gut motility disorder which roots in the aberrance of cranial-caudal neural crest cell migration. In this study, we investigated whether circ-PRKCI may affect cell migration and proliferation in HSCR. Quantitative reverse transcription PCR (qRT-PCR) was performed to detect the expression of circ-PRKCI in 48 HSCR aganglionic tissues and 48 normal bowel tissues. Luciferase reporter assay and RNA immunoprecipitation (RIP) assay verified the direct interaction between miR-1324 and PLCB1 or circ-PRKCI. Cell counting Kit-8 (CCK-8) and Ethynyldeoxyuridine (EdU) assays were employed to appraise the effects of miR-1324 or circ-PRKCI on cell proliferative potential, while transwell was performed to detect the migration in vitro. We found that circ-PRKCI was significantly down-regulated in HSCR aganglionic tissues. Morever, knockdown of circ-PRKCI suppressed cell proliferation and migration in vitro. Mechanistically, we confirmed that circ-PRKCI functioned as a molecular sponge for miR-1324 to upregulate the expression of PLCB1. In conclusion, our present study revealed the important role of circ-PRKCI-miR-1324-PLCB1 regulatory network in HSCR, providing a novel insight for the pathogenesis of HSCR.

Identification of candidate genes for necrotizing enterocolitis based on microarray data.

Necrotizing enterocolitis (NEC) is one of the most serious diseases that could threaten the life of neonates. However the current opinions about the pathogenesis or how to prevent or treat the disease are still ambiguous. The purpose of the present study was to identify the key genes of this disease and provide new insights into the mechanism of NEC. The gene expression data of GSE46619, including 5 specimens from NEC patients and 4 samples from surgical-control infants, were collected from Gene Expression Omnibus (GEO) database. The differentially expressed genes (DEGs) were screened with regard to NEC versus surgical-control group using Limma package in R software and Gene Ontology (GO) enrichment analysis and pathway enrichment analysis were conducted by means of Database for Annotation, Visualization and Integrated Discovery (DAVID) website subsequently. Furthermore the protein-protein interaction (PPI) network for DEGs was constructed using Cytoscape software and the most highly connected module was extracted using MCODE plugin from the PPI network. Moreover, the significantly enriched sub-pathways were identified using iSubpathwayMiner package in R software. A total of 2629 DEGs were screened out between NEC and control samples, including 367 up-regulated genes and 2262 down-regulated genes and they involved in different GO terms and pathways which may be associated with NEC onset and progression. PPI network and module analysis revealed that several genes were defined as hub genes including AGT, IL8 and KNG1. The sub-pathway analysis screened out 189 significantly enriched sub-pathways, including Tryptophan metabolism, Fatty acid metabolism, and Arachidonic acid metabolism. Genes in the corresponding sub-pathway, such as ACACB and CAT were regarded as critical genes in NEC. QRT-PCR was also conducted to identify the expression of the five key genes (AGT, IL8, KNG1, ACACB and CAT) in NEC samples. These findings have identified several hub genes (e.g., AGT, IL8, KNG1, ACACB and CAT) which were presumed to serve critical roles in NEC.

Long non-coding RNA LOC100507600 functions as a competitive endogenous RNA to regulate BMI1 expression by sponging miR128-1-3p in Hirschsprung's disease.

Recently studies reported that long non-coding RNAs (lncRNAs) may take part in a lot of congenital diseases, meanwhile, Hirschsprung's disease (HSCR) is a major congenital digestive tract malformation. Nevertheless whether lncRNAs participate in the occurrence of HSCR and how it contributes to this disease are still unknown. LOC100507600 was selected from our gene expression microarray data obtained from bowel tissues from HSCR patients and negative controls. Subsequently, we used qRT-PCR to prove the result in 64 pairs of HSCR disease bowel stenosis tissues and negative controls. Transwell assay, CCK-8 assay and flow cytometry were employed to explore whether cellular functions change after knocking down the LOC100507600 in SH-SY5Y cell and human 293T cell. Dual-luciferase reporter assay was used to confirm the competitive relationship between BMI1 and LOC100507600 through their association with hsa-miR128-1-3p. Protein extraction and Western blotting were used to further confirm the relationship between LOC100507600 and BMI1. We found that LOC100507600 was obvious reduced in tissues from HSCR patients with noteworthy correlation with BMI1. Furthermore, Downregulation of LOC100507600 repressed cell migration and proliferation and didn't affect cell apoptosis or cycle. Dual-luciferase reporter assay, qRT-PCR and Western blotting assay verified that LOC100507600 serves as a competitive endogenous RNA of miR128-1-3p and down-regulates BMI1 expression by sponging miR128-1-3p in HSCR. In sum, our study researches the potential diagnostic value of LOC100507600 in HSCR and deduces that LOC100507600 can contributes to HSCR as a competitive endogenous RNA to regulate BMI1 expression by sponging miR128-1-3p.

Identification of two novel PCDHA9 mutations associated with Hirschsprung's disease.

Hirschsprung's disease (HSCR) is a complex disorder with multiple pathogenic gene mutations. Protocadherin alpha 9 (PCDHA9) was identified as a potential candidate gene for HSCR by whole-exome sequencing in a Chinese family. Sanger sequencing in 298 HSCR cases revealed two sporadic Chinese patients with a novel missence PCDHΑ9 mutation (NM_031857; c.1280C > T[p.Ala427Val]) and one sporadic Chinese patient with another novel missence PCDHΑ9 mutation (c.1425C > G[p.Phe475Leu]).The silico predictions and 3D modeling suggest the deleterious effect of identified mutations on protein function. Immunohistochemistry analysis showed PCDHΑ9 was predominantly expressed in the myenteric plexus of human colon tissues. For mouse embryos, PCDHΑ9 was expressed in the stomach but rarely seen in the intestine during E10.5-12.5, then obviously expressed in the intestinal mucosa at E13.5 and extensively expressed in intestinal muscularis and mucosa at E14.5. Moreover, the down-regulation of PCDHΑ9 in the SH-SY5Y cell line promoted the proliferation and migration rate but inhibited the apoptotic rate. In summary, PCDHΑ9 is potentially related to HSCR and the clustered protocadherins (Pcdhs) may involve in the enteric nervous system (ENS) ontogeny.