摘要
目的研究星形胶质细胞瘤异柠檬酸脱氢酶1(isocitrate dehydrogenase 1,IDH1)突变对TAZ(transcriptional coactivator with PDZ-binding motif)蛋白的影响,并探讨相关机制。方法采用稳定转染突变型IDH1-132H(132H)及野生型IDH1-132R(WT)的胶质母细胞瘤(U87MG)细胞,通过Western blot法检测细胞内TAZ蛋白表达;免疫组化法检测14例IDH1突变阴性和7例IDH1突变阳性的人脑胶质母细胞瘤组织样本中TAZ蛋白及其胞质结合蛋白14-3-3e的表达差异;qRT-PCR观察两组细胞TAZ在mRNA水平有无差异;Western blot方法检测Hippo信号通路核心激酶LATS1及磷酸化TAZ蛋白的表达,以及14-3-3e蛋白的表达。结果 Western blot结果表明IDH1突变的星形胶质细胞瘤中TAZ蛋白表达降低;人体胶质细胞瘤组织的免疫组织化学结果与Western blot一致,证实了IDH1突变的星形胶质细胞瘤中TAZ胞浆结合蛋白14-3-3e表达升高;qRT-PCR发现IDH1突变细胞TAZ mRNA水平相比野生型细胞表达明显降低;IDH1突变细胞中,LATS1以及TAZ磷酸化水平升高且细胞14-3-3e蛋白表达升高。结论星形胶质细胞瘤IDH1突变导致TAZ mRNA及蛋白水平表达降低,并通过活化Hippo信号通路影响TAZ蛋白的磷酸化水平从而影响TAZ表达。
Objective To investigate the effects of mutated isocitrate dehydrogenase 1 （IDHl） on TAZ （transcriptional coactivator with PDZ-binding motif protein） expression in human astrocytoma and to explore the relevant mechanisms. Methods Glioblastoma （GBM） U87MG cells transfected with mutated IDH1-132H and wild type IDH1-132R （WT） were used to detected the TAZ protein expression by Western blot;The expression of TAZ protein and its cytoplasmic binding protein 14-3-3e were investigated in a cohort of GBM cases,which including 14 IDH1 wild type cases and 7 IDH1 mutated cases.In vitro,qRT-PCR was used to detect TAZ mRNA expression;Western blot was used to detect the levels of LATS1,phosphorylated TAZ and 14-3-3e protein. Results We found that the expression of TAZ protein was decreased in IDH1 mutated cells compared with those of IDH1 wild type cells.This result was further verified in vivo by using immunohistochemistry to detect the expression of TAZ in human GBM samples with or without IDH1 mutation.Immunohistochemistry also showed that TAZ cytoplasmic binding protein 14-3-3e was increased;qRT-PCR showed that TAZ mRNA level was decreased in IDH1 mutated cells;and subsequent Western blot showed the levels of LATS1,TAZ phosphorylation and the 14-3-3e were increased in IDH1 mutant cells. Conclusions IDH1 mutation in gliomacould reduce TAZ mRNA and its protein expression,increase the phosphorylation level of TAZ protein by activating Hippo signaling pathway,thereby furtherinhibit TAZ expression.
出处
《复旦学报（医学版）》
CAS
CSCD
北大核心
2016年第4期379-384,共6页
Fudan University Journal of Medical Sciences
基金
国家自然科学基金面上项目(81272796)~~
