tourt-affy-human-131547. Homo sapiens

Nerve growth factor (NGF) is a neurotrophin that plays an important role in regulating the survival, growth, and differentiation of sympathetic neurons. Many in vitro studies indicate that Egr transcription factors are coupled to NGF signaling and are essential signaling mediators of NGF-dependent differentiation of sympathetic neurons, such as neuroblastoma cells and pheochromocytoma cells. Mice that are deficient for both Egr1 and Egr3 have profound sympathetic nerve system defects, including abnormal neuron degeneration and impaired differentiation (unpublished observations). To further understand the role of Egr genes in sympathetic neuron development, it is necessary to examine the signal transduction pathways involved in NGF-mediated Egr-dependent gene regulation. The results will be helpful in understanding the pathobiology of those diseases related to aberrant sympathetic neuron differentiation, such as neuroblastoma and dysautonomias, and may provide new insights into therapies for these refractory diseases. To identify NGF-mediated Egr-dependent target genes in human SH-SY5Y/TrkA neuroblastoma cells: Many potential Egr target genes have been described over the years. However, very few have been characterized to be involved in NGF-mediated sympathetic neuron differentiation. In order to further understand the role of Egr genes in sympathetic neuron development, it is necessary to examine the signal transduction pathways involved in NGF-mediated Egr-dependent gene regulation. Egr1 and Egr3 are rapidly induced after NGF treatment and Egr1 is involved in activation of the differentiation marker gene NPY in SH-SY5Y/TrkA cells. Therefore, SH-SY5Y/TtrkA cells appear to be an excellent model system to study the role of Egr transcription factors in sympathetic neuron differentiation in vitro. A dominant negative Egr molecule that specifically blocks transcriptional activity mediated by Egr transcription factors will be used in this study to identify Egr-dependent target genes. Egr1 and Egr3 are rapidly induced after NGF treatment in human SH-SY5Y/TrkA neuroblastoma cells, which in turn differentiate into sympathetic-like neurons. We hypothesize that Egr transcription factors are involved in activating downstream signaling pathways during NGF mediated differentiation of SH-SY5Y/TrkA cells. Moreover, we hypothesize that by using a dominant negative Egr (dnEgr) molecule that blocks all Egr mediated gene transcription and Affymetrix microarray analysis, it will be possible to identify NGF-mediated Egr transcription dependent gene regulatory networks that may be involved in growth and differentiation of neuroblastoma. An unbiased approach to understanding these gene regulatory networks may lead to new insights relating to NGF signaling involved in neuronal growth and differentiation. Human neuroblastoma SH-SY5Y/TrkA cells will be infected with either dnEgr-expressing adenovirus (SH-SY5Y/TrkA-dnEgr) or with EGFP-expressing control adenovirus (SH-SY5Y/TrkA-EGFP). Equivalent infection efficiency and lack of viral toxicity will be verified by EGFP fluorescence microscopy 24 hours after infection and the cells will be treated with NGF (100 ng/ml). Total RNA will be extracted from SH-SY5Y/TrkA (uninfected), SH-SY5Y/TrkA-dnEgr, and SH-SY5Y/TrkA-EGFP cells treated with NGF for 0, 1 hour and 3 hours. Total RNA will be prepared from all of the samples and a portion subjected to real-time PCR analysis to ensure that NGF mediated Egr gene induction was not altered by the context of viral infection. Pilot experiments demonstrate that Egr genes are still induced in the context of viral infection greater than 100-fold. Egr1 mRNA peak expression is known to occur at 1 hour and decrease by 3 hours after NGF treatment in all of the samples. The peak expression of Egr target genes is expected to occur later than Egr1 peak expression since Egr1 proteins need to be expressed first to initiate the transcription of target promoters. Therefore, the RNA samples from SH-SY5Y/TrkA-dnEgr and SH-SY5Y/TrkA-EGFP treated with NGF for 3 hours will be used to probe Affymetrix high-density human genome U133 Plus 2.0 Arrays to identify differentially expressed genes. RNA amplification for probe synthesis should not be necessary since we will provide 10 ug of intact total RNA for each sample. We will provide three sets of samples to perform the comparative microarray analysis twice from different starting materials and a nine-way comparative analysis of the data will be performed. We expect that cells containing high levels of dnEgr will inhibit NGF mediated Egr-dependent target gene expression and that these gene networks should be identifiable when compared to EGFP infected cells that have normal Egr gene transcriptional activity. Keywords: time-course Overall design: as above

神经生长因子（Nerve growth factor, NGF）是一种神经营养因子（neurotrophin），在调控交感神经元的存活、生长与分化过程中发挥重要作用。诸多体外研究表明，早期生长反应转录因子（Egr transcription factors）与NGF信号通路存在耦联，且是NGF依赖的交感神经元（如神经母细胞瘤细胞与嗜铬细胞瘤细胞）分化过程中必不可少的信号介导因子。同时缺失Egr1与Egr3的小鼠会出现严重的交感神经系统缺陷，包括神经元变性异常与分化受损（尚未发表的观察结果）。为进一步阐明Egr基因在交感神经元发育中的作用，有必要探究NGF介导的、依赖Egr的基因调控所涉及的信号转导通路。该研究结果将有助于理解与交感神经元分化异常相关疾病的病理生物学机制，例如神经母细胞瘤与自主神经功能障碍，并可为这些难治性疾病的治疗提供全新的研究思路。
为鉴定人类SH-SY5Y/TrkA神经母细胞瘤细胞中NGF介导的依赖Egr的靶基因：多年来已有诸多潜在的Egr靶基因被报道，但极少有被证实参与NGF介导的交感神经元分化过程。鉴于前述研究目的，仍需深入探究NGF介导的依赖Egr的基因调控所涉及的信号转导通路。在人类SH-SY5Y/TrkA细胞中，Egr1与Egr3会在NGF处理后快速被诱导表达，且Egr1参与分化标记基因NPY的激活过程。因此，SH-SY5Y/TrkA细胞堪称研究体外交感神经元分化过程中Egr转录因子作用的理想模型系统。
本研究将采用一种可特异性阻断Egr转录因子介导的转录活性的显性负效Egr分子（dominant negative Egr, dnEgr），以鉴定依赖Egr的靶基因。在人类SH-SY5Y/TrkA神经母细胞瘤细胞中，NGF处理后Egr1与Egr3会被快速诱导，进而使细胞分化为交感样神经元。我们提出如下假说：在NGF介导的SH-SY5Y/TrkA细胞分化过程中，Egr转录因子参与激活下游信号通路。此外，我们还假说：通过使用可阻断所有Egr介导的基因转录的dnEgr分子，结合Affymetrix微阵列分析，有望鉴定出可能参与神经母细胞瘤生长与分化的、NGF介导的依赖Egr转录的基因调控网络。
通过无偏倚的方法解析这些基因调控网络，或可加深对神经元生长与分化相关NGF信号通路的认识。本研究将使用分别携带dnEgr表达盒的腺病毒（SH-SY5Y/TrkA-dnEgr）与携带增强型绿色荧光蛋白（enhanced green fluorescent protein, EGFP）表达盒的对照腺病毒（SH-SY5Y/TrkA-EGFP）感染人类神经母细胞瘤SH-SY5Y/TrkA细胞。感染后24小时，将通过EGFP荧光显微镜验证等效的感染效率与无病毒毒性，随后用NGF（100 ng/ml）处理细胞。
将从NGF处理0小时、1小时与3小时的未感染SH-SY5Y/TrkA细胞、SH-SY5Y/TrkA-dnEgr细胞以及SH-SY5Y/TrkA-EGFP细胞中提取总RNA。对所有样本制备总RNA，并选取一部分进行实时聚合酶链反应（real-time PCR）分析，以确认病毒感染的环境不会改变NGF介导的Egr基因诱导过程。预实验结果显示，在病毒感染的环境下，Egr基因的诱导倍数仍可超过100倍。已知在所有样本中，Egr1 mRNA的表达峰值出现在NGF处理后1小时，并在3小时时下降。由于Egr1蛋白需先被表达才能启动靶基因启动子的转录，因此Egr靶基因的表达峰值预计会晚于Egr1的表达峰值。因此，我们将使用NGF处理3小时的SH-SY5Y/TrkA-dnEgr与SH-SY5Y/TrkA-EGFP细胞的RNA样本，进行Affymetrix高密度人类基因组U133 Plus 2.0芯片杂交，以鉴定差异表达基因。由于我们将为每个样本提供10 μg完整的总RNA，因此无需进行RNA扩增以制备探针。
我们将设置三组样本，使用不同的起始材料进行两次比较微阵列分析，并对数据进行九组比较分析。我们预计，高表达dnEgr的细胞会抑制NGF介导的依赖Egr的靶基因表达，与保留正常Egr基因转录活性的EGFP感染细胞相比，这些基因调控网络将可被识别。
关键词：时间进程 整体实验设计：如上所述