转录因子GTF2B对垂体生长激素腺瘤中AIP表达调控及对肿瘤表型影响的分子机制研究

Aryl hydrocarbon receptor-interacting protein (AIP) is an important tumor suppressor in GH-secreting pituitary adenoma. Clinically, there is a correlation between the expression of AIP and GHPA phenotype, such as tumor proliferation, invasiveness and drug resistence. Until now, it has not been clear about the mechanism how the expression of AIP is regulated transcriptionally. With the results of bioinformatics analysis and the preliminary experiments, the transcription factor GTF2B is indicated to be able to bind two different non-coding evolutionary conserved regions (ncECR) respectively, and to promote the transcription activity and expression of AIP consequently. Hence, based on these information, first of all, the correlation analysis of expression between AIP and GTF2B is going to be performed, using GHPA samples and xenografts in mice; Then, the combination of the predicted ncECR in AIP 5’UTR and GTF2B will be tested and verified in SH-SY5Y and 293T cells, followed by the research on the effect of it on the expression of AIP and tumor phenotypes in rat GHPA cell line GH3, in vitro and in vivo. It is expected that the transcriptional mechanism about AIP expression in GHPA could be revealed via this study, in order to provide theoretical basis and new therapeutic methods against low AIP-expression GHPA.

芳香烃受体相互作用蛋白(AIP)是垂体生长激素腺瘤(GHPA)中的一类重要抑癌因子。临床观察发现AIP表达水平与GHPA增殖、侵袭、及耐药等表型存在相关性。目前影响AIP表达水平的转录因子调控机制尚未见报道。前期生物信息学分析及预实验结果提示，转录因子GTF2B可与AIP的5’UTR非编码进化保守序列(ncECR)结合，促进AIP转录表达。本项目拟在此基础上，收集临床GHPA标本，并通过动物实验，观察分析GTF2B与AIP表达相关性；利用SH-SY5Y和293T工具细胞，验证GTF2B与AIP5’UTR中ncECR的相互结合作用以及对AIP表达调控的影响；最后，以大鼠GH3细胞为实验细胞，通过体内外实验，验证GTF2B对GHPA增殖、侵袭及耐药等肿瘤表型的影响。本项目旨在揭示GHPA中AIP低表达的转录调控机制，为将来临床治疗此类肿瘤提供思路和前期实验数据。

芳香烃受体相互作用蛋白(AIP)是垂体生长激素腺瘤(GHPA)中一类重要的抑癌因子。临床观察发现AIP表达水平与GHPA增殖、侵袭、及耐药等表型存在相关性。目前影响AIP表达水平的分子调控机制尚不十分清楚。我们经前期生物信息学分析及预实验结果提示，泛转录因子IIB (general transcript factor II B, GTF2B)可与AIP的5’UTR非编码进化保守序列(ncECR)结合，促进AIP转录表达。本项目在此基础上，收集临床GHPA标本56例，验证GTF2B与AIP表达量存在正相关性，同时，侵袭组、稀疏颗粒亚型组或Ki67≥3%组GHPA中GTF2B与AIP表达水平明显低于非侵袭组、致密颗粒亚型或Ki67<3%组，提示GTF2B和AIP表达水平与GHPA肿瘤发生发展呈负相关性；接下来，通过动物实验，利用Western Blot、qRT-PCR及免疫组化等验证发现GTF2B与AIP表达相关性及其对肿瘤增殖/侵袭等表型影响；并进一步利用SH-SY5Y工具细胞和GH3大鼠GHPA细胞系，通过ChIP和双荧光素酶报告实验验证GTF2B与AIP5’UTR中ncECR的相互结合作用以及对AIP表达调控的影响，而GTF2B (S65A)或AIP SNP (rs12271280或 rs1049506)组则未见明显调控变化；最后，以GH3细胞为实验细胞，通过体内外实验，验证GTF2B可通过促进AIP表达增加GHPA对奥曲肽等生长抑素类似物(SSAs)的敏感性(包括肿瘤体积缩小及相关蛋白表达变化)。综上，本项目研究结果揭示了GHPA中转录因子GTF2B对AIP表达的转录调控机制以及对GHPA肿瘤表型的影响。今后，希望能进一步开展中国GHPA人群中GTF2B基因/蛋白翻译后修饰等筛查研究，研发相关抗体、检测试剂盒及基因治疗方法等，用于基础研究、临床病例风险筛查及肿瘤药物增敏治疗，为丰富GHPA分子机制和完善临床GHPA诊治流程提供扎实的前期理论基础。