CFTR is a tumor suppressor gene in murine and human intestinal cancer [RNA-seq]

Analysis of the cystic fibrosis gene Cftr in the colon and small intestine of Cftr-deficient murine model. The hypothesis was loss of Cftr altered expression of genes important in intestinal homeostasis and oncogenic signaling pathways. The results identified potential roles of Cftr in up- or down-regulating major gene clusters that belong to groups of immune response, ion channel, intestinal stem cell and other growth regulators. The experiments were designed to analyze the role of Cftr-deficiency in tumorigenesis. The goal of this study was to identify genes and pathways associated with Cftr-deficiency in Apc wildtype and ApcMin mice. Total RNAs were isolated from mice, and subjected to deep sequencing, in duplicates, using Illumina HiSeq 2500. Samples that were sequenced in the same batch were analyzed in pair-wise using Tophat-Cuffdiff pipeline as outlined in Nature Protocol from Trapnell C. et al, 2012. The results indicated that Cftr-deficiency overlapped with genes and pathways involved in immune and inflammatory signaling, stem cell regulation, and Wnt/beta catenin signaling. Total RNA was isolated from multiple colon tumors and multiple small intestine tumors from Apc wildtype Cftr-deficient mice, ApcMin Cftr-deficient mice, and ApcMin Cftr wildtype mice. Total RNA was also obtained from Apc wildtype normal colon (epithelial cells) and normal duodenum (whole duodenum minus villi) from three Cftr wildtype and three Cftr-deficient mice. RNA Seq was then conducted on all samples with at least two replicates for each biological sample. Please note that 1) The 23 mice were processed in several batches, and two sequencing runs were carried out at two different dates. To control for the batch effect of sequencing, some samples were included in both runs (run1 and run2). 2) To reach the desired sequencing depth and to keep loading balance, each sample was split into halves, and sequenced on two lanes (L007 and L008 for run1, L006 and L007 for run2). therefore, for 11 samples, there are 4 technical replicates, including the 2-batches and 2-lane sequencing method. For the remaining 12 samples, there are 2 technical replicates, referring to the 2-lane sequencing. 3) some of the mice are heterozygous mutant of CFTR gene (CFTRhet), named as "CFTR knockdown".本研究针对Cftr缺陷型小鼠模型的结肠与小肠组织，开展囊性纤维化基因Cftr（CFTR）的相关分析。本研究提出假说：Cftr的缺失会改变肠道稳态及致癌信号通路中关键基因的表达。研究结果揭示了Cftr在调控免疫应答、离子通道、肠道干细胞及其他生长调节因子相关核心基因簇的表达上调或下调中的潜在作用。本实验旨在分析Cftr缺失在肿瘤发生发展中的作用，本研究的目标为在Apc（腺瘤性结肠息肉病蛋白）野生型及ApcMin突变小鼠中，明确与Cftr缺失相关的基因及通路。 研究人员从小鼠体内分离总RNA，采用Illumina HiSeq 2500平台开展双平行深度测序。同批次测序的样本采用Trapnell C等人于2012年发表于《Nature Protocol》的Tophat-Cuffdiff分析流程，进行组间两两比对分析。结果显示，Cftr缺失所影响的基因与通路，与免疫及炎症信号、干细胞调控以及Wnt/β-连环蛋白信号通路存在重叠。 研究人员从Apc野生型Cftr缺陷小鼠、ApcMin突变且Cftr缺陷小鼠及ApcMin突变且Cftr野生型小鼠的多个结肠肿瘤与小肠肿瘤组织中分离总RNA；同时从3只Cftr野生型小鼠及3只Cftr缺陷型小鼠的Apc野生型正常结肠（上皮细胞）与正常十二指肠组织（去除绒毛的完整十二指肠）中获取总RNA。随后对所有样本开展RNA测序（RNA-seq），每个生物学样本至少设置2个重复。 请注意： 1) 本次实验共处理23只小鼠，分为多个批次开展，并于两个不同日期完成两轮测序。为控制测序批次效应，部分样本同时参与了两轮测序（run1与run2）。 2) 为达到预设测序深度并维持上样均衡，每个样本被分为两份，分别在两个测序通道进行测序（run1的通道为L007与L008，run2的通道为L006与L007）。因此，11个样本共包含4个技术重复，涵盖2个批次与2个通道的测序方案；剩余12个样本则包含2个技术重复，仅采用2个通道的测序方式。 3) 部分小鼠为CFTR基因杂合突变体（CFTRhet），被命名为‘CFTR敲低模型’。