Hirschsprung disease (HSCR) is a congenital disorder caused by the defective function of the embryonic enteric neural crest. The impaired migration of embryonic enteric neural crest plays an important role in the pathogenesis of this disease. Recent studies showed that the ARP2/3 complex and RAC isoforms had effects on actin cytoskeleton remodelling, which contributes to migration. Moreover, some regulatory relationships were identified between ARP2/3 complex and RAC isoforms. Although microRNAs (miRNAs) have been known to modulate target gene expression on the post-transcriptional level, little is known about the regulation among miRNAs, ARP2/3 complex and RAC isoforms. Here, we report that down-regulation of ARP2 and ARP3, two main subunits of ARP2/3 complex, suppressed migration and proliferation in 293T and SH-SY5Y cell lines via the inhibition of RAC1 and RAC2. Meanwhile, as the target genes, ARP2 and ARP3 are reduced by increased miR-24-1* and let-7a*, respectively, in 70 HSCR samples as compared with 74 normal controls. Co-immunoprecipitation showed that aberrant reduction in ARP2 and ARP3 could weaken the function of ARP2/3 complex. Our study demonstrates that the miR-24-1*/let-7a*-ARP2/3 complex-RAC isoforms pathway may represent a novel pathogenic mechanism for HSCR.

Hirschsprungs disease (HSCR), a congenital gastrointestinal disorder, is one of the most common causes of neonatal bowel obstruction. Without an early screening and diagnosis, some patients develop serious complications, such as toxic megacolon or acute enterocolitis. We sought to identify specific serum microRNAs (miRNAs) that can serve as novel early, non-invasive screening signature and then to test their specificity and sensitivity in diagnosing Hirschsprungs disease. We obtained serum samples from 95 HSCR cases and 104 matched controls. An initial screening of miRNA expression was performed through TaqMan Low Density Array. The candidate miRNAs were validated by individual reverse transcription quantitative real-time PCR arranged in the training and a two-stage validation set. Additional double-blind testing was performed in 23 patients with clinically suspected HSCR to evaluate the diagnostic value and accuracy of the serum miRNA profile in predicting HSCR. Following a multi-stage evaluation approach, five miRNAs were significantly increased in HSCR cases compared with controls. The areas under the receiver operating characteristic (ROC) curve of this five-serum miRNA signature were 0.895, 0.893 and 0.925 in training set and two validation sets, respectively. The accuracy rate of the five-miRNA profile as HSCR signature was 82.6%, which, in the double-blind testing set, was markedly higher than that of contrast enema (70%), the most commonly used test performed to diagnose HSCR. Our results indicate that a five-serum miRNA signature may be linked to HSCR, representing a potential, novel, non-invasive diagnostic approach for early screening of HSCR.

Hirschsprung disease (HSCR) is a birth defect with an approximate incidence of 1/5,000 live births, and up to one-third of HSCR patients develop Hirschsprung-associated enterocolitis (HAEC), the leading cause of HSCR-related death. Very little is known about the pathogenesis, prevention, and early diagnosis of HAEC. Here, we used a prospective study to investigate the enteric microbiome composition at the time of surgery as a predictor for developing postoperative HAEC. We identified a microbiome signature containing 21 operational taxonomic units (OTUs) that can potentially predict postoperative HAEC with ~85% accuracy. Furthermore, we identified exclusive breastfeeding as a novel protective factor for total HAEC (i.e., preoperative and postoperative HAEC combined). In addition, we discovered that breastfeeding was associated with a lowered risk for HAEC potentially mediated by modulating the gut microbiome composition characterized by a lower abundance of Gram-negative bacteria and lower LPS concentrations. In conclusion, modulating the gut microbiome by encouraging breastfeeding might prevent HAEC progression in HSCR patients.