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Numerous patients with anorectal malformations (ARMs) continue to experience fecal incontinence and constipation following surgical procedures. One of the most important factors that influences defecation is the striated muscle complex (SMC). Wnt signaling regulates the expression of myogenic regulatory factors, which serve an important role in muscle development. Therefore, the present study aimed to investigate the expression pattern of Wnt3a protein (by immunohistochemistry and western blot analysis) and mRNA [by reverse transcription‑quantitative polymerase chain reaction (RT-qPCR)] in the SMC of ARM model rats that were exposed to ethylenethiourea. Immunostaining revealed that the expression of Wnt3a exhibits space‑ and time‑dependent changes in the developing SMC of ARM model rat embryos. Immunohistochemistry demonstrated that on embryonic day 17 (E17), Wnt3a‑positive cells were observed in the SMC in normal embryos, and expression levels gradually increased as the rat embryos developed. Similar changes in Wnt3a protein expression were detected in ARM model rat embryos; however, the expression of Wnt3a was significantly reduced compared with the normal rat embryos. Western blotting and RT‑qPCR also revealed lower expression levels of Wnt3a protein and mRNA, respectively, in the SMC of ARMs model rat embryos compared with normal rat embryos. These data revealed that the expression of Wnt3a in ARM embryos was notably reduced, indicating a potential role for Wnt3a in the maldevelopment of the SMC in patients with ARMs.

Hedgehog (HH) is one of the key morphogens expressed in the gut mesenchyme that control animal development and tissue homeostasis. The HH gene has been shown to be closely associated with Hirschsprung's disease (HSCR) and anorectal malformations (ARMs); thus, it was hypothesized that HH signaling pathway‑associated genes may be candidate genes for HSCR and ARMs. The present study aimed to evaluate whether polymorphisms in the HH gene were associated with HSCR and ARM in a Chinese population. HH gene variants (rs61730970, rs200798148 and rs146535482) were analyzed in whole blood samples from patients with HSCR and ARMs, as well as normal children (control group). The results suggested that, when the rs61730970, rs200798148 and rs146535482 alleles of the HH gene lacked particular single nucleotide polymorphisms (SNPs), the patients were associated with a greater risk of HSCR and/or ARM [HSCR: odds ratio (OR)=1.543, P=0.004; OR=1.494, P=0.007; rs146535482: OR=1.556, P=0.003, respectively. ARM: OR=1.528, P=0.045; OR=1.800, P=0.007; OR=1.743, P=0.009, respectively). Sequencing of rs61730970 and rs200798148 revealed a loss of heterozygosity and SNPs at these loci in patients with HSCR. Similarly, the sequencing of rs61730970 and rs146535482 revealed a loss of heterozygosity and SNPs at these loci in patients with ARM. Although preliminary, these results suggested that the HH gene may be involved in genetic interactions associated with the pathogenesis of HSCR and ARM.

Hirschsprung's disease (HSCR) is a congenital disorder characterized by an absence of enteric ganglion cells in the terminal regions of the gut during development. To date, the cause of HSCR remains unclear, although the pathogenesis of this complex disease is hypothesized to be influenced by numerous genetic and environmental factors. Dishevelled‑2 (DVL‑2) is a subtype of the dishevelled protein, which is known to be involved in embryonic development. In the present study, the pathogenesis of HSCR was investigated by measuring the expression of the DVL‑2 gene and protein using reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR), western blotting and immunohistochemistry staining in the aganglionic and ganglionic segments of colonic tissues in patients with HSCR. The results showed that the level of DVL‑2 mRNA in the aganglionic segments was 0.28 fold that of the ganglionic segments. Similarly, the protein expression of DVL‑2 was lower (11.31±2.23) in the aganglionic segments than that of the ganglionic segments (35.21±2.66), as assessed by western blot analysis. Furthermore, immunohistochemical staining demonstrated that DVL‑2 expression was significantly higher in the mucosal and submucosal layers from ganglionic colon segments compared with that from the aganglionic segments. The data suggest that the expression of DVL‑2 in colon tissue segments may be important in the pathogenesis of HSCR.

Hirschsprung's disease (HSCR) is a polygenic disease, of which the cause remains to be elucidated. It has been suggested that SRY-related HMG-box 9 (SOX9) is fundamental for the correct development of oligodendrocytes and astrocytes; however, not the development of neurons. There are currently no reports regarding SOX9 expression in patients with HSCR; therefore, the present study aimed to investigate the expression of microRNA-124 (miR-124) and its target gene, SOX9, in HSCR. Quantitative polymerase chain reaction (qPCR), western blot analysis and immunohistochemistry were used to detect the mRNA and protein expression of miR-124 and SOX9 in patients with HSCR. miR-124 expression was observed to be markedly higher in stenotic colon segment tissues compared with normal colon segment tissues in patients with HSCR. Furthermore, mRNA and protein analyses revealed that SOX9 expression was also higher in the stenotic colon segment tissues compared with the normal colon segment tissues. In conclusion, these data suggest that miR-124 and its target gene, SOX9, are overexpressed in the stenotic colon segment of patients with HSCR, and may have a significant role in the development of HSCR.