Comparative gene expression profiling in livers of A1cf-transgenic and wild-type mice

Rationale: RNA binding protein Apobec1 Complementation Factor (A1CF) regulates posttranscriptional ApoB mRNA editing but the range of RNA targets and long-term impact of altered A1CF expression on liver function are unknown. Objective: We studied hepatocyte-specific A1cf transgenic (A1cf +/Tg), A1cf+/Tg Apobec1– /– and A1cf –/– mice fed chow or high fat/high fructose diets using RNA-Seq, RNA-CLIP Seq and tissue microarrays from human hepatocellular cancer (HCC). Findings: A1cf +/Tg mice exhibited increased hepatic proliferation and steatosis, with increased lipogenic gene expression (Mogat1, Mogat2, Cidea, Cd36) associated with shifts in polysomal RNA distribution. Aged A1cf +/Tg mice developed spontaneous fibrosis, dysplasia and HCC, which was accelerated on a high fat/fructose diet and independent of Apobec1. RNA-Seq revealed increased expression of mRNAs involved in oxidative stress (Gstm3, Gpx3, Cbr3), inflammatory response (Il19, Cxcl14, Tnfα, Ly6c), extracellular matrix organization (Mmp2, Col1a1, Col4a1), proliferation (Kif20a, Mcm2, Mcm4, Mcm6) with a subset of mRNAs (including Sox4, Sox9, Cdh1) identified in RNA CLIP-Seq. Increased A1CF expression in human HCC correlated with advanced fibrosis and with reduced survival in a subset with nonalcoholic fatty liver disease. Conclusions: Hepatic A1CF overexpression selectively alters polysomal distribution and mRNA expression, promoting lipogenic, proliferative and inflammatory pathways leading to HCC. Samples from A1cf-transgenic mice were compared to samples from age-matched wild-type C57BL/6NJ mice as a reference. For each genotype, pools were prepared containing RNAs from 3-4 separate mice, labeled, and hybridized to Agilent microarrays.

研究背景：RNA结合蛋白Apobec1互补因子（Apobec1 Complementation Factor, A1CF）可调控转录后ApoB信使RNA（mRNA）的编辑，但目前尚未明确其RNA靶标的覆盖范围，以及A1CF表达异常对肝脏功能产生的长期影响。
研究目的：本研究针对喂食普通饲料或高脂高果糖饮食的肝细胞特异性A1cf转基因（A1cf +/Tg）、A1cf+/Tg Apobec1–/–以及A1cf –/–小鼠，结合RNA测序（RNA-Seq）、RNA交联免疫沉淀测序（RNA-CLIP Seq）以及人类肝细胞癌（hepatocellular cancer, HCC）组织微阵列展开相关分析。
研究结果：A1cf +/Tg小鼠表现出肝细胞增殖增强与脂肪变性，其脂肪生成相关基因（Mogat1、Mogat2、Cidea、Cd36）的表达上调，且该现象与多聚核糖体RNA分布的改变相关。老龄A1cf +/Tg小鼠可自发出现肝纤维化、异型增生以及肝细胞癌，该进程在高脂高果糖饮食条件下会被进一步加速，且不依赖于Apobec1。RNA测序（RNA-Seq）结果显示，参与氧化应激（Gstm3、Gpx3、Cbr3）、炎症应答（Il19、Cxcl14、肿瘤坏死因子α（Tnfα）、Ly6c）、细胞外基质重塑（Mmp2、Col1a1、Col4a1）以及细胞增殖（Kif20a、Mcm2、Mcm4、Mcm6）的信使RNA表达均出现上调，其中部分信使RNA（包括Sox4、Sox9、Cdh1）经RNA交联免疫沉淀测序（RNA-CLIP Seq）得以鉴定。人类肝细胞癌组织中A1CF表达上调与肝纤维化进展相关，且在合并非酒精性脂肪性肝病的患者亚群中，A1CF高表达与生存期缩短存在关联。
研究结论：肝脏A1CF过表达可选择性改变多聚核糖体分布与信使RNA表达谱，激活脂肪生成、细胞增殖与炎症通路，最终促进肝细胞癌的发生发展。本研究以同周龄野生型C57BL/6NJ小鼠的样本作为对照，与A1cf转基因小鼠的样本进行比对。针对每种基因型，我们将3~4只独立小鼠的RNA混合制备样本，经标记后与安捷伦（Agilent）基因芯片进行杂交。