Background：Hirschsprung’s disease (HSCR) is a congenital colon disease characterized by the lack of ganglion cells which is closely related to impaired migration and proliferation of enteric neural crest cells (ENCCs). Long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) have been authenticated as an important class of regulators of biological functions. In this study, a microarray analysis was conducted and found that Rhabdomyosarcoma 2-associated transcript (RMST), a newly defined lncRNA, was down-regulated in the stenotic segment of HSCR patients. MiR-1251 is transcribed from the intron region of RMST and was also low expressed in the aganglionic tract. This study aimed to clarify the roles they played in the occurrence of HSCR.
Methods：qRT-PCR was applied to validate the mRNA expression of RMST, miR-1251, SOX2 and AHNAK, while western blotting was employed to evaluate the protein level of SOX2 and AHNAK in clinical samples and cells. CCK-8 and transwell assays were used to detect cell proliferation and migration after transfection. Chromatin immunoprecipitation (CHIP) was applied to confirm SOX2 could bind to the promoter region of miR-1251. RNA binding protein immunoprecipitation (RIP) was used to demonstrate the interaction between RMST and SOX2. Dul-luciferase reporter assay was performed to confirm miR-1251 could interact with AHNAK.
Results: When the expression of RMST or miR-1251 was reduced, cell proliferation and migration was attenuated. However, RMST not to influence the expression of miR-1251 directly. Through bioinformatic analysis, transcription factor SOX2 was predicted to bind to the promoter region of miR-1251 which was confirmed by CHIP assay. We also demonstrated that RMST exerted as a co-regulator of SOX2 via RIP assay. AHNAK was predicted as the downstream gene of miR-1251 confirmed by the dual-luciferase reporter assay. Furtherly, rescue experiments showed RMST functioned as a transcription co-regulator of SOX2 to upregulate the expression of downstream gene AHNAK by strengthening the regulation of SOX2 on miR-1251 in HSCR.
Conclusions: These findings uncovered the role of RMST/SOX2/miR-1251/AHNAK pathway during the development of Hirschsprung's disease and presented potential therapeutic targets for HSCR.