先天性小睑裂综合征中p.Y225X突变对FOXL2功能的影响  

Functional influence of p.Y225X mutation on FOXL2 in Blepharophimosis-ptosis-epicanthus-inversus syndrome

作　　者：耿玉磊[1] 王平章[2] 李冬梅[1] 

机构地区：[1]首都医科大学附属北京同仁医院 北京同仁眼科中心 北京市眼科学与视觉科学重点实验室,100730 [2]国家人类基因组北方研究中心

出　　处：《眼科》2013年第3期209-213,共5页Ophthalmology in China

基　　金：北京市自然科学基金(7102032)

摘　　要：目的研究p.Y225X突变对FOXL2功能的影响,探讨先天性小睑裂综合征(BPES)的可能发病机制。设计实验研究。研究对象正常和p.Y225X突变的FOXL2基因。方法克隆正常和p.Y225X突变的FOXL2基因编码序列,连接入绿色荧光蛋白表达载体pEGFP-N1,构建融合绿色荧光蛋白的表达载体,转染COS7细胞,在共聚焦显微镜下比较正常和突变型FOXL2在细胞内定位的差异。将正常和突变基因的编码序列克隆入pcDNA3.1-myc-his(-)B真核表达载体,转染HEK-293T细胞和KGN细胞后提取蛋白,用蛋白印迹法验证其过表达;同时提取细胞RNA和蛋白,用RT-PCR和蛋白印迹法分别检测OSR2和StAR在RNA和蛋白水平的改变。主要指标荧光蛋白在细胞内的定位,目的 RNA和蛋白的表达量。结果成功构建了FOXL2及其突变体的绿色荧光蛋白融合表达质粒和融合myc/his标签的真核表达质粒,经蛋白印迹法验证了FOXL2及其突变体在HEK-293T和KGN细胞内的过表达。正常FOXL2均匀分布于细胞核内,而发生p.Y225X突变的FOXL2突变体蛋白则均匀分布于细胞质和细胞核中。正常FOXL2可降低KGN细胞内StAR的RNA和蛋白表达量,而p.Y225X突变使其对StAR转录表达的抑制作用明显减弱。正常FOXL2可提高HEK-293T细胞内OSR2的RNA和蛋白表达量,而突变型FOXL2对OSR2的促进作用明显减弱。结论 p.Y225X突变使FOXL2分子在细胞内异常定位于细胞质,对靶基因OSR2和StAR的调控作用减弱,可能是导致I型BPES的重要因素。Objective To investigate the effect of p.Y225X mutation on FOXL2, and explore the potential mechanism of Blepharophimosis-ptosis-epicanthus-inversus syndrome （BPES）. Design Experimental study. Participants The wild-type and p.Y225X mutant FOXL2 genes. Method Wild-type and mutant sequence of FOXL2 genes were amplified, and then cloned into green fluorescence protein expression vector （pEGFP-N1） to construct GFP labeled expression vectors, which were later transfected into COS7 cells. The confocal microscope was used to locate FOXL2 in COS7 cells and compare the difference between them. Wild-type and mutant FOXL2 genes were cloned into pcDNA3.1-myc-his （-）B vector, then transfected into HEK-293T and KGN cells. Proteins were extracted to make sure that wild-type and mutant FOXL2 were expressed after transient transfection. And then total RNA and protein were extracted to analyze OSR2 and StAR expression on transcriptional and protein levels by RT-PCR and Western blotting. Main Outcome Measures GFP locations, target RNA and protein expression. Results The expression plasmids of wild-type and mutant FOXL2 labeled by GFP and myc/his flags were constructed successfully. The expression of FOXL2 was identified by Western blot assay in transfected HEK-293T and KGN cells. Under the eonfocal microscope, wild type FOXL2 evenly located in the nucleus, while the mutant FOXL2 located in both cytoplasm and nucleus. Furthermore, wild-type FOXL2 inhibited StAR in transcriptional and protein levels in KGN cells, while this inhibition effect in p.Y225X mutant were dramatically attenuated. Besides, wild-type FOXL2 enhanced OSR2 transcription and expression in HEK-293T cells, while the promotion of OSR2 expression in FOXL2 mutants was also reduced. Conclusion p.Y225X mutation, leading to FOXL2 dislocation in the cytoplasm, attenuates its regulation function, disturbs theexpression of target genes, OSR2 and STAR, which contribute to impaired development of eyelid and ovary, and probably eventually lead to type I BPES.

关 键 词：先天性小睑裂综合征 FOXL2 突变 

分 类 号：R777.1[医药卫生—眼科]