Differential expression produced by Pdrg1 gene silencing in rat hepatoma H35 cells

RTqPCR results suggested a relevant role for Pdrg1 in rat hepatoma H35 cells, where its expression was dramatically enhanced. Hence, these cells were chosen as a suitable model for stable Pdrg1 silencing. For this purpose, H35 cells were transfected with appropriate shRNA plasmids against Pdrg1 and stable clones isolated. Among those exhibiting reproducible behavior, clones CN-10 (negative control), 3-44 (shRNA3) and 4-18 (shRNA4) were selected for further analysis. Pdrg1 expression analyzed by RT-qPCR was reduced by 50% and 70% in 3-44 and 4-18 clones, respectively, as compared to CN-10. RNAs of CN-10, 3-44 and 4-18 clones, as well as, RNA of an enriched pool of shRNA3 transiently transfected cells (shRNA3T) were used for expression analysis using Agilent one-color microarrays. Genes exhibiting changes =2-fold with FDR<0.05, according to LIMMA analysis, were identified. Pathway analysis was performed with BioProfiling using data of 114 genes (74 upregulated and 40 downregulated) exhibiting similar behavior in the three silenced samples. Selected genes, at least two of pathways with p=0.02, were used for verification of expression changes by RT-qPCR, together with Pdrg1.逆转录实时定量聚合酶链反应（RT-qPCR）结果显示，Pdrg1在大鼠肝癌H35细胞中发挥相关调控作用，该细胞内Pdrg1的表达水平显著上调。因此，我们选取该细胞作为稳定沉默Pdrg1的合适模型。为此，我们将靶向Pdrg1的短发夹RNA（shRNA）质粒转染至H35细胞，并分离获得稳定克隆株。在表现出稳定可重复实验行为的克隆株中，我们选取CN-10（阴性对照）、3-44（shRNA3）以及4-18（shRNA4）用于后续分析。与阴性对照CN-10相比，3-44和4-18克隆株中的Pdrg1表达水平分别下调了50%和70%。我们收集了CN-10、3-44、4-18克隆株的总RNA，以及富集后的shRNA3瞬时转染细胞池（shRNA3T）的总RNA，采用Agilent单通道微阵列进行表达谱分析。通过线性模型分析微阵列数据（LIMMA）工具筛选，我们鉴定出表达变化倍数≥2倍且错误发现率（False Discovery Rate, FDR）<0.05的差异表达基因。我们采用BioProfiling工具，针对3个沉默样本（3-44、4-18及shRNA3T）中表达趋势一致的114个差异基因（其中74个上调，40个下调）进行通路富集分析。我们选取至少富集于2个通路且校正P值为0.02的基因，连同Pdrg1一同通过RT-qPCR验证其表达变化。