Gene expression during neuronal differentiation in two subtypes of SH-SY5Y. Homo sapiens

Background: SH-SY5Y cells exhibit a neuronal phenotype when treated with all-trans retinoic acid (RA), but the molecular mechanism of activation in the signaling pathway mediated by phosphatidylinositol 3-kinase (PI3K) is not sufficiently understood. To shed new light on the mechanism, we comprehensively compared the gene expression profiles between SK-N-SH cells and two subtypes of SH-SY5Y cells (SH-SY5Y-A and SH-SY5Y-E), each of which showed a different phenotype during RA-mediated differentiation. Results: SH-SY5Y-A cells differentiated in the presence of RA, whereas RA-treated SH-SY5Y-E cells required additional treatment with brain-derived neurotrophic factor (BDNF) for full differentiation. In combination with perturbation using a PI3K inhibitor, LY294002, we identified 386 genes and categorized them into two clusters dependent on the PI3K signaling pathway during RA-mediated differentiation in SH-SY5Y-A cells. Transcriptional regulation of the gene cluster was greatly reduced in SK-N-SH cells or partially impaired in SH-SY5Y-E cells in coincidence with a defect in the neuronal phenotype of these cell lines. Additional stimulation with BDNF induced a set of neural genes which were down-regulated in RA-treated SH-SY5Y-E cells but were abundant in the differentiated SH-SY5Y-A cells. Conclusions: We identified the gene clusters controlled by PI3K- and TRKB-mediated signaling pathways during differentiation in two subtypes of SH-SY5Y cells. TRKB-mediated bypass pathway compensates for the impaired neural functions generated by defects in several signaling pathways including PI3K in SH-SY5Y-E cells. The expression profiling data are useful for further studies to elucidate the signal transduction-transcriptional network including PI3K and/or TRKB. Keywords: Cell type comparison, time course Overall design: Human neuroblastomas, SK-N-SH (HTB-11) and SH-SY5Y-A cells (CRL-2266) were obtained from the American Type Culture Collection (ATCC). We also obtained SH-SY5Y-E cells (EC94030304) from the European Collection of Cell Cultures (ECACC). Tissue culture cells were maintained in D-MEM/F12 1:1 mixture supplemented with 15% FBS (Fetal Bovine Serum) and 1% NEAA (Non-essential amino acid) in a 5% CO2 humidified incubator at 37oC. The culture medium was changed twice a week. For the RA-inducible experiment, random culture cells from two clone subtypes of SH-SY5Y and SK-N-SH were seeded in laminin coated culture dishes (BioCoat Laminin Cellware; BD Biosciences, Billerica, MA, USA) for 1 day and then transferred to a medium containing 10 μM of RA in the presence or the absence of LY294002 (10μM) for five days. For BDNF-induced sequential differentiation of the SH-SY5Y-E strain, cells were washed with D-MEM/F12 twice after five days in the presence of RA and then incubated with 50 ng/ml of BDNF in D-MEM/F12 without serum for three days.

背景：SH-SY5Y细胞经全反式维甲酸（all-trans retinoic acid, RA）处理后可呈现神经元表型，但磷脂酰肌醇3-激酶（phosphatidylinositol 3-kinase, PI3K）介导的信号通路激活的分子机制尚未完全阐明。为深入解析该机制，我们对SK-N-SH细胞与两种SH-SY5Y细胞亚型（SH-SY5Y-A及SH-SY5Y-E）的基因表达谱进行了全面比较——这两种亚型在RA介导的分化过程中呈现出不同的表型特征。
结果：在RA存在的条件下，SH-SY5Y-A细胞可顺利完成分化；而经RA处理的SH-SY5Y-E细胞则需额外添加脑源性神经营养因子（brain-derived neurotrophic factor, BDNF）才能实现完全分化。结合使用PI3K抑制剂LY294002开展的扰动实验，我们在SH-SY5Y-A细胞的RA介导分化过程中，鉴定出386个基因并将其划分为两个依赖于PI3K信号通路的基因簇。该基因簇的转录调控在SK-N-SH细胞中大幅减弱，在SH-SY5Y-E细胞中则部分受损，这与这两种细胞系的神经元表型缺陷相契合。额外添加BDNF刺激后，诱导了一组神经基因的表达：这类基因在RA处理的SH-SY5Y-E细胞中表达下调，但在已分化的SH-SY5Y-A细胞中丰度较高。
结论：我们在两种SH-SY5Y细胞亚型的分化过程中，鉴定出了受PI3K及TRKB介导的信号通路调控的基因簇。TRKB介导的旁路通路可补偿SH-SY5Y-E细胞中包括PI3K在内的多条信号通路缺陷所导致的神经功能受损。本次表达谱数据可为后续阐明包含PI3K和/或TRKB的信号转导-转录调控网络提供有效支撑。
关键词：细胞类型比较，时间进程
整体实验设计：人神经母细胞瘤细胞SK-N-SH（HTB-11）与SH-SY5Y-A（CRL-2266）均购自美国典型培养物保藏中心（American Type Culture Collection, ATCC）；SH-SY5Y-E细胞（EC94030304）则购自欧洲细胞培养物保藏中心（European Collection of Cell Cultures, ECACC）。所有组织培养细胞均培养于含15%胎牛血清（Fetal Bovine Serum, FBS）及1%非必需氨基酸（Non-essential amino acid, NEAA）的D-MEM/F12 1:1混合培养基中，于37℃、5%CO₂饱和湿度培养箱内培养，每周更换两次培养基。
针对RA诱导实验：随机接种SH-SY5Y的两种克隆亚型及SK-N-SH细胞至层粘连蛋白包被的培养皿（BioCoat层粘连蛋白细胞培养板；BD Biosciences，美国马萨诸塞州比勒里卡）中培养1天，随后转移至含10μM RA的培养基中，分别添加或不添加10μM LY294002，持续培养5天。
针对SH-SY5Y-E细胞的BDNF诱导序贯分化实验：经RA处理5天后，用D-MEM/F12培养基洗涤细胞两次，随后更换为不含血清的D-MEM/F12培养基，添加50ng/ml BDNF继续培养3天。