Transcriptomic profiling of non-alcoholic fatty liver induced by a high-fat and high-sucrose diet in the genetically diverse Collaborative Cross mice.

Identification of molecular determinants of the inter-individual and sex variability in the development of nonalcoholic fatty liver disease (NAFLD) is of great importance. Next-generation sequencing (NGS) derived transcriptome profiling (RNA-seq) is a powerful technique to investigate gene transcripts and gene variants for the systems-based analysis of molecular and cellular pathways. The goal of this study was to determine transcriptomic profiles in the livers of male and female Collaborative Cross (CC) mice fed a high-fat and high-sucrose (HF/HS) diet or a control diet for 12 weeks and to identify inter-individual- and sex-specific gene expression differences during the development of NAFLD. Liver mRNA profiles of 10 CC mouse strains (CC003/Unc, CC011/Unc, CC013/GeniUnc, CC019/TauUnc, CC032/GeniUnc, CC040/TauUnc, CC041/TauUnc, CC042/GeniUnc, CC043/GeniUnc, and CC060/Unc) fed an HF/HS diet were generated by deep sequencing, in triplicate, using Illumina NextSeq 500 NGS system. The sequence reads that passed quality filters were analyzed at the transcript level using TopHat followed by Cufflinks. The total number of differentially expressed genes identified by RNA-seq varied between sexes and individual mouse strains and correlated with the severity of liver steatosis. Pathway analysis of differentially expressed genes showed dysregulation of genes involved in lipid metabolism, cell proliferation and death, inflammation, and fibrogenesis pathways in the livers of males and females CC mice. The RNA-seq data were validated and confirmed by qRT-PCR analysis of 27 selected genes associated with lipid metabolism and fibrogenesis using TaqMan assays. Our results show that RNA-seq based transcriptome analysis offers a comprehensive, quantitative, and qualitative evaluation of whole-genome expression that can be used for the identification of unique genes associated with the inter-individual and sex differences in susceptibility to NAFLD at the population level. Overall design: Liver total RNA profiles of 10 CC mouse strains fed an HF/HS diet were generated by deep sequencing, in triplicate, using Illumina NextSeq 500 NGS system.

鉴定非酒精性脂肪性肝病（nonalcoholic fatty liver disease, NAFLD）发生过程中个体间与性别差异的分子决定因素，至关重要。下一代测序（next-generation sequencing, NGS）衍生的转录组谱分析（RNA-seq）是一项强大的技术，可用于研究基因转录本与基因变异，以开展分子及细胞通路的系统级解析。本研究的目标为：对喂食高脂高糖（high-fat and high-sucrose, HF/HS）饮食或对照饮食12周的雌雄协作杂交小鼠（Collaborative Cross, CC）的肝脏转录组特征进行全面解析，并鉴定NAFLD发生进程中个体间及性别特异性的基因表达差异。
本研究通过Illumina NextSeq 500 NGS系统，对10株CC小鼠（CC003/Unc、CC011/Unc、CC013/GeniUnc、CC019/TauUnc、CC032/GeniUnc、CC040/TauUnc、CC041/TauUnc、CC042/GeniUnc、CC043/GeniUnc及CC060/Unc）喂食HF/HS饮食后的肝脏mRNA进行深度测序，每个品系设置三次生物学重复。对通过质量过滤的测序读段，采用TopHat及Cufflinks工具在转录本水平开展分析。研究鉴定得到的差异表达基因总数因性别与小鼠品系而异，且与肝脏脂肪变性的严重程度呈显著相关。对差异表达基因的通路分析显示，雌雄CC小鼠肝脏中涉及脂质代谢、细胞增殖与死亡、炎症及纤维化通路的基因均存在调控异常。本研究通过TaqMan探针法，对27个与脂质代谢及纤维化相关的候选基因进行实时定量聚合酶链反应（quantitative real-time polymerase chain reaction, qRT-PCR）验证，证实了RNA-seq数据的可靠性。
本研究结果表明，基于RNA-seq的转录组分析可实现全基因组表达的全面、定量与定性评估，能够用于在群体水平鉴定与NAFLD易感性个体间及性别差异相关的特有基因。
整体实验设计：采用Illumina NextSeq 500 NGS系统，对10株喂食HF/HS饮食的CC小鼠的肝脏总RNA进行深度测序，每个品系设置三次生物学重复。