RNA sequencing of WT and Uhrf1 KO iNKT cells

Deletion of Uhrf1 resulted in stage 1-specific defects during iNKT cell development. To investigate the molecular mechanism, we sorted WT and Uhrf1-KO stage 1 iNKT cells and performed RNA-seq. By comparing gene expression profile, we found metabolic defects in Uhrf1-KO stage 1 iNKT cells. The expression of CD71 (Tfrc), two subunits of CD98 (Slc3a2 and Slc7a5) and Glut3 (Slc2a3) was reduced in stage 1 iNKT cells. Besides, the downstream pathways of AKT-mTOR axis were significantly reduced. Collectively, our results suggest that Uhrf1 is required for iNKT cell development by regulating the Akt-mTOR signaling pathway. We first sorted WT and Uhrf1-KO stage 1 iNKT cells, extracted the mRNA and performed RNA-seq. We then analyzed the differentially expressed genes and performed KEGG pathway analysis. We used RT-PCR to verify the expression of the key nutrient related genes (Tfrc, Slc3a2, Slc7a5 and Slc2a3) and used flow cytometry to test the protein level of metabolic related molecules. Besides, we also analyzed the expression of genes of mTOR downstream pathways to demonstrate that Uhrf1 mediated AKt-mTOR axis regulates iNKT cell development.敲除Uhrf1会在恒定自然杀伤T细胞（iNKT细胞）发育过程中引发第1阶段特异性缺陷。为探究其分子机制，我们分选了野生型（WT）与Uhrf1基因敲除型（Uhrf1-KO）的第1阶段iNKT细胞，并开展了RNA测序（RNA-seq）实验。通过对比基因表达谱，我们发现Uhrf1-KO的第1阶段iNKT细胞存在代谢缺陷。CD71（Tfrc）、CD98的两个亚基（Slc3a2与Slc7a5）以及Glut3（Slc2a3）的表达在第1阶段iNKT细胞中均出现下调。此外，AKT-mTOR信号轴的下游通路活性显著降低。综上，我们的研究结果表明，Uhrf1通过调控AKT-mTOR信号通路，在iNKT细胞发育过程中发挥必需作用。 我们首先分选WT与Uhrf1-KO的第1阶段iNKT细胞，提取总mRNA并开展RNA-seq实验。随后我们分析了差异表达基因，并开展了京都基因与基因组百科全书（KEGG）通路富集分析。我们采用逆转录聚合酶链式反应（RT-PCR）验证了关键营养相关基因（Tfrc、Slc3a2、Slc7a5与Slc2a3）的表达水平，并通过流式细胞术（flow cytometry）检测了代谢相关分子的蛋白表达量。此外，我们还分析了mTOR下游通路相关基因的表达，以验证Uhrf1通过介导AKT-mTOR信号轴调控iNKT细胞发育这一结论。