目的:探讨GATA3与FOXA1在luminal亚型乳腺癌细胞中的相互作用。方法:基于cBioportal For Cancer Genomics公共数据库中乳腺癌临床病例数据集,分析luminal A、luminal B和basal-like亚型乳腺癌组织中GATA3与FOXA1mRNA表达水平的相关性;...目的:探讨GATA3与FOXA1在luminal亚型乳腺癌细胞中的相互作用。方法:基于cBioportal For Cancer Genomics公共数据库中乳腺癌临床病例数据集,分析luminal A、luminal B和basal-like亚型乳腺癌组织中GATA3与FOXA1mRNA表达水平的相关性;利用蛋白-蛋白相互作用数据库STRING和分析工具Cytoscape预测GATA3与FOXA1的相互作用。以luminal亚型的MCF-7和T-47D乳腺癌细胞以及basal-like亚型的MDA-MB-231和SUM-149PT乳腺癌细胞为研究对象,采用RT-qPCR和Western印迹检测GATA3和FOXA1的mRNA和蛋白表达水平;细胞免疫荧光检测GATA3和FOXA1的细胞定位;蛋白质免疫共沉淀验证GATA3与FOXA1之间的相互作用。结果:临床病例数据分析显示GATA3与FOXA1在luminal A、luminal B和basal-like亚型乳腺癌组织中mRNA的表达均呈正相关(r=0.4047、0.4761、0.5876,均P<0.0001)。蛋白质相互作用预测显示GATA3与FOXA1存在潜在的相互作用关系。Luminal乳腺癌细胞中GATA3和FOXA1的mRNA(t=80.95、79.73、33.84、33.60,均P<0.0001;t=15.24、5.21、14.95、14.93,均P<0.001)和蛋白(t=29.63、28.48、36.60、35.60,均P<0.0001;t=34.06、35.30、75.01、74.32,均P<0.0001)表达水平显著高于basal-like细胞。GATA3与FOXA1在MCF-7和T-47D细胞核中共定位。GATA3与FOXA1存在蛋白相互作用。结论:GATA3与FOXA1可能通过相互作用维持luminal亚型乳腺癌表型稳态,抑制肿瘤恶性进展。展开更多
Hepatocyte nuclear factor 1 alpha(HNF1A),hepatocyte nuclear factor 4 alpha(HNF4A),and forkhead box protein A2(FOXA2)are key transcription factors that regulate a complex gene network in the liver,cre-ating a regulator...Hepatocyte nuclear factor 1 alpha(HNF1A),hepatocyte nuclear factor 4 alpha(HNF4A),and forkhead box protein A2(FOXA2)are key transcription factors that regulate a complex gene network in the liver,cre-ating a regulatory transcriptional loop.The Encode and ChIP-Atlas databases identify the recognition sites of these transcription factors in many glycosyltransferase genes.Our in silico analysis of HNF1A,HNF4A.and FOXA2 binding to the ten candidate glyco-genes studied in this work confirms a significant enrich-ment of these transcription factors specifically in the liver.Our previous studies identified HNF1A as a master regulator of fucosylation,glycan branching,and galactosylation of plasma glycoproteins.Here,we aimed to functionally validate the role of the three transcription factors on downstream glyco-gene transcriptional expression and the possible effect on glycan phenotype.We used the state-of-the-art clus-tered regularly interspaced short palindromic repeats/dead Cas9(CRISPR/dCas9)molecular tool for the downregulation of the HNF1A,HNF4A,and FOXA2 genes in HepG2 cells-a human liver cancer cell line.The results show that the downregulation of all three genes individually and in pairs affects the transcrip-tional activity of many glyco-genes,although downregulation of glyco-genes was not always followed by an unambiguous change in the corresponding glycan structures.The effect is better seen as an overall change in the total HepG2 N-glycome,primarily due to the extension of biantennary glycans.We propose an alternative way to evaluate the N-glycome composition via estimating the overall complexity of the glycome by quantifying the number of monomers in each glycan structure.We also propose a model showing feedback loops with the mutual activation of HNF1A-FOXA2 and HNF4A-FOXA2 affecting glyco-genes and protein glycosylation in HepG2 cells.展开更多
文摘目的:探讨GATA3与FOXA1在luminal亚型乳腺癌细胞中的相互作用。方法:基于cBioportal For Cancer Genomics公共数据库中乳腺癌临床病例数据集,分析luminal A、luminal B和basal-like亚型乳腺癌组织中GATA3与FOXA1mRNA表达水平的相关性;利用蛋白-蛋白相互作用数据库STRING和分析工具Cytoscape预测GATA3与FOXA1的相互作用。以luminal亚型的MCF-7和T-47D乳腺癌细胞以及basal-like亚型的MDA-MB-231和SUM-149PT乳腺癌细胞为研究对象,采用RT-qPCR和Western印迹检测GATA3和FOXA1的mRNA和蛋白表达水平;细胞免疫荧光检测GATA3和FOXA1的细胞定位;蛋白质免疫共沉淀验证GATA3与FOXA1之间的相互作用。结果:临床病例数据分析显示GATA3与FOXA1在luminal A、luminal B和basal-like亚型乳腺癌组织中mRNA的表达均呈正相关(r=0.4047、0.4761、0.5876,均P<0.0001)。蛋白质相互作用预测显示GATA3与FOXA1存在潜在的相互作用关系。Luminal乳腺癌细胞中GATA3和FOXA1的mRNA(t=80.95、79.73、33.84、33.60,均P<0.0001;t=15.24、5.21、14.95、14.93,均P<0.001)和蛋白(t=29.63、28.48、36.60、35.60,均P<0.0001;t=34.06、35.30、75.01、74.32,均P<0.0001)表达水平显著高于basal-like细胞。GATA3与FOXA1在MCF-7和T-47D细胞核中共定位。GATA3与FOXA1存在蛋白相互作用。结论:GATA3与FOXA1可能通过相互作用维持luminal亚型乳腺癌表型稳态,抑制肿瘤恶性进展。
基金the European Structural and Investment Funded Grant"Cardio Metabolic"(#KK.01.2.1.02.0321)the Croatian National Centre of Research Excellence in Personalized Healthcare Grant(#KK.01.1.1.01.0010)+2 种基金the European Regional Development Fund Grant,project"CRISPR/Cas9-CasMouse"(#KK.01.1.1.04.0085)the European Structural and Investment Funded Project of Centre of Competence in Molecular Diagnostics(#KK.01.2.2.03.0006)the Croatian National Centre of Research Excellence in Personalized Healthcare Grant(#KK.01.1.1.01.0010).
文摘Hepatocyte nuclear factor 1 alpha(HNF1A),hepatocyte nuclear factor 4 alpha(HNF4A),and forkhead box protein A2(FOXA2)are key transcription factors that regulate a complex gene network in the liver,cre-ating a regulatory transcriptional loop.The Encode and ChIP-Atlas databases identify the recognition sites of these transcription factors in many glycosyltransferase genes.Our in silico analysis of HNF1A,HNF4A.and FOXA2 binding to the ten candidate glyco-genes studied in this work confirms a significant enrich-ment of these transcription factors specifically in the liver.Our previous studies identified HNF1A as a master regulator of fucosylation,glycan branching,and galactosylation of plasma glycoproteins.Here,we aimed to functionally validate the role of the three transcription factors on downstream glyco-gene transcriptional expression and the possible effect on glycan phenotype.We used the state-of-the-art clus-tered regularly interspaced short palindromic repeats/dead Cas9(CRISPR/dCas9)molecular tool for the downregulation of the HNF1A,HNF4A,and FOXA2 genes in HepG2 cells-a human liver cancer cell line.The results show that the downregulation of all three genes individually and in pairs affects the transcrip-tional activity of many glyco-genes,although downregulation of glyco-genes was not always followed by an unambiguous change in the corresponding glycan structures.The effect is better seen as an overall change in the total HepG2 N-glycome,primarily due to the extension of biantennary glycans.We propose an alternative way to evaluate the N-glycome composition via estimating the overall complexity of the glycome by quantifying the number of monomers in each glycan structure.We also propose a model showing feedback loops with the mutual activation of HNF1A-FOXA2 and HNF4A-FOXA2 affecting glyco-genes and protein glycosylation in HepG2 cells.
