The butyrophilin 1a1 knock-out mouse revisited: Ablation of Btn1a1 leads to concurrent cell death and renewal in the mammary epithelium during lactation

Purpose: To assess global changes to the levels of mRNA and protein in butyrophilin-null mice (Btn1a1-/-), which were previously shown to be defective in regulated milk-lipid secretion in the lactating mammary gland. Methods: mRNA profiles of wild-type (WT) and Btn1a1-/-mice were generated by mRNA/cDNA library preparation (Illumina), qPCR quantitation, cluster generation and pair-end deep sequencing (Illumina HiSeq 4000 sequencer) using an Illumina HiSeq SBS kit. Changes in mRNA levels (null/WT ≥ ± 2-fold; adjusted p ≀ 0.05) were assesed by GSEA and IPA analysis and compared with a proteomic analysis of the same sample set.. Results: Samples had 95-167 million pass filter reads (92% above Q30). Mapping of all samples was > 97% (mm10 reference genome). Significant changes in mRNA amounts accounted for 1.4% of 11,834 transcipts (113 up and 53 down regulated). Only 10 mRNA/protein pairs were significantly upregulated and one (Btn1a1) downregulated. Pathways and networks associated with inflammation, apoptosis and the cell cycle were upregulated and lipid synthesis and metabolism were downregulated. Biochemical and histochemical analysis confirmed that lactation is maintained in null animals, in the face of significant apoptosis, by the continual regeneration of secretory epithelial cells. Cell death proceeded through several pathways including activation of caspases 8 and 3 and not by the lysosomal lysis pathway predominant in WT cells Conclusions: Ablation of Btn1a1 has multiple effects on milk-secreting mammary cells, including disruption of lipid secretion, increased cell damage and death. Lactation is maintained by continuous regeneration of the epithelium. Expression of functional Btn1a1 is essential for the maintainance of terminally differentiated mammary cells and optimal milk production throughout lactation. RNA-seq analysis of 12 mice (5 WT and 7 Btn1a1 KO).研究目的：评估嗜乳脂蛋白（butyrophilin）敲除小鼠（Btn1a1-/-）体内mRNA与蛋白质水平的全局变化，既往研究已证实该模型在泌乳乳腺的调控性乳汁脂分泌功能存在缺陷。 研究方法：采用Illumina HiSeq SBS试剂盒，通过mRNA/cDNA文库构建、qPCR定量、簇生成及双端深度测序（Illumina HiSeq 4000测序仪），获取野生型（WT）与Btn1a1-/-小鼠的mRNA表达谱。以（敲除型/野生型）比值≥±2倍、校正后p值≤0.05为阈值，通过GSEA与IPA分析评估mRNA水平的变化，并与同一样本组的蛋白质组学分析结果进行对比。 研究结果：所有样本的有效过滤读数为9500万至1.67亿条，其中92%的读数质量值高于Q30。所有样本的基因组比对率均超过97%（以mm10为参考基因组）。在11834个转录本中，1.4%的mRNA水平发生显著变化（113个上调、53个下调）。仅10组mRNA/蛋白配对呈现显著上调，仅1组（Btn1a1）呈现显著下调。炎症、细胞凋亡与细胞周期相关的通路及调控网络呈上调状态，而脂质合成与代谢通路则呈下调状态。生化与组织化学分析证实，尽管存在显著细胞凋亡，敲除型小鼠仍可通过分泌上皮细胞的持续再生维持泌乳功能。细胞死亡通过包括半胱天冬氨酸蛋白酶8与3激活在内的多条通路发生，而非野生型细胞中占主导的溶酶体裂解通路。 研究结论：Btn1a1基因的敲除对泌乳乳腺细胞具有多重影响，包括破坏脂质分泌、加重细胞损伤与死亡。机体可通过上皮细胞的持续再生维持泌乳功能。功能性Btn1a1的表达对于维持终末分化乳腺细胞的稳态以及泌乳全程的最优乳汁产出至关重要。本研究共对12只小鼠开展RNA测序分析，其中野生型5只、Btn1a1敲除型7只。