Abstract： Objective:To explore the mechanism of maternally expressed gene 3(MEG3)and Rac1/Cdc42/PAK1 signaling pathway in the pathogenesis of Hirschsprung disease(HSCR).Methods:From January 2016 to December 2018, colonic tissue specimens of 30 operated children with pathologically confirmed HSCR were collected as experimental group(HSCR group). There were 23 boys and 7 girls with an age of(18.86±1.78)months and a body weight of(4.56±0.19)kg.Fourteen normal intestinal specimens from children with non-congenital digestive tract malformations and non-intestinal nerve-related diseases, including necrotizing enterocolitis, incarcerated hernia and intussusception fistula, were taken as control group(normal group). There were 10 boys and 4 girls with an age of(16.73±1.03)months and a weight range of(5.01±0.20)kg.Quantitative real-time polymerase chain reaction(qRT-PCR)and Western blot were employed for detecting the expressions of MEG3 and Rac1/Cdc42/PAK1 signal pathway proteins in HSCR and normal groups.And cell model was constructed by transfecting MEG3 over-expression plasmid and adding pathway inhibitor AZA1 in SH-SY5Y cell.There were four groups of blank control(only SH-SY5Y cell), empty vector(pcDNA3.1, SH-SY5Y cell + pcDNA3.1 plasmid), EG3 over-expression(MEG3, SH-SY5Y cells + pcDNA3.1-MEG3 plasmid)and inhibitor(MEG3+ AZA1, SH-SY5Y cell + pcDNA3.1-MEG3+ AZA1 inhibitor). The protein expressions of Rac1, Cdc42, PAK1 and P-PAK1 and the capabilities of cell proliferation and migration were detected by Western blot, CCK-8 and Transwell methods.For measurement data with normal distribution or approximate normal distribution, two groups of data were compared by two independent samples T test after variance homogeneity test.And one-way ANOVA was utilized for comparing means among multiple groups.If population variance was non-uniform, modified limit value or Tamhane's T2 test was adopted; For data of abnormal distribution, rank sum test of multiple independent samples was adopted.Results:The expression of MEG3 and protein expression levels of Rac1, Cdc42, PAK1 and P-PAK1 were lower in HSCR group than those in normal group(Rac1: 0.20±0.05 vs 0.78±0.05, t=8.37; Cdc42: 0.38±0.08 vs 1.03±0.08, t=5.68; AK1: 0.27±0.02 vs 1.10±0.06, t=13.52; P-PAK1: 0.24±0.03 vs 1.07±0.06, t=12.99, all P<0.01). In the cell model, the protein expression levels of Rac1, Cdc42, PAK1 and P-PAK1 were higher in MEG3 group than those in control and MEG3+ AZA1 groups(Rac1: 0.96±0.05 vs 0.36±0.11 vs 0.75±0.20, F=3.46, P<0.05; Cdc42: 1.12±0.08 vs 0.53±0.19 vs 0.68±0.10, F=5.37, P<0.01; PAK1: 1.02±0.09 vs 0.55±0.14 vs 0.47±0.14, F=5.60, P<0.05; P-PAK1: 0.97±0.07 vs 0.66±0.08 vs 0.47±0.03, F=15.66, P<0.05). The expression of MEG3 was significantly higher in MEG3 group than that in control and pcDNA3.1 groups(1.00±0.20 vs 0.14±0.04 vs 0.02±0.01, F=20.56, P<0.01). The expression of MEG3 was not significantly different in MEG3 group from that in control/pcDNA3.1 group( P>0.05). Cell proliferation capability was higher in MEG3 group than that in control/(MEG3+ AZA1)group(12 h: 0.19±0.00 vs 0.19±0.00 vs 0.18±0.00, P>0.05; 24 h: 0.21±0.00 vs 0.19±0.01 vs 0.17±0.00, F=12.00, P<0.01; 48 h: 0.30±0.02 vs 0.26±0.00 vs 0.22±0.01, F=9.60, P<0.05). The number of cell migration was higher in MEG3 group than that in control group(141.67±11.93 vs 96.33±8.02, t=3.15, P<0.05). The number of cell migration was lower in MEG3+ AZA1 group than that in MEG3/control group(71.00±7.00 vs 141.67±11.93 vs 96.33±8.02, F=15.04, P<0.01). Conclusions:A down-regulation of MEG3 probably suppresses the proliferation and migration of neural crest cells through Rac1/Cdc42/PAK1 signaling pathway in the pathogenesis of HSCR.