突变对转录因子DEC家族蛋白质结构和DNA结合能力影响作用及LIPF与DGKA对胃癌预后的影响的研究

发布时间：2018-02-14 12:12

本文关键词： 突变转录因子家族蛋白质结构结合能力影响作用 LIPF DGKA 胃癌后的响的研究　出处：《山东大学》2016年博士论文　论文类型：学位论文

【摘要】：人类DEC (Differentially Expressed In Chondrocytes)超家族包含高度保守的两个成员DEC1和DEC2,属于basic helix-loop-helix (BHLH)类型转录因子。此保守家族在不同的物种中广泛存在,在胃癌、生物节律等多个关键信号通路中发挥重要的调控作用。目前,缺乏实验途径证实的DEC家族蛋白质的三维结构。由于此家族的重要功能,本研究通过比较建模的方法建立了DEC家族蛋白BHLH区域同源二聚体,以及DEC家族蛋白和HES-1蛋白组成的异二聚体三维结构。本研究建立包含DEC三个可能与蛋白质-DNA结合和蛋白质稳定性相关的突变位点(DEC1:H57A, R65A, LL7879AA; DEC2:LL7071AA)的蛋白质空间结构,并对野生型和突变型蛋白进行分子动力学(Molecular Dynamics, MD)模拟。通过MD模拟比较突变型分子在水溶液中的运动模式与野生型分子的差异。通过将蛋白质单体划分亚结构域的方式研究各个结构域的不同行为。突变蛋白Basic区中的波动高于野生型蛋白。携带R65A突变的蛋白质,其蛋白质-DNA之间的绑定结合能(binding energy)高于其他类型,可能导致蛋白质-DNA结构的不稳定性增加,提示该突变影响了蛋白质-DNA的亲和性。研究野生型和突变型的DEC家族蛋白质空间结构以及通过分子动力学研究突变残基对蛋白质溶剂中运动模式的影响,为未来潜在的靶向药物治疗提供了靶点。胃癌是世界范围广泛发生的高致死率胃肠道疾病。然而,由于其临床表现复杂,症状不典型,其诊断和治疗常常被不完整的主诉误导。研究基因表达谱,确定肿瘤中具有相似变化趋势的基因对于疾病的诊疗具有重要的意义。本研究分析了NCBI-GEO数据库中的RNA测序数据集(GEO:SRP049809),通过对高加索人胃癌及其癌旁正常组织转录本的分析,以及综合四个独立的表达谱芯片数据库(GEO:GSE13911, GSE19826, GSE29272, GSE33335)的结果,筛选出373个差异表达的基因(differentially expressed gene, DEG),其中下调的DEG主要与脂类代谢功能相关。本研究发现胃脂酶(lipase, gastric) LIPF与多种脂类代谢过程相关,并且在所有数据集中均出现显著下调。通过对胃癌组织中的LIPF和其下游基因二酰基甘油激酶a (DGKA)进行免疫组织化学染色,结果显示两者在胃癌组织中的表达[LIPF,59.1%(53/90); DGKA,77.8%(70/90)]相对于正常组织LIPF,94.4%(85/90); DGKA,90%(81/90)]均出现显著下调。LIPF和DGKA的表达与肿瘤本地侵染和疾病分期有关。Cox回归分析显示DGKA表达水平(HR,0.49; 95% CI,0.26-0.94; P=0.03)与更好的预后相关,LNM状态(HR,4.63; 95% CI,1.39-15.51; P=0.01)与更差的预后相关。LIPF-DGKA在胃癌中的表达水平下调,可能作为胃癌潜在的诊断标记,为胃癌的发生、进展研究提供新的研究角度。
[Abstract]:The human DEC differential Expressed in Chondrocytes superfamily consists of two highly conserved members, DEC1 and Dec2, which belong to the basic helix-loop-helix BHLH type transcription factors. Biological rhythms and other key signaling pathways play an important role in regulation. At present, there is a lack of experimental approaches to confirm the three-dimensional structure of proteins in the DEC family. In this study, the homologous dimer of BHLH region of DEC family proteins was established by comparative modeling. And the heterodimer structure of DEC family protein and HES-1 protein. In this study, we established the protein spatial structure of DEC containing three mutant sites (DEC1: H57A, R65A, LL7879AAA; Dec2: LL7071AA), which may be related to protein-DNA binding and protein stability. The molecular dynamics molecular dynamics (MDD) simulation of wild and mutant proteins was carried out. The difference between the motion patterns of mutant molecules in aqueous solution and that of wild type molecules was compared by MD simulation. The protein monomers were divided into subdomains. The volatility in the Basic region of the mutant protein is higher than that in the wild-type protein. Proteins carrying the R65A mutation, The binding energy between proteins and DNA is higher than that of other types, which may increase the instability of protein-DNA structure. The results indicated that the mutation affected the affinity of protein to DNA. The spatial structure of DEC family of wild type and mutant type was studied, and the effect of mutant residues on the motility pattern in protein solvent was studied by molecular dynamics. Gastric cancer is a worldwide high mortality gastrointestinal disease. However, because of its complex clinical manifestations, the symptoms are not typical. Its diagnosis and treatment are often misled by incomplete master complaints. It is important to identify genes with similar trends in cancer for diagnosis and treatment of diseases. This study analyzed the RNA sequencing data set in NCBI-GEO database, GEO: SRP049809, by analyzing the transcripts of gastric cancer and its adjacent normal tissues in Caucasians. And synthesizing the results of four independent expression microarray databases, namely, GEO: GSE13911, GSE19826, GSE29272, GSE33335). A total of 373 differentially expressed expressed genes were screened, among which down-regulated DEG was mainly related to lipid metabolism. This study found that gastric lipase lipase (restriction) LIPF was associated with many lipid metabolism processes. There was a significant down-regulation in all data sets. Immunohistochemical staining was performed on LIPF and its downstream gene diacylglycerol kinase a (DGKAA) in gastric cancer tissues. The results showed that the expression of LIPF and DGKA were significantly down-regulated in gastric cancer tissues [LIPF 59.1T 53 / 90; DGKA 77.8% 70 / 90] compared with normal tissue LIPF 94.4 + 85 / 90; DGKA90T 81P / 90).] the expression of LIPF and DGKA was correlated with the local invasion and disease stage. Cox regression analysis showed that DGKA expression level was 0.49; 95% CI0.26-4; P0.03) and 0.939; 95% CI0.26-4; P0.03). Better prognosis was correlated with HRN 4.63; 95% CI 1.39-15.51; P0. 01) and lower expression of LIPF-DGKA in gastric cancer. It may be a potential diagnostic marker for gastric cancer and provide a new perspective for the development and progression of gastric cancer.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.2

【相似文献】