RNA-Seq analysis of genes differentially regulated in naïve Cd4 T cells from wide-type and PIK3CD GOF mice

Objective:mRNA sequencing of sorted naive CD4+ T cells from the spleens of PIK3CD GOF mice and WT littermate mice.Methods:Naive CD4+ T cells were negatively isolated ex vivo(Stemcell Technologies).Total RNA from isolated naive CD4+ T cells was extracted using RNeasy Plus Mini Kit (Qiagen, GmBH, Germany) according to the manufacture’s instruction and checked for RNA integrity by an Agilent Bioanalyzer 2100 (Agilent technologies, Santa Clara, US). Qualified total RNA was further purified by RNeasy micro kit (QIAGEN) and RNase-Free DNase Set (QIAGEN). Paired-end libraries were constructed using the TruSeq® RNA Sample Preparation Kit (Illumina, USA) according to the manufacturer’s instructions. The products are purified, enriched, quantified by Qubit® 2.0 Fluorometer (Life Technologies, USA) and validated by Agilent 2100 bioanalyzer to confirm insert size and calculate the mole concentration. Cluster was generated using cBot with the library diluted to 10 pM and then were sequenced on the Illumina HiSeq 2500 (Illumina, USA). The library construction and sequencing was performed at Shanghai Biotechnology Corporation (Shanghai, China).Results:Compared to the expression profiles for the WT cells, those for the mutant TNaive showed 2,307 genes with differential expression, of which 1,749 were upregulated and 558 were downregulated. Gene ontology analysis showed that PIK3CD GOF TNaive upregulated the expression of genes encoding molecules involved in the cell cycle and mitosis, including E2f1, E2f2, cyclin A2, cyclin B2, Cdk1, Hells and Nuf2, all of which might synergistically promote entry into the cycling phase. In addition, the genes encoding T cell activation, cytokines and cytokines receptors, chemokines and chemokine receptors, transcription factors, and metabolic regulators were differentially regulated in PIK3CD GOF mice compared to WT mice. Kyoto Encyclopedia of Genes and Genomes analysis showed that the altered genes in TNaive from PIK3CD GOF mice displayed significant enrichment of several sets associated with infection, inflammation and autoimmune diseaseConlusion:PIK3CD GOF results in a loss of quiescence-associated gene expression patterns in naive T cells by collectively coupling the cell cycle, nutrient metabolism, cell trafficking and signal transduction.

研究目标：对PIK3CD功能获得性（gain-of-function, GOF）小鼠及其野生型（wild type, WT）同窝对照小鼠的脾脏中分选得到的初始CD4+ T细胞进行mRNA测序。
实验方法：采用体外负向分选法分离初始CD4+ T细胞（Stemcell Technologies）。使用RNeasy Plus Mini试剂盒（Qiagen, 德国GmBH）按照制造商说明书提取分离所得初始CD4+ T细胞的总RNA，并通过安捷伦Bioanalyzer 2100（Agilent technologies, 美国圣克拉拉）检测RNA完整性。合格总RNA经RNeasy微量试剂盒（QIAGEN）及无RNase DNase套装（QIAGEN）进一步纯化。采用TruSeq® RNA样本制备试剂盒（Illumina, 美国）按照制造商说明书构建双端测序文库。对文库产物进行纯化、富集，通过Qubit® 2.0荧光计（Life Technologies, 美国）定量，并经Agilent 2100生物分析仪验证插入片段大小并计算摩尔浓度。将文库稀释至10 pM后使用cBot生成簇，随后在Illumina HiSeq 2500（Illumina, 美国）平台上完成测序。文库构建与测序工作均由上海生物科技有限公司（中国上海）完成。
实验结果：与野生型细胞的表达谱相比，PIK3CD功能获得性小鼠的初始CD4+ T细胞（TNaive）共存在2307个差异表达基因，其中1749个基因上调，558个基因下调。基因本体（Gene Ontology, GO）分析显示，PIK3CD功能获得性初始CD4+ T细胞中上调表达的基因编码参与细胞周期与有丝分裂过程的分子，包括E2f1、E2f2、细胞周期蛋白A2、细胞周期蛋白B2、Cdk1、Hells及Nuf2，上述基因可能协同促进细胞进入增殖周期。此外，与野生型小鼠相比，PIK3CD功能获得性小鼠的初始CD4+ T细胞中，编码T细胞活化相关分子、细胞因子及细胞因子受体、趋化因子及趋化因子受体、转录因子及代谢调控因子的基因均存在差异表达。京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）分析显示，PIK3CD功能获得性小鼠初始CD4+ T细胞中的差异表达基因显著富集于与感染、炎症及自身免疫性疾病相关的多个通路。
研究结论：PIK3CD功能获得性突变通过协同调控细胞周期、营养代谢、细胞迁移及信号转导过程，使初始T细胞丧失静息状态相关的基因表达模式。