HSCR is a common congenital disease in infants, which is characterized by the disturbance of intestinal motility[14]. However, there is no precise mechanism to explain the related pathophysiology of HSCR[15]. In this study, the differentially expressed miRNAs were screened out by comparing plasma-derived exosomes of healthy groups and HSCR groups, using comprehensive bioinformatics analysis to investigate the pathogenesis of HSCR. The results showed that 62 DEMs were screened in HSCR. Moreover, by comparing the TCA group and the other HSCR types group, miR-106b-5p, miR-205-5p, miR-375-3p, and miR-34a-5p were identified as main miRNAs that may participate in the pathogenesis of TCA.
Among the 62 differentially expressed miRNAs in the HSCR group, 31 miRNAs showed up-regulation and 31 miRNAs showed down-regulation. Among them, miR-205-5p was significantly up-regulated that was significantly enriched in nine KEGG pathways, including cell cycle and prostate cancer. In different malignancies, miR-205-5p is a well-studied tumor suppressor miRNA that may trigger cell cycle arrest and apoptosis, reducing tumor cell chemotolerance[16]. Previously, it was found that miR-205-5p inhibits the proliferation of pulmonary artery smooth muscle cells by targeting MICAL2 that activates ERK1/2 signaling[17]. It is noteworthy in our study that the tendency of miR-205-5p expression was lower in the control, moderately expressed in other types of HSCR, and highly expressed in TCA. MiR-34a-5p, another miRNA that follows a similar tendency, has been demonstrated to impede cell migration, proliferation, and invasion[18]. Therefore, miR-34a-5p and miR-205-5p may play a critical role in TCA formation by disrupting the cell cycle or other cancer-related pathways.
The findings demonstrated that miR-106b-5p was significantly decreased in the TCA group. In addition, miR-106b-5p was also found to be significantly enriched in the prion disease pathway, which promotes proliferation and inhibits apoptosis by regulating BTG3[19]. The most intricate interacting network of predicted targeted genes was found in the interaction network analysis of three miRNAs, suggesting that miR-106b-5p may play a role in TCA development.
miR-205-5p and miR-106b-5p shared the four target genes: AFF4, MTMR9, POU2F1, and WW-and-C2-domain-containing(WWC2). AFF4, a member of the super elongation complex, recruits other factors through direct interactions with ELL proteins and the P-TEFb complex as a central scaffold[20]. The encoded protein MTMR9 has a double-helical motif that is similar to the SET interaction domain, and it regulates cell proliferation[21]. In human herpes simplex virus (HSV) infection, the POU2F1 forms a multiprotein-DNA complex with the viral trans-activator proteins HCFC1 and VP16[22]. It has the ability to enhance viral gene transcription in the early stages. WWC2 is a meiotic and early mitotic cell division regulator that controls the fate of mouse blastocyst cells[23]. The function of these genes reveals that the invasion of exogenous pathogens and abnormal cell differentiation may be one of the pathogenesis of TCA.
Most of the prior research employed blood or colon tissue as samples for bioinformatics analysis and to study the mechanism of miRNAs[24, 25]. However, we believe that the use of plasma-derived exosomes for mechanistic studies has its unique advantages. The most important thing is to obtain miRNA from blood samples, which can overcome the spatial heterogeneity and inaccessibility of tissue samples and can detect the progression of HSCR in time, that is lower expression in normal children and moderately expressed in other types of HSCR, and highly expressed in TCA. On the other hand, it has been reported that miRNAs in exosomes are more stable than those in the blood due to the double-membrane structure of exosomes[26]. However, due to the complex origin of miRNAs in blood, their variability will be higher which will lead to statistical uncertainty[27]. In the future, we intend to conduct more functional research and mechanism exploration of selected miRNAs and genes in human colon tissues and animal models.