Gene expression profiling of GPR86 (P2RY13) KO mice

The purinergic receptor P2Y13 (P2RY13) has been shown to play a role in the uptake of holo-HDL particles in in vitro hepatocyte experiments. In order to determine the role of P2Y13 in lipoprotein metabolism in vivo, we ablated the expression of this gene in mice and found that P2Y13 knockout mice have lower plasma HDL (17%) and LDL (27%) levels as well as lower fecal concentrations of neutral sterols. In addition, significant decreases were detected in serum levels of fatty acids, glycerol and triglycerides in P2Y13 knockout mice. mRNA profiling analyses showed that ablation of P2Y13 affects hepatic gene expression in a gender-specific manner. Non-supervised agglomerative cluster analysis showed that expression changes in mice with the same genotype (KO or WT) cluster together and that distinct gene expression clusters can be observed for males and females. Subsequent gene set enrichment analysis showed increased expression of cholesterol and fatty acid biosynthesis genes, while fatty acid beta-oxidation genes were significantly decreased. Liver gene signatures also identified changes in SREBP-regulated and PPARgamma-regulated transcript levels. Differential gene expression upon P2RY13 gene ablation. Livers were isolated from male and female wildtype and P2RY13 knockout mice (6 per gender/genotype group, except for 5 per male/WT group). Isolated RNA was subjected to microarray analyses. The results of a one-way ANOVA analysis (p<0.05) showed 3471 transcripts whose relative expression changes were more than 20%.已有研究证实，嘌呤能受体P2Y13（purinergic receptor P2Y13，P2RY13）在体外肝细胞实验中参与完整高密度脂蛋白（holo-HDL）颗粒的摄取过程。为明确P2RY13在体内脂蛋白代谢中的调控作用，我们构建了P2RY13基因敲除小鼠模型，结果显示，该基因敲除小鼠的血浆高密度脂蛋白（HDL）、低密度脂蛋白（LDL）水平分别较对照组降低17%和27%，粪便中性固醇浓度亦显著下降。此外，P2RY13敲除小鼠的血清脂肪酸、甘油及甘油三酯水平均出现明显降低。mRNA表达谱分析（mRNA profiling analyses）结果表明，P2RY13基因敲除对肝脏基因表达的调控具有性别特异性。无监督聚集聚类分析（non-supervised agglomerative cluster analysis）显示，相同基因型（基因敲除型KO或野生型WT）小鼠的基因表达变化可聚为一类，且雌雄小鼠各自呈现出独特的基因表达聚类模式。后续基因集富集分析（gene set enrichment analysis）结果显示，胆固醇与脂肪酸生物合成相关基因的表达上调，而脂肪酸β氧化基因（fatty acid beta-oxidation genes）的表达则显著下调。肝脏基因特征分析还发现，受固醇调节元件结合蛋白（SREBP）与过氧化物酶体增殖物激活受体γ（PPARγ）调控的转录本水平发生了显著改变。P2RY13基因敲除后的差异基因表达实验：分别从雄性和雌性野生型、P2RY13敲除型小鼠中分离肝脏组织（每个性别/基因型组设置6只样本，雄性野生型组为5只），提取总RNA后进行基因芯片分析（microarray analyses）。单因素方差分析（one-way ANOVA）结果（p<0.05）显示，共有3471个转录本（transcripts）的相对表达量变化幅度超过20%。