Binyu Wang1, Xiang Fang1, Xinhe Sun2, Chunxia Du1, Lingling Zhou1, Xiurui Lv3, Yuhan Li1, Hongxing Li2, Weibing Tang4. 1. Department of Pediatric Surgery, Children's Hospital of Nanjing Medical University, Nanjing 210000, Jiangsu, China; State Key Laboratory of Reproductive Medicine, Center for Global Health, Key Laboratory of Modern Toxicology of Ministry of Education, School of Public Health, Nanjing Medical University, Nanjing 210029, Jiangsu, China. 2. Department of Pediatric Surgery, Children's Hospital of Nanjing Medical University, Nanjing 210000, Jiangsu, China. 3. Department of Pediatric Surgery, Children's Hospital of Nanjing Medical University, Nanjing 210000, Jiangsu, China; State Key Laboratory of Reproductive Medicine, Center for Global Health, Key Laboratory of Modern Toxicology of Ministry of Education, School of Public Health, Nanjing Medical University, Nanjing 210029, Jiangsu, China; School of Medicine & Dentistry, University of Rochester NY 14642, NY, USA. 4. Department of Pediatric Surgery, Children's Hospital of Nanjing Medical University, Nanjing 210000, Jiangsu, China. Electronic address: twbcn@njmu.edu.cn.
Abstract
OBJECTIVES: This study aims to investigate the role of demethylase ALKBH5 mediated demethylation of TAGLN mRNA in the occurrence of Hirschsprung's disease (HSCR), and to clarify how ALKBH5 reduces the m6A level of TAGLN mRNA and inhibits its degradation, thereby inhibiting the proliferation and migration of neural crest cells, and potentially contributing to the occurrence of HSCR. MATERIAL AND METHODS: Quantitative real-time PCR (qRT-PCR) and Western-Blot (WB) were conducted to test the expression level of ALKBH5 and TAGLN genes. Cell function assays were adopted to detect cell phenotypes. The qRT-PCR and methylated RNA immunoprecipitation (MeRIP-qPCR) were used to test the regulation of TAGLN by ALKBH5. RESULTS: 1. Compared with control intestinal tissue, the expression level of TAGLN and ALKBH5 in the aganglionic intestinal tissue of HSCR is increased. 2. The MeRIP-PCR and dualluciferase report confirmed that ALKBH5 could bind to m6A sites of TAGLN mRNA and reduce the m6A level of TAGLN mRNA. 3. In vitro cell experiments confirmed that overexpression of ALKBH5 can inhibit the degradation of TAGLN mRNA, increase the expression of TAGLN, thereby inhibiting cell proliferation and migration. 4. A zebrafish model of ALKBH5 overexpression was constructed. Studies have shown that ALKBH5 could inhibit the proliferation and migration of zebrafish enteric neurons. CONCLUSIONS: ALKBH5 could demethylate TAGLN mRNA and up-regulate TAGLN expression, leading to the inhibition of proliferation and migration of enteric neural crest cells and contributing to the occurrence of HSCR.
OBJECTIVES: This study aims to investigate the role of demethylase ALKBH5 mediated demethylation of TAGLN mRNA in the occurrence of Hirschsprung's disease (HSCR), and to clarify how ALKBH5 reduces the m6A level of TAGLN mRNA and inhibits its degradation, thereby inhibiting the proliferation and migration of neural crest cells, and potentially contributing to the occurrence of HSCR. MATERIAL AND METHODS: Quantitative real-time PCR (qRT-PCR) and Western-Blot (WB) were conducted to test the expression level of ALKBH5 and TAGLN genes. Cell function assays were adopted to detect cell phenotypes. The qRT-PCR and methylated RNA immunoprecipitation (MeRIP-qPCR) were used to test the regulation of TAGLN by ALKBH5. RESULTS: 1. Compared with control intestinal tissue, the expression level of TAGLN and ALKBH5 in the aganglionic intestinal tissue of HSCR is increased. 2. The MeRIP-PCR and dualluciferase report confirmed that ALKBH5 could bind to m6A sites of TAGLN mRNA and reduce the m6A level of TAGLN mRNA. 3. In vitro cell experiments confirmed that overexpression of ALKBH5 can inhibit the degradation of TAGLN mRNA, increase the expression of TAGLN, thereby inhibiting cell proliferation and migration. 4. A zebrafish model of ALKBH5 overexpression was constructed. Studies have shown that ALKBH5 could inhibit the proliferation and migration of zebrafish enteric neurons. CONCLUSIONS:ALKBH5 could demethylate TAGLN mRNA and up-regulate TAGLN expression, leading to the inhibition of proliferation and migration of enteric neural crest cells and contributing to the occurrence of HSCR.