INPP5E 基因影响小鼠胚胎神经管闭合的 分子机制研究

Molecular mechanism of INPP5E gene on embryonic neural tube closure in mice

摘要:

目的 探讨肌醇多聚磷酸5- 磷酸酶（INPP5E）基因影响小鼠胚胎神经管闭合的分子机制。 方法 采用前期复制的神经管缺陷（NTD）小鼠模型，在体视显微镜下观察胚胎表型、苏木精- 伊红染色情况， 测量胚胎顶臀长、体重等指标；重亚硫酸盐测序法检测NTD 组及对照组胚胎发育第11.5 天小鼠胚胎神经组 织中INPP5E 基因启动子区DNA 甲基化情况；实时荧光定量聚合酶链反应和Western blotting 检测胚胎神经组 织中INPP5E 蛋白和mRNA 相对表达量；超高效液相色谱串联质谱法检测胚胎神经组织中叶酸（FA）、5- 甲 基四氢叶酸（5-MeTHF）、5- 甲酰四氢叶酸（5-FoTHF）及同型半胱氨酸（Hcy）水平。结果 NTD 组小 鼠胚胎神经组织INPP5E 基因启动子区甲基化水平、INPP5E 蛋白和mRNA 相对表达量在胚胎发育第11.5 天 时较对照组低（P <0.05）。NTD 组胚胎神经组织的FA 和Hcy 水平较对照组升高（P <0.05），而5-MeTHF 和5-FoTHF 水平较对照组低（P <0.05）。结论 INPP5E 基因在调节小鼠胚胎神经管闭合中起重要作用。FA 代谢紊乱引起的INPP5E 基因启动子区甲基化水平降低，可能通过影响其表达水平参与NTD 的发生。

Abstract:

Objective To investigate the molecular mechanism of inositol polyphosphate 5-phosphtase IV (INPP5E) gene on embryonic neural tube closure in mice. Methods All NTD embryos were examined carefully using a dissecting microscope. The structure changes of neural tube tissues were detected by HE stain, the crownrump length and body weight of embryos were measured. Bisulphite sequencing (BS) was used to detect the DNA methylation level within the promoter region of INPP5E in embryonic neural tissues of NTD and control group on embryonic day 11.5 (E 11.5). Real-time quantitative polymerase chain reaction (RT-PCR) and western blot was used to detect the transcription and expression levels of the INPP5E gene in neural tube tissues on E 11.5. UPLC-MS/ MS was used to detect the level of folic acid (FA), 5-methyltetrahydrofolate (5-MeTHF), 5-formyltetrahydrofolate (5-FoTHF) and homocysteine (Hcy) in embryonic neural tissues. Results Compared with control group, the methylation level of INPP5E promoter region in embryonic neural tissues were lower in NTD group on E 11.5 (P < 0.05), and the transcription level and the expression level of INPP5E were decreased (P < 0.05). Meanwhile, the concentrations of FA (2.76±0.98 VS 4.39±1.41 mg/g) and Hcy (4.33±1.42 VS 7.06±1.38 mg/g) increased (P < 0.05), and the concentrations of 5-MeTHF (9.55±2.38 VS 4.62±1.98 mg/g) and 5-FoTHF (4.86±1.02 VS 3.19±0.75 mg/g) decreased in embryonic neural tissues in the NTD group compared with the control group (P < 0.05). Conclusions The INPP5E gene plays an important role in regulation of embryonic neural closure. Reduced methylation level in INPP5E promoter region caused by folic acid metabolic disorder may be involved in the etiology of NTD by affecting its expression.