Background HeberFERON is a co-formulation of α2b and γ interferons, based on their synergism, that have shown its clinical superiority over individual interferons in basal cell carcinomas. In Glioblastoma (GBM), HeberFERON has shown promising preclinical and clinical results. This motivated us to design a microarray experiment aimed to identify the molecular mechanisms involved into the distinctive effect of HeberFERON compared with individual interferons. Methods Transcriptional expression profiling including a control (untreated) and three groups receiving α2b-interferon, γ-interferon and HeberFERON was performed using an Illumina HT-12 microarray platform. Unsupervised methods for gene and sample grouping, identification of differentially expressed genes, functional enrichment and network analysis computational biology methods were applied to identify distinctive patterns of HeberFERON action. Validation of most distinctive genes was performed by qPCR. Cell Cycle analysis of cell treated by HeberFERON for 24h, 48h and 72h was carried out by flow cytometry. Results The three treatments show different behavior based on the gene expression profiles. Enrichment analysis identified several Mitotic Cell Cycle related events, in particular from Prometaphase to Anaphase, that are exclusively targeted by HeberFERON. FOXM1 transcription factor network which is involved in several Cell Cycle phases and is highly expressed in GBMs is significantly down regulated by HeberFERON. Flow cytometry experiments corroborated the action of HeberFERON over Cell Cycle in a dose and time dependent manner with a clear cellular arrest since 24h post-treatment. Despite the fact that p53 was not down-regulated by HeberFERON several genes involved in its regulatory activity were functionally enriched. Network analysis also revealed a strong relation of p53 with genes targeted by HeberFERON. We propose a mechanistic model to explain HeberFERON distinctive action, based on the simultaneous activation of PKR and ATF3, p53 phosphorylation changes as well as its reduced MDM2 mediated ubiquitination and export from nucleus to cytoplasm. PLK1, AURKB, BIRC5 and CCNB1 genes, all regulated by FOXM1, also play central roles in this model. These and other interactions could explain a G2/M arrest and the effect of HeberFERON over the proliferation of U-87MG. Conclusions We proposed molecular mechanisms underlying the distinctive behavior of HeberFERON compared to individual interferon treatments, where Cell Cycle related events showed the highest relevance.

中文翻译：

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HeberFERON 独特地靶向胶质母细胞瘤衍生细胞系 U-87MG 中的细胞周期。

背景 HeberFERON 是 α2b 和 γ 干扰素的联合制剂，基于它们的协同作用，在基底细胞癌中显示出其优于单个干扰素的临床优势。在胶质母细胞瘤 (GBM) 中，HeberFERON 已显示出有希望的临床前和临床结果。这促使我们设计了一个微阵列实验，旨在确定与单个干扰素相比，HeberFERON 的独特作用所涉及的分子机制。方法 使用 Illumina HT-12 微阵列平台进行转录表达谱分析，包括对照（未处理）和接受 α2b-干扰素、γ-干扰素和 HeberFERON 的三组。基因和样本分组的无监督方法，差异表达基因的鉴定，应用功能丰富和网络分析计算生物学方法来识别 HeberFERON 作用的独特模式。通过 qPCR 对大多数独特基因进行验证。通过流式细胞术对HeberFERON处理24h、48h和72h的细胞进行细胞周期分析。结果 三种处理根据基因表达谱显示出不同的行为。富集分析确定了几个有丝分裂细胞周期相关事件，特别是从前中期到后期，这是 HeberFERON 专门针对的。参与多个细胞周期阶段并在 GBM 中高度表达的 FOXM1 转录因子网络被 HeberFERON 显着下调。流式细胞术实验证实了 HeberFERON 在细胞周期中以剂量和时间依赖性方式的作用，自治疗后 24 小时后细胞明显停滞。尽管 p53 没有被 HeberFERON 下调，但参与其调节活动的几个基因在功能上得到了丰富。网络分析还显示 p53 与 HeberFERON 靶向的基因密切相关。我们提出了一个机制模型来解释 HeberFERON 的独特作用，基于 PKR 和 ATF3 的同时激活、p53 磷酸化变化以及其减少的 MDM2 介导的泛素化和从细胞核到细胞质的输出。PLK1、AURKB、BIRC5 和 CCNB1 基因均受 FOXM1 调控，在该模型中也发挥核心作用。这些和其他相互作用可以解释 G2/M 停滞和 HeberFERON 对 U-87MG 增殖的影响。结论 我们提出了与单个干扰素治疗相比，HeberFERON 独特行为的分子机制，其中细胞周期相关事件显示出最高的相关性。