LncRNA-RMST Functions as a Transcriptional Co-regulator of SOX2 to Regulate miR-1251 in the Progression of Hirschsprung's Disease.

Hirschsprung's disease (HSCR) is a congenital disorder characterized by the absence of enteric neural crest cells (ENCCs). LncRNA rhabdomyosarcoma 2-associated transcript (RMST) is essential for the growth and development of neuron. This study aimed to reveal the role of RMST in the pathogenesis of HSCR. The expression level of RMST, miR-1251, SOX2, and AHNAK was evaluated with qRT-PCR or western blot. CCK-8 and transwell assays were applied to detect cell proliferation and migration. CHIP and RIP assays were applied to determine the combination relationship between SOX2 and promoter region of miR-1251 or RMST and SOX2, respectively. Dual-luciferase reporter assay was performed to confirm miR-1251 targeted AHNAK. As results have shown, RMST was downregulated in the aganglionic colon of HSCR patients. The knockdown of RMST attenuated cell proliferation and migration significantly. MiR-1251, the intronic miRNA of RMST, was also low expressed in HSCR, but RMST did not alter the expression of miR-1251 directly. Furthermore, SOX2 was found to regulate the expression of miR-1251 via binding to the promoter region of miR-1251, and RMST strengthened this function by interacting with SOX2. Moreover, AHNAK was the target gene of miR-1251, which was co-regulated by RMST and SOX2. In conclusion, our study demonstrated that RMST functioned as a transcriptional co-regulator of SOX2 to regulate miR-1251 and resulted in the upregulation of AHNAK, leading to the occurrence of HSCR. The novel RMST/SOX2/miR-1251/AHNAK axis provided potential targets for the diagnosis and treatment of HSCR during embryonic stage.

Apoptotic neuron-secreted HN12 inhibits cell apoptosis in Hirschsprung's disease.

Perturbation in apoptosis can lead to Hirschsprung's disease (HSCR), which is a genetic disorder of neural crest development. It is believed that long noncoding RNAs (lncRNAs) play a role in the progression of HSCR. This study shows that apoptotic neurons can suppress apoptosis of nonapoptotic cells by secreting exosomes that contain high levels of HN12 lncRNA. Elevated exogenous HN12 in nonapoptotic cells effectively inhibited cell apoptosis by maintaining the function of mitochondria, including the production of ATP and the release of cytochrome C. These results demonstrate that secreted lncRNAs may serve as signaling molecules mediating intercellular communication in HSCR. In addition, high HN12 levels in the circulation worked as a biomarker for predicting HSCR, providing a potential, novel, noninvasive diagnostic approach for early screening of HSCR.

Negative feedback circuitry between MIR143HG and RBM24 in Hirschsprung disease.

Hirschsprung disease (HSCR) is a genetic disorder of neural crest development. It is also believed that epigenetic changes plays a role in the progression of this disease. Here we show that the MIR143 host gene (MIR143HG), the precursor of miR-143 and miR-145, decreased cell proliferation and migration and forms a negative feedback loop with RBM24 in HSCR. As RBM24 mRNA is a target of miR-143, upregulation of RBM24 upon an increase in the level of MIR143HG could be attributed to sequestration of miR-143 by MIR143HG (sponge effect). The RBM24 protein was shown to bind to MIR143HG, and subsequently, accelerated its degradation by destabilizing its transcript and facilitating its interaction with Ago2, thus forming a negative feedback between MIR143HG and RBM24. In addition, experiments using siRNA against DROSHA indicated that RBM24 could promote the biogenesis of miR-143. This feedback loop we describe here represents a novel mode of autoregulation, with implications in HSCR pathogenesis.

Suppressive action of miRNAs to ARP2/3 complex reduces cell migration and proliferation via RAC isoforms in Hirschsprung disease.

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.

Long none coding RNA HOTTIP/HOXA13 act as synergistic role by decreasing cell migration and proliferation in Hirschsprung disease.

Long noncoding RNAs (lncRNAs) have been confirmed to be associated with various human diseases. However, whether they are associated with Hirschsprung disease (HSCR) progression remains unclear. In this study, we designed the experiment to explore the relationship between lncRNA HOTTIP and HOXA13, and their pathogenicity to HSCR. Quantitative real-time PCR and Western blot were performed to detect the levels of lncRNA, mRNAs, and proteins in colon tissues from 79 patients with HSCR and 79 controls. Small RNA interference transfection was used to study the function experiments in human 293T and SK-N-BE cell lines. The cell viability and activities were detected by the transwell assays, CCK8 assay, and flow cytometry, respectively. LncRNA HOTTIP and HOXA13 were significantly down-regulated in HSCR compared to the controls. Meanwhile, the declined extent of their expression levels makes sense between two main phenotype of HSCR. SiRNA-mediated knock-down of HOTTIP or HOXA13 correlated with decreased levels of each other and both reduced the cell migration and proliferation without affecting cell apoptosis or cell cycle. Our study demonstrates that aberrant reduction of HOTTIP and HOXA13, which have a bidirectional regulatory loop, may play an important role in the pathogenesis of HSCR.

Nidogen-1 is a common target of microRNAs MiR-192/215 in the pathogenesis of Hirschsprung's disease.

Recent studies have emphasized the important role of microRNA (miRNA) clusters and common target genes in disease progression. Despite the known involvement of the miR-192/215 family in many human diseases, its biological role in Hirschsprung disease (HSCR) remains undefined. In this study, we explored the role of the miR-192/215 family in the pathogenesis of HSCR. Quantitative real-time PCR and western blotting measured relative expression levels of miRNAs, mRNAs, and proteins in 80 HSCR patients and 77 normal colon tissues. Targets were evaluated by dual-luciferase reporter assays, and the functional effects of miR-192/215 on human 293T and SH-SY5Y cells were detected by the Transwell assay, CCK8 assay and flow cytometry. MiR-192/215 was significantly down-regulated in HSCR tissue samples, and their knockdown inhibited cell migration and proliferation in the human 293T and SH-SY5Y cell lines. Nidogen 1 (NID1) was confirmed as a common target gene of miR-192/215 by dual-luciferase reporter gene assay and its expression was inversely correlated with that of miR-192/215 in tissue samples and cell lines. Silencing of NID1 could rescue the extent of the suppressing effects by miR-192/215 inhibitor. The down-regulation of miR-192/215 may contribute to HSCR development by targeting NID1. We proposed the following cascade for the proposed mechanism of miR-192/215 in the pathogenesis of Hirschsprung disease (HSCR) by targeting Nidogen 1 (NID1). Aberrant expression of miR-192/215 inhibits cell migration and cell proliferation via NID1. We think the miR-192/miR-215/NID1 signaling pathway may play an important role in the pathogenesis of HSCR.

Associations Between CYP2B6 rs707265, rs1042389, rs2054675, and Hirschsprung Disease in a Chinese Population.

BACKGROUND: Previous studies suggested that cytochrome P450 participated in the tumor metastasis and migration. CYP2B6 also acts as an important enzyme which metabolize partially or primarily metabolism of drugs, environmental contaminants, and mutagens. The objective of this study was to investigate the influence of CYP2B6 polymorphism on susceptibility of Hirschsprung disease. METHODS: TaqMan assay was performed to determine the genotypes of CYP2B6 rs707265, rs1042389, rs2054675 in 262 cases and 290 control subjects. Logistic regression was used to assess the associations between these polymorphisms and HSCR. RESULTS: We observed a significant association of CYP2B6 rs707265 (G>A) polymorphism and HSCR susceptibility (p < 0.001). Besides, rs707265 A presented a significant risk of HSCR (p < 0.001). CONCLUSIONS: Our result suggested that CYP2B6 rs707265 modified the risk of HSCR.