Smad4 modulates expression of chemokine receptor Ccr6 in mouse colon stroma

Purpose: To understand how loss of epithelial Smad4 alters gene expression in mouse colon stroma Methods: To understand the extent of Smad-mediated gene regulation in the colon, we isolated colon epithelium from three Smad4ΔLrig1 and from three SMAD4+ control mice (either mice lacking a CreERT allele and treated with tamoxifen, or mice bearing a CreERT allele but treated with vehicle only) and analyzed the colonic stroma by RNAseq. Results: A comparison of gene expression within the sub-epithelial stroma of Smad4Lrig1 mice relative to SMAD4+ control mice demonstrated a marked increase in inflammatory gene signaling. Of the 95 genes that were upregulated by at least 2.5-fold (≥1.32 Log2 Fold Change) and a false discovery rate <0.05, 51 genes are known to be expressed on immune cells or have immune-related functions, including Ccr6. Conclusions: SMAD4 modulates inflammatory signaling in colonic stroma to repress colitis-associated tumor formation Three Smad4Lrig1 and three SMAD4+ control mice were dissected 1 month after administration of tamoxifen. Colon crypts were chelated using Ethylenediaminetetraacetic acid (EDTA). Following crypt chelation, the sub-epithelial stroma was removed from the underlying muscle layer by scraping. RNA was then made from the sub-epithelial stroma using RNeasy kit (Qiagen, Germantown, MD) and RNA-seq performed by the Vanderbilt Technologies for Advanced Genomics (VANTAGE) core facility. 32–37 million 51–base pair, single-end reads were generated per sample. Reads were mapped to the mouse genome mm10 using TopHat-2.1.0, uniquely mapping 86%–95% single-end reads to the genome, depending on the study. The number of reads that fell into annotated genes were counted using samtools-1.3.1 and HTSeq-0.5.4p5. Count-based differential expression analysis was performed using edgeR_3.4.2.

研究目的：探究上皮细胞Smad4缺失如何改变小鼠结肠间质的基因表达模式。
为明确Smad介导的结肠基因调控范围，我们从3只Smad4ΔLrig1小鼠及3只SMAD4+对照小鼠中分离结肠上皮细胞；其中对照小鼠分为两类：一类不携带CreERT等位基因且经他莫昔芬处理，另一类携带CreERT等位基因但仅给予溶媒处理。随后通过RNA测序（RNA-seq）分析结肠间质组织。
研究结果：相较于SMAD4+对照小鼠，Smad4ΔLrig1小鼠上皮下间质的基因表达对比分析显示，炎症相关基因信号通路显著激活。在95个上调幅度≥2.5倍（对应Log2倍数变化≥1.32）且错误发现率（false discovery rate, FDR）<0.05的基因中，有51个已知可在免疫细胞中表达或具有免疫相关功能，包括Ccr6。
研究结论：SMAD4可调控结肠间质的炎症信号通路，从而抑制结肠炎相关肿瘤的发生。
本实验于他莫昔芬给药1个月后，对3只Smad4ΔLrig1小鼠及3只SMAD4+对照小鼠进行解剖。采用乙二胺四乙酸（Ethylenediaminetetraacetic acid, EDTA）螯合结肠隐窝，隐窝螯合完成后，通过刮取操作将上皮下间质从下方的肌层中分离出来。随后使用RNeasy试剂盒（Qiagen，美国马里兰州日耳曼敦）从上皮下间质中提取总RNA，并由范德堡大学先进基因组技术核心实验室（Vanderbilt Technologies for Advanced Genomics, VANTAGE）完成RNA测序。每个样本均生成3200万至3700万条51碱基单端测序读段。使用TopHat-2.1.0将读段比对至小鼠基因组mm10，不同样本的单端读段唯一比对率为86%~95%。使用samtools-1.3.1与HTSeq-0.5.4p5统计比对至注释基因的读段数目。采用edgeR_3.4.2进行基于读段计数的差异表达分析。