HNF4alpha P1 and P2 isoforms regulate different gene networks in the colorectal cancer cell line Caco2/15.

The nuclear receptor HNF4alpha is a master regulator of gene transcription in intestinal epithelial cells where P1 and P2 isoforms are expressed. HNF4alpha has been associated with colorectal cancer where its role remains controverisal since both tumor suppressor and oncogene characteristics have been observed. Evidences gathered from mouse models suggested however that this controversy on HNF4alpha function in colorectal cancer could be explained by the different functions of its two isoforms classes. In order to clarify this hypothesis in a human context, we downregulated the expression of HNF4alpha P1 or P2 isoforms using isoforms specific shRNA in the colorectal cancer cell line Caco2/15. Transcriptomic data were obtained by RNA-seq from these shRNA cell populations and compared to a non-target shRNA control. Overall design: Colorectal cancer cell line Caco2/15 (clone of Caco-2) was infected for 48 hours with lentiviral shRNAs targeting HNF4alpha P1 or P2 isoforms classes or a control shRNA targeting no human genes. Lentiviruses were removed and cells incubated for another 24 hours before extracting RNA. Cells reached one day post-confluence at the time RNA was extracted. RNA librairies were prepared using TrueSeq mRNA kit (Illumina) and paired reads generated using Illumina Hiseq 2000/2500 sequencer. Base calls were made using Illumina CASAVA pipeline. Three biological independant replicates were analyzed in each condition.核受体HNF4α（nuclear receptor HNF4alpha）是肠上皮细胞基因转录的主调控因子，该类细胞中表达P1与P2两种亚型。HNF4α与结直肠癌密切相关，但其功能却存在争议：研究中既观察到其肿瘤抑制因子的特性，也发现了其作为癌基因的特征。不过，来自小鼠模型的证据显示，这种关于HNF4α在结直肠癌中功能的争议，可通过其两类亚型的不同功能加以解释。为在人类背景下阐明这一假说，研究人员在结直肠癌细胞系Caco2/15（Caco-2的克隆株）中，利用亚型特异性短发卡RNA（shRNA）下调HNF4α P1或P2亚型的表达。随后通过RNA测序（RNA-seq）获取这些转染shRNA的细胞群的转录组数据，并与非靶向shRNA对照样本进行比较。实验整体设计：将结直肠癌细胞系Caco2/15（Caco-2的克隆株）用靶向HNF4α P1或P2亚型的慢病毒shRNA，或靶向人类无同源基因的对照shRNA感染48小时；移除慢病毒后，将细胞继续培养24小时再提取RNA，提取RNA时细胞处于汇合后1天的状态。RNA文库采用TrueSeq mRNA试剂盒（Illumina）构建，使用Illumina Hiseq 2000/2500测序仪生成双端读段。碱基识别通过Illumina CASAVA流程完成。每个实验条件均设置3次独立生物学重复。