Meg3在全反式视黄酸诱发小鼠腭裂中的作用  

Role of maternally expressed gene 3 in all-trans retinoic acid-induced cleft palate in mice

作　　者：刘小转 张军喜 余增丽 王国旭 何志东 宋帅星 李雪 LIU Xiaozhuang;ZHANG Junxi;YU Zengli;WANG Guoxu;HE Zhidong;SONG Shuaixing;LI Xue(Clinical Medical Research Center,Henan Provincial People′s Hospital,Zhengzhou 450003;NHC Key Laboratory of Birth Defects Prevention,Henan Key Laboratory of Population Defects Prevention,Zhengzhou 450002;Department of Nutrition and Food Hygiene,College of Public Health,Zhengzhou University,Zhengzhou 450001)

机构地区：[1]河南省人民医院临床医学研究中心,郑州450003 [2]国家卫生健康委员会出生缺陷预防重点实验室,河南省人口缺陷干预技术研究重点实验室,郑州450002 [3]郑州大学公共卫生学院营养与食品卫生学教研室,郑州450001

出　　处：《郑州大学学报（医学版）》2023年第3期332-337,共6页Journal of Zhengzhou University(Medical Sciences)

基　　金：国家自然科学基金项目(81801547);河南省科技攻关省部共建重点项目(SBGJ202102014);国家卫生健康委员会出生缺陷预防重点实验室&河南省人口缺陷干预技术研究重点实验室开放基金项目(ZD202203)。

摘　　要：目的:探索母系印迹基因3(Meg3)在全反式视黄酸(atRA)诱发腭裂中的作用。方法:妊娠第10天(GD10),将90只孕鼠随机分为atRA组和对照组(每组45只),分别给予100 mg/kg atRA和等体积的玉米油灌胃。HE染色观察GD13、GD14、GD15胎鼠腭部发育情况,BrdU荧光染色观察GD13、GD14、GD15胎鼠腭突间充质细胞增殖情况。在GD14,荧光原位杂交和实时荧光定量PCR法检测两组胎鼠腭突间充质细胞中Meg3的定位和表达,蛋白印迹法检测Smad通路相关蛋白磷酸化Smad2(p-Smad2)、Smad2、Smad4和Smad7的表达,RNA结合蛋白免疫沉淀实验检测Meg3基因与Smad2蛋白结合情况。结果:Meg3基因主要定位于胎鼠腭突间充质细胞的细胞核。与对照组相比,在GD14,atRA组细胞中Meg3 mRNA表达量升高(P<0.001),p-Smad2和Smad4蛋白表达水平降低(P<0.05),而Smad7蛋白表达水平增加(P<0.001);两组细胞中Meg3基因均可与Smad2蛋白结合,Meg3 mRNA相对表达量atRA组大于对照组(P<0.001)。结论:atRA可能通过促进Meg3与Smad2的结合,影响TGF-β/Smad信号通路的激活,抑制胎鼠腭突间充质细胞的增殖,从而导致腭裂的发生。Aim:To explore the effect of maternally expressed gene 3(Meg3)in all-trans retinoic acid(atRA)-induced cleft palate.Methods:At gestation day 10(GD10),ninety pregnant mice were randomly allocated into atRA group and control group(45 mice in each group),100 mg/kg atRA and equal volume of corn oil were given by gavage,respectively.At GD13,GD14,GD15,the development of fetal mice palate was observed by HE staining,embryonic palatal mesenchymal(MEPM)cells proliferation was detected by BrdU immunofluorescence staining.The localization and expression of Meg3 in MEPM cells at GD14 were detected by fluorescence in situ hybridization and real-time quantitative PCR.The phospho-Smad2(p-Smad2),Smad2,Smad4 and Smad7 protein expressions in MEPM cells were analyzed by Western blot.The interaction of Meg3 gene and Smad2 protein was examined by RNA binding protein immunoprecipitation(RIP).Results:Meg3 was mainly expressed in the nuclei of MEPM cells.Compared with those of control group,at GD14,the expression of Meg3 in atRA group was significantly higher(P<0.001),the protein expression levels of p-Smad2 and Smad4 were significantly lower(P<0.05),while the expression level of Smad7 was significantly higher(P<0.001).RIP results revealed that atRA significantly promoted the binding of Meg3 gene and Smad2 protein in MEPM cells(P<0.001).Conclusion:AtRA might suppress MEPM cell proliferation via suppressing Meg3 interacting with Smad2 protein,and thereby suppressing TGF-β/Smad signaling pathway,and induce cleft palate.

关 键 词：腭裂 全反式视黄酸 母系印迹基因3 小鼠 腭突间充质细胞 SMAD蛋白 

分 类 号：R782[医药卫生—口腔医学]