Transcriptional network analysis reveals that AT1 and AT2 Angiotensin II receptors are both involved in the regulation of genes essential for glioma progression

Gliomas are aggressive primary brain tumors, presenting surgery limitations due to their highly infiltrative potential. The expression of Angiotensin II (Ang II) receptors in human astrocytomas was previously associated with a poor prognosis. Accordingly, this study was undertaken to reveal the molecular mechanisms underlying Ang II actions in gliomas through the transcriptomic analysis of glioma cells exposed to Ang II. C6 glioma cells were treated with Ang II and specific antagonists of AT1 and AT2. Total RNA was isolated at three and six hours intervals and submitted to oligonucleotide microarray protocol. The differentially expressed genes were obtained by paired t-tests with p<0.05 and interpreted using Venn diagrams, functional enrichment and protein interaction network analyses. Validation of microarray results was carried out through qPCR experiments of selected genes. We found a high number of significant genes with low fold changes in gene expression at the time intervals studied. These genes were regulated in a time dependent-manner, with most gene expression changes being exclusive to one of the time intervals evaluated. Our results indicated that blocking AT1 or AT2 changed the expression of genes involved in regulation of transcription, cell cycle, cell proliferation, differentiation, apoptosis, cell adhesion, cell migration, regulation of actin cytoskeleton, protein transport and protein ubiquitination. Additionally, the signaling pathways over-represented by the significant genes were ErbB, mTOR, MAPK, neurotrophin, insulin and Wnt. Finally, interactome analyses revealed hub genes associated with cell proliferation, survival, migration, transport, structural support, neurotrophin pathway, MAPK signaling and Wnt signaling. Taken together, our findings implicate Ang II-transcriptional regulation in glioma progression by means of the modulation of genes participating in protumoral functions. This transcriptome pattern is observed upon Ang II activation of either AT1 or AT2 receptors, thereby highlighting the relevance of both receptor subtypes in glioma progression. Interactome analyses disclosed hub genes regulated by Ang II which may present higher control over their networks. These genes participate in biological functions that could enhance the degree of malignancy in gliomas and thus should be further explored. C6 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 Units/ml penicillin and 100 µg/ml streptomycin. Cells were seeded in cell culture dishes and incubated at 37°C/ 5% CO2 until becoming confluent. Then, these cells were pre-treated (30 minutes) with either AT1 receptor antagonist (Losartan: 10-5M) or AT2 receptor antagonist (PD123319: 10-5M) followed by Ang II treatment (10-7 M) according to the treatment scheme: Group 1 – control; Group 2 – cells only treated with Ang II; Group 3 – cells pre-treated (30 minutes) with Losartan and then treated with Ang II; Group 4 – cells pre-treated (30 minutes) with PD123319 and then treated with Ang II. To identify which genes were significantly differentially expressed, paired t-tests (p<0.05) were performed in the following comparisons: Ang II x Control (3h); Ang II x Control (6h); Ang II +Los x Ang II (3h); Ang II +Los x Ang II (6h); Ang II +PD123319 x Ang II (3h); Ang II +PD123319 x Ang II (6h).

神经胶质瘤（Gliomas）是一类侵袭性原发性脑肿瘤，因其具有高度浸润性而面临手术治疗的局限性。此前已有研究证实，人星形细胞瘤（astrocytomas）中血管紧张素II（Angiotensin II, Ang II）受体的表达与不良预后密切相关。基于此，本研究通过对暴露于Ang II的胶质瘤细胞开展转录组分析，旨在揭示Ang II在胶质瘤中发挥作用的分子机制。

本研究采用Ang II以及AT1、AT2特异性拮抗剂处理C6胶质瘤细胞。分别于3小时和6小时两个时间点提取总RNA，开展寡核苷酸微阵列（oligonucleotide microarray）实验。通过配对t检验（p<0.05）筛选得到差异表达基因，并借助维恩图（Venn diagrams）、功能富集分析及蛋白质相互作用网络分析对结果进行解读。此外，通过对选定基因进行qPCR实验，验证了微阵列实验结果的可靠性。

本研究在所评估的时间点内发现了大量表达倍数变化较低的显著差异基因。这些基因的表达呈现时间依赖性调控特征，多数基因表达变化仅局限于某一个被评估的时间点。研究结果显示，阻断AT1或AT2受体会改变参与转录调控、细胞周期、细胞增殖、分化、凋亡、细胞黏附、细胞迁移、肌动蛋白细胞骨架调控、蛋白质运输及蛋白质泛素化过程的基因的表达水平。此外，显著差异基因富集的信号通路包括ErbB、mTOR、MAPK、神经营养因子（neurotrophin）、胰岛素及Wnt通路。

最后，相互作用组（interactome）分析揭示了与细胞增殖、存活、迁移、运输、结构支持、神经营养因子通路、MAPK信号通路及Wnt信号通路相关的核心基因（hub genes）。

综上，本研究结果表明，Ang II通过调控参与促肿瘤功能的基因，参与胶质瘤进展的转录调控过程。该转录组特征在Ang II激活AT1或AT2受体时均能观测到，因此凸显了这两种受体亚型在胶质瘤进展中的重要相关性。相互作用组分析还揭示了受Ang II调控的核心基因，这类基因可能对其所在的基因调控网络具有更强的调控能力。这些基因所参与的生物学功能可能会增强胶质瘤的恶性程度，因此有待进一步深入研究。

C6细胞在添加了10%胎牛血清、100 U/ml青霉素及100 µg/ml链霉素的达尔伯克改良伊格尔培养基（Dulbecco's modified Eagle's medium, DMEM）中培养。将细胞接种于细胞培养皿中，于37°C、5% CO₂条件下孵育至细胞汇合。随后，将细胞分别用AT1受体拮抗剂（氯沙坦：10⁻⁵M）或AT2受体拮抗剂（PD123319：10⁻⁵M）预处理30分钟，再按照以下处理方案加入Ang II（10⁻⁷M）：组别1为对照组；组别2为仅用Ang II处理的细胞组；组别3为先以氯沙坦预处理30分钟，再用Ang II处理的细胞组；组别4为先以PD123319预处理30分钟，再用Ang II处理的细胞组。

为鉴定显著差异表达的基因，我们在以下几组比较中实施了配对t检验（p<0.05）：Ang II处理组vs对照组（3小时）；Ang II处理组vs对照组（6小时）；Ang II+氯沙坦处理组vs Ang II单独处理组（3小时）；Ang II+氯沙坦处理组vs Ang II单独处理组（6小时）；Ang II+PD123319处理组vs Ang II单独处理组（3小时）；Ang II+PD123319处理组vs Ang II单独处理组（6小时）。