MITF基因突变致Ⅱ型Waardenburg综合征发病的实验研究

发布时间：2018-04-26 03:08

本文选题：Waardenburg综合征 + 小眼球畸形相关转录因子　；参考：《听力学及言语疾病杂志》2014年04期

【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(P0.01),而T192fsX18蛋白则完全失去调控TYR启动子转录活性作用(P0.01);二者均未对野生MITF蛋白功能产生显性负效应(P0.05)。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。
[Abstract]:Objective to investigate the molecular mechanism of the pathogenesis of Waardenburg syndrome (WS) induced by Waardenburg syndrome (Waardenburg syndrome, WS) by studying the function of microphthalmia-associated transcription factor (MITF) mutation gene in vitro. Methods the expression plasmid pCMV-MITF-Flag of the wild type MITF eukaryotic expression plasmid was used as a model. Two new MITF gene mutation R217I and T192fsX18 eukaryotic expression plasmids were constructed respectively. Wild MITF and mutant R217I and T192fsX18 expressed plasmids transiently transfected melanoma cells or mouse embryonic fibroblasts (NIH3T3 cells). Western blot and cell immunofluorescence were used to detect their expression and subcells respectively. Localization; using the luciferase activity detection system to detect the transcriptional activity of the wild / mutant MITF protein on its target gene TYR through the detection of tyrosinase (tyrosinase, TYR) gene activity, and the effect of the two mutant proteins on the function of the wild MITF protein, and the DNA oligoside containing the E box motif (CATGTG) labeled with biotin The acid chain probes precipitated MITF, R217I and T192fsX18 proteins to detect the binding force between the wild / mutant MITF protein and the target gene TYR promoter. The mutant R217I, the T192fsX18 eukaryotic recombinant expression plasmid pCMV-R217I-Flag and pCMV-T192fsX18-Flag, MITF protein and R217I, and the T192fsX18 mutant protein in the melanoma cells were successfully constructed. It was confirmed that the recombinant plasmid was correct in the construction of the recombinant plasmid. The mutant R217I protein was only distributed in the nucleus as in the wild MITF protein, while the T192fsX18 protein appeared abnormal subcellular localization and only in the cytoplasm. Although the residual function of the R217I protein could increase the transcriptional activity of the TYR promoter, it was associated with the wild MITF protein. The differences of the two were statistically significant (P0.01), while T192fsX18 protein completely lost the transcriptional activity of TYR promoter (P0.01); the two had no dominant negative effect on the function of wild MITF protein (P0.05). The mutant R217I protein and the wild MITF protein could be combined with the TYR promoter specific DNA sequence, and the mutant T192fs eggs were mutated. White can not be combined with it. Conclusion R217I and T192fsX18 mutant proteins can reduce the expression of TYR by affecting the transcriptional activity of the target gene, decrease the synthesis of melanin, and cause the type II WS. by the effect of the haploid dose deficiency.

【作者单位】：新疆医科大学第一附属医院耳鼻咽喉科;中南大学湘雅医院耳鼻咽喉科;中南大学医学遗传学国家重点实验室;
【基金】：国家自然科学基金(81260160,81170923)资助
【分类号】：R764.43

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