Global Transcript Changes In Human Keratinocyte Cells Induced by Apratyramide, a Marine-Derived Peptidic Stimulator of VEGF-A and Other Growth Factors. Homo sapiens

To elucidate the mode of action of apratyramide, we performed microarray profiling using the Affymetrix GeneChip® Human Transcriptome Array 2.0 to determine global changes in transcript levels in HaCaT cells treated with apratyramide. Comparativel analysis identified 371 differentially expressed genes after 12 h treatment with 30 μM apratyramide (p 1.5 or <0.67). Consistent with our previous data, VEGF-A appeared to be one of the most up-regulated genes. To examine the molecular functions and genetic networks, the microarray data was analyzed using Ingenuity Pathways Aanalysis (IPA).The global changes of transcript levels are associated with increased downstream phenotypic effects including angiogenesis, mitogenesis, differentiation of epithelial tissue and formation of skin, and decreased effects such as apoptosis of liver cells and hypoplasia of organs. IPA analysis of 371 microarray hits indicated the unfolded protein response (UPR) as the top canonical pathway with a p-value of 1.45 × 10-16. The IPA also elucidated that the 371 hits were most related to a molecular network associated with the function of cellular compromise and cellular maintenance. The network contains molecular components from UPR pathway, NRF2-mediated oxidative stress response signaling as well as glucocorticoid receptor signaling. Overall design: Four samples were analyzed in biological duplicates. Transcriptome profile of arpatyramide was compared to control.

为阐明apratyramide的作用机制，我们采用Affymetrix GeneChip®人类转录组芯片2.0（Affymetrix GeneChip® Human Transcriptome Array 2.0）开展微阵列表达谱分析，以检测经apratyramide处理的HaCaT细胞中转录本水平的整体变化。比较分析显示，使用30 μM apratyramide处理12小时后，共筛选得到371个差异表达基因（变化倍数≥1.5或≤0.67）。与本团队此前的研究数据一致，血管内皮生长因子A（VEGF-A）是上调幅度最显著的基因之一。为探究其分子功能与遗传调控网络，我们采用Ingenuity通路分析（Ingenuity Pathways Analysis, IPA）对微阵列数据进行分析。转录本水平的整体变化与多种下游表型效应的改变密切相关：其中血管生成、细胞有丝分裂活化、上皮组织分化及皮肤形成等效应显著增强，而肝细胞凋亡、器官发育不全等效应则显著减弱。针对371个差异表达基因的IPA分析显示，未折叠蛋白反应（unfolded protein response, UPR）为排名首位的经典通路，其p值为1.45×10^-16。IPA分析同时揭示，该371个差异表达基因最显著富集于与细胞损伤应答及细胞稳态维持功能相关的分子网络中。该网络包含未折叠蛋白反应通路、核因子红细胞2相关因子2（NRF2）介导的氧化应激应答通路以及糖皮质激素受体信号通路的分子组分。实验整体设计：共设置4份样本，均进行生物学重复检测；将apratyramide处理组的转录组表达谱与对照组进行比较。