The M~6A Methyltransferase METTL3 Affects Cell Proliferation And Migration By Regulating YAP Expression In Hirschsprung Disease

Background:Hirschsprung disease(HSCR)is a neurocristopathy that occurs in approximately 1 in 5,000 newborns.This disorder has a complex multifactorial or polygenic etiology,and displays a tendency for non-Mendelian,sex-modified inheritance.The N6-methyladenosine(m6A)modification,catalyzed by a RNA methyltransferase complex comprising METTL3,METTL14,and WTAP,is the most common internal mRNA modification in eukaryotes and is present in over 50%of all methylated ribonucleotides.METTL3,a mRNA m6A methyltransferase,has been implicated in various steps of mRNA metabolism,such as stabilization,splicing,nuclear transportation,translation,and degradation.Yes-associated protein(YAP),a downstream effector of the Hippo pathway,is activated in early,pre-migratory neural crest cells and is associated with terminal neuronal differentiation in humans.In this study,we quantified m6A levels in stenotic and dilated tissues from patients with HSCR and explored the function of METTL3 and YAP in HSCR in vitro,finally further confirm the relation between METTL3 and YAP.Methods:12 children with HSCR were included.Stenotic segments(HSCR-S)and dilated segments(HSCR-D)were obtained from the intestines.The gene expression levels of YAP and several methyltransferases,demethylases,and effectors were evaluated by RT-qPCR.Protein levels were evaluated by western blot and immunohistochemistry.Cell proliferation and migration were detected by CCK-8 and Transwell assays,respectively.The overall levels of m6A modification were determined by colorimetry.Results:We found that m6A levels were reduced in stenotic intestinal tissue of patients with HSCR.When METTL3 was knocked down in SH-SY5Y and HEK293T cells,the proliferative and migratory abilities of the cells were inhibited,m6A modification levels were reduced,and YAP expression was increased.Importantly,YAP and METTL3 expression displayed a negative correlation in both cell lines as well as in HSCR tissue.Conclusion:Our results provide evidence for an interaction between METTL3 and YAP in HSCR,and further suggest that METTL3 is involved in the pathogenesis of HSCR by regulating neural crest cell proliferation and migration upstream of YAP.