Quantitative Analysis of PPARD RNA-Seq Transcriptomes of Mouse Gastric Corpus Epithenial Cells by Next Generation Sequencing (NGS)

Purpose: NGS has revolutionized systems-based analysis of cell signaling pathways. The goal of this study is to determine the effects of PPARD in gastric corpus epithenial cell transcriptomes in relation to gastric progenitor cell transfromation and gastric tumorigenesis. Methods: NGS-derived mRNA transcriptome profiles of gastric corpus epithelial cells from villin-PPARD and their age and sex matched WT mice at 10, 25 and 55 weeks were generated by deep sequencing (4-5 mice per group) using Illumina HiSeq4000 .The transcriptomes of villin-PPARD and WT mice were compared to determine the differentially expressed genes. Differentially expressed genes in the top canonical pathways will be examined and validated by qRT-PCR. Results: The raw data was aligned to the MM10 genome with Tophat2/2.0.14, and the number of reads per gene was counted with HTSeq/0.6.1.The read counts were normalized with "DESeq2". We used cutoff: FDR< 0.01 and fold change larger than 2 to identify 407 differentially expressed genes (DEGs) for 10 weeks, 717 DEGs for 25 weeks and 2694 DEGs for 55 weeks. Among those DEGs, 255 are shared by the 3 ages, in which 219 were upregulated and 36 were downregulated in villin-PPARD mice compared with the WT mice. Ingenuity Pathway Analysis for the 255 DGEs among the 3 age groups showed that IFN-g signaling was the top canonical pathway that PPAR-d activated, with an overlap ratio of 36.1% of the known genes involved in this pathway. The next two top canonical pathways, with overlap ratios of 23.7% and 15.9%, were the antigen presentation pathway and the pathway for activation of IRF by cytosolic pattern recognition receptors, respectively, both of which are related to IFN-g signaling pathway activation and immune inflammation. Conclusions: Our study represents the detailed analysis of PPARD transcriptomes in gastric corpus epithelial cells regulated by villin-promoter, generated by mRNA-seq technology. Our results show that NGS offers a comprehensive and accurate quantitative and qualitative evaluations of mRNA contents in cells. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions. The RNA-seq transcriptome profile of mouse gastric corpus epithenial cells scraped from the villin-PPAR-d mice and their WT littermates at ages 10,25 and 55 weeks (4-5 mice per group) were generated by deep sequencing using Illumina HiSeq4000.研究背景：下一代测序（Next-Generation Sequencing, NGS）革新了细胞信号通路的基于系统的分析方法。本研究旨在探究过氧化物酶体增殖物激活受体δ（PPARD）对胃体上皮细胞转录组的影响，及其与胃祖细胞转化和胃肿瘤发生的关联。 方法：本研究通过Illumina HiSeq4000平台开展深度测序，获取了villin-PPARD转基因小鼠及其同年龄、同性别匹配的野生型（Wild Type, WT）小鼠在10、25、55周龄时的胃体上皮细胞NGS来源mRNA转录组图谱（每组4-5只小鼠）。通过对比villin-PPARD与WT小鼠的转录组，筛选差异表达基因（Differentially Expressed Genes, DEGs）。后续将针对核心经典通路中的差异表达基因，采用定量实时聚合酶链反应（quantitative Real-Time Polymerase Chain Reaction, qRT-PCR）进行检测与验证。 结果：原始测序数据通过Tophat2/2.0.14比对至MM10小鼠参考基因组，利用HTSeq/0.6.1统计每个基因的读段计数。读段计数通过DESeq2完成标准化。本研究以错误发现率（False Discovery Rate, FDR）<0.01且折叠变化（Fold Change, FC）>2作为筛选阈值，分别在10、25、55周龄组中鉴定出407、717和2694个差异表达基因。其中，255个差异表达基因在三个年龄组中均存在；相较于WT小鼠，villin-PPARD小鼠中219个基因上调，36个基因下调。对三个年龄组共有的255个差异表达基因进行Ingenuity通路分析（Ingenuity Pathway Analysis, IPA）显示，干扰素-γ（Interferon-gamma, IFN-γ）信号通路是PPARδ激活的首要经典通路，该通路覆盖该已知通路中36.1%的相关基因。紧随其后的两个经典通路分别为抗原呈递通路（覆盖比例23.7%）以及胞质模式识别受体激活干扰素调节因子（Interferon Regulatory Factor, IRF）通路（覆盖比例15.9%），二者均与IFN-γ信号通路激活及免疫炎症反应相关。 结论：本研究详细分析了由villin启动子调控的胃体上皮细胞中PPARD的转录组，该转录组数据通过mRNA测序技术获取。研究结果表明，下一代测序可对细胞内mRNA含量进行全面且精准的定量与定性评估。综上，基于RNA测序的转录组表征可加速遗传网络分析，并助力解析复杂的生物学功能。本研究生成了villin-PPARδ转基因小鼠及其野生型同窝幼鼠在10、25、55周龄时的胃体上皮细胞刮取样本的RNA-seq转录组图谱（每组4-5只小鼠），测序采用Illumina HiSeq4000平台完成深度测序。