溴区包含蛋白1在脑硫脂调控整合蛋白αV亚基中的作用研究

Integrin αV subunit forms the adhesion molecule αVβ3 heterodimers through interaction with integrin β3 subunit, which is one of the most important molecules in tumor metastasis and angiogenesis. Integrin αV expression is functionally involved in the maintenance of a highly migratory, mesenchymal cellular phenotype as well as the acquisition of a stem/progenitor phenotype in human cancer cells with metastasis-initiating capacity. A change of the expression and distribution of integrin αV subunit in the tumor cells can modulate the strength of cell adhesion, migration, and metastasis..Our group first observed that abnormally elevated sulfatide in hepatocellular carcinoma (HCC) can promote integrin αV gene expression, but the detail mechanism of the regulation by sulfatide remains largely unknown. Through our preliminary investigation, we observed that sulfatide produced by cerebroside sulfotransferase, can stimulate the expression of integrin αV which forms αVβ3 on the membrane of HCC cells. An analysis of the human integrin αV subunit gene promoter based on Transfac website predicted Sp1 binding sites. Further experiments demonstrated that Sp1 is recruited to the promoter of integrin αV subunit gene and involved in the regulation of the promoter activity. Interestingly in our preliminary experiments, we observed that histone 3 K9/14 and Sp1 acetylation were regulated after sulfatide treatment in HCC. The acetylation events of histones and transcription factors are important in the transcription regulation for a coding gene. Next in our preliminary study, we cross-linked sulfatide to the beads and incubated them with the proteins from whole cell lysate. In the precipitation we identified bromodomain-containing protein 1 (BRD1) via mass spectrometer , which is reported to belong to the family of BRPF protein which is a subunit of histone acetyltransferase (HAT) complex. BRD1, the member of BRPF family bridges MOZ/MORF with ING5 and EAF6, respectively, to constitute histone acetyltransferase (HAT) complex. Therefore, it is possible that sulfatide interacts with BRD1, a subunit of HAT, to regulate acetylation on histone and Sp1 proteins. Acetylated histones and Sp1 are important for gene expression regulation of integrin protein αV subunit. To test this hypothesis, we will analyze Sp1 and histones H3K9, K14, and H4K12 acetylation after sulfatide treatment. Through the assays of immunoprecipitation, fluorescence co-localization, distribution, and mass spectrometry, we will determine and confirm the interaction between sulfatide and BRD1. By gene mutation, we will determine the functional area of BRD1 molecule that is responsible for the binding of Sp1. Through small interference RNA, we will determine the role of BRD1 in sulfatide regulation of acetylation. This project will be from a new point to uncover molecular mechanism of sulfatide regulating integrin αV expression, and demonstrate BRD1 is a new ligand protein of sulfatide.

整合蛋白αVβ3是参与肿瘤血管形成和转移过程的重要分子，本课题组首先发现了在肝癌细胞中异常升高的脑硫脂能够促进整合蛋白αV基因的表达，但是调控途径不清楚。我们预实验观察到脑硫脂能够影响H3组蛋白以及转录因子Sp1的乙酰化水平，在预实验筛选脑硫脂配体蛋白的实验中，通过质谱鉴定到了溴区包含蛋白1（BRD1）。由于BRD1是一种组蛋白乙酰基转移酶复合物的亚基，有可能脑硫脂通过BRD1影响乙酰化，进而调控整合蛋白αV基因的表达。为了证实这一假说，我们将通过分析确定脑硫脂对组蛋白H3K9、H3K14、H4K12和Sp1乙酰化水平的影响；通过免疫共沉淀、荧光共定位、质谱分析进一步确定脑硫脂与BRD1的结合作用；通过基因突变确定BRD1与Sp1结合的功能区域；通过干扰确定BRD1在脑硫脂调控乙酰化的作用。本课题将从新的视觉阐明脑硫脂调控整合蛋白αV表达的分子机制，证明BRD1为其重要的调控蛋白。

通过本项目的研究，我们观察到了脑硫脂无论是外源性、还是内源性都能促进整合蛋白αV的表达；促进细胞表面整合蛋白αVβ3的表达，增强肝癌细胞与基质玻连蛋白、纤连蛋白的粘附和迁移；发现了脑硫脂促进转录因子Sp1结合到ITGAV基因启动子，进一步深入研究发现脑硫脂促进Sp1蛋白和 ITGAV基因启动子部位组蛋白发生乙酰化修饰，所以脑硫脂促进ITGAV基因启动子的转录活性，增强整合蛋白αV的表达。由于在这种调控中的乙酰化受到脑硫脂的诱导促进；所以我们深入研究这种诱导乙酰化的机制。由于脑硫脂是一种鞘糖脂，其分子本身没有酶活性和催化能力，我们推测脑硫脂由于本身结构的特点可以结合一些特殊的蛋白质而发挥作用。但是从已知的可结合脑硫脂的蛋白质中没有与乙酰化调控有关的蛋白质，因此我们筛选和鉴定新的脑硫脂结合蛋白。通过实验我们把脑硫脂分子固定在磁性珠子上，并与肝癌细胞总蛋白一起孵育，洗去非特异的蛋白质以后，将结合蛋白洗脱下来进行SDS-PAGE电泳，与对照组GalCer的结合蛋白比较，发现在130Kda大小位置有一条特异的蛋白条带，切胶以后进行质谱分析鉴定，发现得分最高的是溴区包含蛋白 1（BRD1）和Sin3B，二者都与乙酰化修饰有关。经过进一步的免疫共沉淀、共定位和荧光分析， 结果证明脑硫脂确实与溴区包含蛋白 1结合，而且我们发现BRD1主要分布在细胞浆中，在脑硫脂的诱导作用下，向细胞核部分转位，Sin3B的转位现象不明显。由于BRD1是乙酰化识别蛋白复合物的主要成员，参与乙酰化的调控。通过免疫共沉淀实验，我们发现BRD1可以与转录因子Sp1以及乙酰基转移酶Hbo1和MOZ形成复合物。而且溴区包含蛋白 1在与脑硫脂结合以后，能够诱导组蛋白H3K9、H3K14、H4K12以及Sp1蛋白发生乙酰化修饰，从而使整合蛋白αV 亚基基因开放而转录。研究结果确立了溴区包含蛋白 1 在脑硫脂调控肝癌细胞整合蛋白αV 亚基转录表达过程中的作用。完成了全部的研究内容，达到了预期的研究目标。