启动子DNA超甲基化与先天性巨结肠患儿结肠组织中GFRα1低表达的关系研究

Promoter DNA hypermethylation leads to a low expression of GFRα1 in Hirschsprung's disease

目的探讨DNA甲基化在调控先天性巨结肠(Hirschsprung's disease,HSCR)患儿肠道组织中GFRα1基因表达及HSCR发病过程中的可能作用。方法对2016年3月至2018年6月收治的24例HSCR患儿(HSCR组)及同期18例新生儿坏死性小肠结肠炎患儿(对照组)的手术样本进行对比分析。采用实时定量PCR及免疫印迹实验检测两组患儿结肠组织内GFRα1、DNMT1、DNMT3A和DNMT3B的表达水平。采用亚硫酸氢盐处理后测序检测两组患儿结肠组织内GFRα1启动子区DNA甲基化水平。结果HSCR组GFRα1 mRNA及蛋白的相对表达量分别为0.38±0.17和0.55±0.13,均较对照组(0.97±0.36和0.99±0.16)显著降低,组间比较,差异有统计学意义(P<0.01)。HSCR组GFRα1启动子区DNA甲基化水平较对照组显著升高(0.67±0.23比0.34±0.18),组间比较,差异有统计学意义(P<0.01)。相关性分析显示,DNA甲基化水平与其mRNA表达水平呈明显负相关(r=-0.477,P<0.05)。HSCR组DNA甲基转移酶DNMT3B mRNA及蛋白的相对表达量分别为2.19±0.54和2.83±0.54,均较对照组(1.02±0.33和1.69±0.57)显著上升,组间比较,差异有统计学意义(P=0.003和0.021)。相关性分析显示,DNMT3B蛋白表达水平与GFRα1启动子区DNA甲基化水平显著正相关(r=0.408,P<0.05)。结论巨结肠患儿结肠组织中DNA甲基转移酶DNMT3B的过度表达可能引起GFRα1启动子区DNA超甲基化,导致GFRα1表达缺陷,这可能是HSCR发病的重要原因。

Objective To reveal explore possible role of DNA methylation in regulating the expression of GFRα1 and the pathogenesis of Hirschsprung's disease(HSCR)by detecting the expression level and promoter DNA methylation of GFRα1 in intestinal tissue of HSCR children.Methods Twenty-four surgical specimens of HSCR children were randomly selected and 18 surgical specimens of necrotizing enterocolitis were used as control.Real-time polymerase chain reaction(PCR)and Western blot were employed for detecting the expression levels of GFRα1,DNMT1,DNMT3A and DNMT3B in colonic tissues of HSCR children and control group.The DNA methylation level of GFRα1 promoter region in colonic tissue of HSCR children and control group was detected by bisulfite pretreatment sequencing(BSP).Results The relative mRNA and protein level of GFRα1 in HSCR group were lower than those in control group[(0.38±0.17&0.55±0.13)vs.(0.97±0.36&0.99±0.16)].The difference was statistically significant(P<0.01).The DNA methylation level of GFRα1 promoter was significantly higher in HSCR group than that in control group(0.67±0.23 vs.0.34±0.18).The inter-group difference was statistically significant(P<0.01)and DNA methylation level was negatively correlated with mRNA expression level(r=-0.477,P<0.05).The relative expression of DNMT3B mRNA and protein was 2.19±0.54 and 2.83±0.54 in HSCR group respectively.Both were significantly higher than those in control group(1.02±0.33&1.69±0.57).The inter-group difference was statistically significant(P=0.003,0.021).And the expression level of DNMT3B protein was positively correlated with the level of DNA methylation in GFRα1 promoter(r=0.408,P<0.05).Conclusions The overexpression of DNA methyltransferase DNMT3B in colonic tissue leads to DNA hypermethylation in promoter region of GFRα1 and results in a defective expression of GFRα1.It may be an important pathogenic cause of HSCR.