Novoa-Herran Susana, Umaña-Perez Adriana, Canals Francesc, Sanchez-Gomez Myriam
Departamento de Química, Grupo de Investigación en Hormonas (Hormone Research Laboratory), Universidad Nacional de Colombia, Sede Bogotá, Facultad de Ciencias, Cra 30 45-03 Ed 451 Of 464, AA 111321 Bogotá, Colombia.
Laboratory of Proteomics, Vall d'Hebron Institute of Oncology (VHIO), Centre Cellex, C Natzaret 115-117, 08035 Barcelona, Spain.
Cell Mol Biol Lett. 2016 Oct 16;21:22. doi: 10.1186/s11658-016-0018-9. eCollection 2016.
How nutrition and growth factor restriction due to serum depletion affect trophoblast function remains poorly understood. We performed a proteomic differential study of the effects of serum depletion on a first trimester human immortalized trophoblast cell line.
The viability of HTR-8/SVneo trophoblast cells in culture with 0, 0.5 and 10 % fetal bovine serum (FBS) were assayed via MTT at 24, 48 and 64 h. A comparative proteomic analysis of the cells grown with those FBS levels for 24 h was performed using two-dimensional electrophoresis (2DE), followed by mass spectrometry for protein spot identification, and a database search and bioinformatics analysis of the expressed proteins. Differential spots were identified using the Kolmogorov-Smirnov test ( = 3, significance level 0.10, D > 0.642) and/or ANOVA ( = 3, < 0.05).
The results showed that low serum doses or serum depletion differentially affect cell growth and protein expression. Differential expression was seen in 25 % of the protein spots grown with 0.5 % FBS and in 84 % of those grown with 0 % FBS, using 10 % serum as the physiological control. In 0.5 % FBS, this difference was related with biological processes typically affected by the serum, such as cell cycle, regulation of apoptosis and proliferation. In addition to these changes, in the serum-depleted proteome we observed downregulation of keratin 8, and upregulation of vimentin, the glycolytic enzymes enolase and pyruvate kinase (PKM2) and tumor progression-related inosine-5'-monophosphate dehydrogenase 2 (IMPDH2) enzyme. The proteins regulated by total serum depletion, but not affected by growth in 0.5 % serum, are members of the glycolytic and nucleotide metabolic pathways and the epithelial-to-mesenchymal transition (EMT), suggesting an adaptive switch characteristic of malignant cells.
This comparative proteomic analysis and the identified proteins are the first evidence of a protein expression response to serum depletion in a trophoblast cell model. Our results show that serum depletion induces specific changes in protein expression concordant with main cell metabolic adaptations and EMT, resembling the progression to a malignant phenotype.
血清缺乏导致的营养和生长因子限制如何影响滋养层细胞功能仍知之甚少。我们对血清缺乏对首孕期人永生化滋养层细胞系的影响进行了蛋白质组学差异研究。
通过MTT法在24、48和64小时检测HTR-8/SVneo滋养层细胞在含0%、0.5%和10%胎牛血清（FBS）培养基中的活力。对在这些FBS水平下培养24小时的细胞进行二维电泳（2DE）比较蛋白质组分析，随后进行质谱分析以鉴定蛋白质斑点，并对表达的蛋白质进行数据库搜索和生物信息学分析。使用Kolmogorov-Smirnov检验（=3，显著性水平0.10，D>0.642）和/或方差分析（=3，<0.05）鉴定差异斑点。
结果表明，低血清剂量或血清缺乏对细胞生长和蛋白质表达有不同影响。以10%血清作为生理对照，在含0.5%FBS培养的蛋白质斑点中有25%出现差异表达，在含0%FBS培养的蛋白质斑点中有84%出现差异表达。在0.5%FBS中，这种差异与通常受血清影响的生物学过程有关，如细胞周期、凋亡调控和增殖。除了这些变化，在血清缺乏的蛋白质组中，我们观察到角蛋白8下调，波形蛋白、糖酵解酶烯醇化酶和丙酮酸激酶（PKM2）以及与肿瘤进展相关的肌苷-5'-单磷酸脱氢酶2（IMPDH2）上调。受总血清缺乏调节但不受0.5%血清生长影响的蛋白质是糖酵解和核苷酸代谢途径以及上皮-间质转化（EMT）的成员，提示具有恶性细胞的适应性转变特征。
这种比较蛋白质组分析和鉴定出的蛋白质是滋养层细胞模型中对血清缺乏的蛋白质表达反应的首个证据。我们的结果表明，血清缺乏诱导蛋白质表达的特定变化，与主要细胞代谢适应和EMT一致，类似于向恶性表型的进展。
