Global expression analysis identified a preferentially NGF-induced transcriptional program regulated by sustained MEK/ERK and AP-1 activation during PC12 differentiation.. Rattus norvegicus

Neuronal differentiation of PC12 cells in response to NGF is a prototypical model in which signal duration determines a biological response. Sustained ERK activity induced by NGF, as compared to transient activity induced by EGF, is critical to the differentiation of these cells. To characterize the transcriptional program activated preferentially by NGF, we compared global gene expression profiles between cells treated with NGF and EGF for 2-4 hrs, when sustained ERK signaling in response to NGF is most distinct from the transient signal elicited by EGF. This analysis identified 69 genes that were preferentially upregulated in response to NGF. Overall design: PC12 cells that were starved in low serum media for 24 hrs were treated with NGF (50ng/mL) or EGF (25ng/mL) for 2 or 4 hrs, or left untreated. Total RNA for 3 independent biological replicates was extracted and subjected to Affymetrix Rat Gene 1.0ST Arrays. The 69 genes that were preferentially upregulated by NGF compared to EGF met the following criteria: NGF/No treatment log2> 1, FDR p value 0.75, FDR p value< 0.01.

PC12细胞响应神经生长因子（NGF）的神经元分化，是信号时长决定生物学应答的经典研究模型。相较于表皮生长因子（EGF）诱导的瞬时细胞外信号调节激酶（ERK）活性，NGF诱导的持续性ERK活性对该细胞的分化至关重要。为了表征NGF优先激活的转录程序，我们对比了经NGF与EGF处理2-4小时的细胞的全局基因表达谱——此时NGF诱导的持续性ERK信号与EGF引发的瞬时信号差异最为显著。本分析共鉴定出69个响应NGF时优先上调的基因。

整体实验设计：将在低血清培养基中饥饿24小时的PC12细胞分为三组：分别以50ng/mL的NGF、25ng/mL的EGF处理2小时或4小时，同时设置未做处理的对照组。提取3次独立生物学重复的总RNA，随后使用Affymetrix大鼠基因1.0ST芯片（Affymetrix Rat Gene 1.0ST Arrays）进行检测。

该69个相较于EGF被NGF优先上调的基因满足以下筛选标准：NGF与未处理组的log₂比值>1，错误发现率（False Discovery Rate, FDR）p值为0.75，FDR p值<0.01。