Involvement of the lncRNA AFAP1-AS1/microRNA-195/E2F3 axis in proliferation and migration of enteric neural crest stem cells of Hirschsprung's disease

New Findings What is the central question of this study?Long non-coding RNAs (lncRNAs) are widely involved in the progression of Hirschsprung's disease (HSCR), but the role of actin filament associated protein 1 antisense RNA1 (AFAP1-AS1), an lncRNA, in HSCR has not been explored before. What is the main finding and its importance?Downregulation ofAFAP1-AS1blocks enteric neural crest stem cell proliferation, differentiation, migration and invasion and promotes the occurrence of HSCR via themiR-195/E2F3axis, indicating thatAFAP1-ASmight be a potential biomarker for HSCR patients. Long non-coding RNAs (lncRNAs) are involved in several human disorders. Nevertheless, it remains unclear whether they are implicated in the phenotypes of enteric neural crest stem cells (ENCSCs) in Hirschsprung's disease (HSCR). Therefore, we designed this study to explore the pathogenicity ofAFAP1-AS1for HSCR. Microarray analysis and bioinformatic tools were used to screen out the differentially lncRNAs and microRNAs (miRNAs) in patients with HSCR. Small interference RNA transfection was applied to carry out functional experiments in ENCSCs. Cellular activities were detected by cell counting kit-8, 5-ethynyl-2 '-deoxyuridine, Transwell assays and flow cytometry. Finally, rescue experiments were performed to examine the cofunction ofAFAP1-AS1andmiR-195and ofmiR-195and E2F transcription factor 3 (E2F3).AFAP1-AS1was reduced in HSCR patients. Meanwhile, knockdown ofAFAP1-AS1reduced the cell migratory and proliferative capacities and facilitated cell apoptosis along with G0/G1 phase arrest.E2F3was diminished whenmiR-195was upregulated, andAFAP1-AS1inhibition reduced its ability to bind tomiR-195. Altogether,AFAP1-AS1silencing acts as an endogenous RNA by interacting withmiR-195to alterE2F3expression, thus conferring repressive effects on ENCSC activity and promoting HSCR progression.