摘要
目的通过体外实验研究小眼球畸形相关转录因子(microphthalmia-associated transcription factor,MITF)突变基因功能,初步探讨其致Ⅱ型Waardenburg综合征(Waardenburg syndrome,WS)发病的分子机制。方法以野生型MITF基因真核细胞表达质粒pCMV-MITF-Flag为模板分别构建二个致Ⅱ型WS的MITF基因新发突变R217I和T192fsX18的真核细胞表达质粒。野生MITF和突变R217I和T192fsX18表达质粒瞬时转染黑色素瘤细胞或小鼠胚胎成纤维细胞(NIH3T3细胞),应用Western blot和细胞免疫荧光分别检测其表达和亚细胞定位;应用荧光素酶活性检测系统通过对酪氨酸酶(tyrosinase,TYR)报告基因活性检测观察野生/突变MITF蛋白对其靶基因TYR转录活性的调控作用,以及二个突变蛋白对野生MITF蛋白功能的影响;应用生物素标记的含E box基序(CATGTG)的DNA寡核苷酸链探针分别沉淀MITF、R217I及T192fsX18蛋白,检测野生/突变MITF蛋白与靶基因TYR启动子的结合力。结果成功构建了突变型R217I、T192fsX18真核细胞重组表达质粒pCMV-R217I-Flag和pCMV-T192fsX18-Flag,MITF蛋白与R217I、T192fsX18突变蛋白在黑色素瘤细胞中正确表达,进一步验证了重组质粒构建的正确性。突变R217I蛋白与野生MITF蛋白一样仅在细胞核中分布,而T192fsX18蛋白则出现异常亚细胞定位,仅在细胞质中分布。尽管R217I蛋白仍残余部分功能可增加TYR启动子转录活性,但与野生MITF蛋白相比,二者差异有显著统计学意义(P<0.01),而T192fsX18蛋白则完全失去调控TYR启动子转录活性作用(P<0.01);二者均未对野生MITF蛋白功能产生显性负效应(P>0.05)。突变R217I蛋白与野生MITF蛋白均可与TYR启动子特异DNA序列E-box结合,而突变T192fs X18蛋白则不能与之结合。结论 R217I和T192fsX18突变蛋白通过影响靶基因TYR转录活性,使其表达下调、黑色素合成减少,以单倍体剂量不足效应致Ⅱ型WS。
Objective To investigate molecular mechanism of Waardenburg syndrome type Ⅱ resulting from microphthalmia--associated transcription factor （MITF） gene mutations via its functional analysis in vitro. Methods We generated two mutant expression plasmids of novel mutations R217I and T192fsX18 of MITF resulting in Waardenburg syndrome type Ⅱ by using eukaryotic expression plasmid pCMV--MITF--Flag as a templet. The melanoma cells were transient transfected with wild type （WT） MITF and mutant type （MT） R217I, T192fsX18 plasmids. We observed their exogenous expressions by Western blot and subcelluar localization by immunofluores- cence, analyse regulation effects of WT/MT MITF on the transcriptional activities of tyrosinase （TYR） gene and the influence of the two mutants on WT MITF function using luciferase activity assays, detected DNA binding ca- pacity of WT/MT MITF to TYR gene promoter using a biotinylated double--stranded oligonucleotide probe contai- ning MITF binding sequence E box （CATGTG） to precipitate MITF,R217I and TlO2fsX18, respectively. Results We successfully generated mutant expression plasmids pCMV--R217I--Flag and pCMV--T192fsX18--Flag, which were detected at the expected size in the melanoma cells, and further verified the mutant plasmids. WT MITF and R217I mutant were only localized in the nucleus of NIH3T3 cells, whereas T192fsX18 mutant showed localization only in the cytoplasm. R217I mutant was partially functional and was able to transactivate the TYR promoter in part, but it was dramatically reduced as compared with WT MITF （P〈0.01）. T192fsX18 mutant was loss--of-- function and failed to transactivate the TYR promoter as compared with WT MITF （P〈0. 01）. None of them showed dominant--negative effect on WT MITF（P〉0.05）. WT MITF and R217I mutant were able to bind specif- ically to the E box motif on the TRY promoter, whereas T192fsX18 MITF lost the DNA--binding ability. Conclu- sion The mutations R217I and T192fsX18 made down--regulation of TYR transcripti
出处
《听力学及言语疾病杂志》
CAS
CSCD
北大核心
2014年第4期378-385,共8页
Journal of Audiology and Speech Pathology
基金
国家自然科学基金(81260160
81170923)资助
