Argonaute-miRNA complexes reveal concerted action in disease related pathways of a human, glioblastoma cell line. Argonaute-miRNA complexes reveal concerted action in disease related pathways of a human, glioblastoma cell line

Glioblastoma is the most common primary malignant brain tumor occurring in the central nervous system and is characterized by rapid proliferation, genetic aberrations and poor response to treatment. The classical genetic alterations in glioblastoma target pathways governing cellular proliferation, cellular survival, invasion and angiogenesis. In this study, the regulation of glioblastoma-relevant pathways by small non-coding RNAs was analyzed by identification of Argonaute protein-associated microRNAs and mRNAs. We utilized a highly controlled method termed PAR-CLIP-Array by biochemical isolation of cross-linked ribonucleoprotein complexes with monoclonal antibodies specific for individual Argonaute proteins followed by microarray detection. We demonstrate here that the different Argonaute proteins bind different subsets of miRNAs and mRNAs in a glioblastoma cell line model. By extending our study generating miRNA-mRNA network interaction models we could further show, that different Argonaute proteins act in concert to regulate glioblastoma-relevant pathways. We therefore provide novel insights into glioblastoma regulation by microRNAs. Overall design: Co-immunoprecipitation in the glioblastoma cell line model SNB19 of three different Argonaute complexes using monoclonal antibodies and stringent washing conditions. We performed photo-activated UV cross-linking using 4'-thioruidine before cell lysis. Unspecific binding to the bead matrix and to the Fc part of the monoclonal rat antibody were recorded and corrected by an antibody isotype control and empty bead controls. Argonaute-associated miRNAs/mRNAs and uspecific bound miRNAs/mRNAs of the isotype control were identified by microarray hybridization. The empty bead controls were not hybridized to the microarrays, but only used for correction of unspecific RNA binding to the bead matrix during Western Blot and RT-PCR validation. The total RNA of SNB19 cells was hybridized to microarrays for comparison with Ago-associated RNAs. These experiments were performed in triplicates.

胶质母细胞瘤（Glioblastoma）是中枢神经系统中最常见的原发性恶性脑肿瘤，以快速增殖、遗传畸变及治疗响应不佳为核心特征。该肿瘤的经典遗传改变靶向调控细胞增殖、细胞存活、侵袭与血管生成的信号通路。本研究采用一种名为PAR-CLIP-Array的高度可控方法，通过靶向结合单个Argonaute蛋白（Argonaute）的单克隆抗体生化分离交联核糖核蛋白复合物，随后开展微阵列检测，分析了非编码小RNA对胶质母细胞瘤相关通路的调控作用，具体工作为鉴定Argonaute蛋白结合的微RNA（microRNA，miRNA）与信使RNA（mRNA）。我们在胶质母细胞瘤细胞系模型中证实，不同的Argonaute蛋白可结合不同子集的miRNA与mRNA。通过拓展本研究并构建miRNA-mRNA网络互作模型，我们进一步证明，不同的Argonaute蛋白可协同调控胶质母细胞瘤相关通路。因此，本研究为miRNA介导的胶质母细胞瘤调控机制提供了全新的学术见解。
整体实验设计：在胶质母细胞瘤细胞系SNB19中，使用单克隆抗体与严格洗涤条件，对三种不同的Argonaute复合物开展免疫共沉淀实验。我们在细胞裂解前，采用4'-硫代尿苷进行光激活紫外交联。针对磁珠基质与单克隆大鼠抗体Fc段的非特异性结合，我们通过抗体同型对照与空白磁珠对照进行记录与校正。通过微阵列杂交，我们鉴定出Argonaute结合的miRNA/mRNA以及同型对照中的非特异性结合miRNA/mRNA。空白磁珠对照未进行微阵列杂交，仅用于校正蛋白质印迹（Western Blot）与逆转录聚合酶链反应（RT-PCR）验证过程中RNA与磁珠基质的非特异性结合。将SNB19细胞的总RNA与微阵列杂交，以用于与Argonaute结合的RNA进行比对。上述实验均重复开展三次。