Transcriptomic profiling of untreated and oxidative stress treated Wild-type and IPO13-/- mouse embryonic stem cells to study the role of IPO13 in transcriptional response to stress.. Transcriptomic profiling of untreated and oxidative stress treated Wild-type and IPO13-/- mouse embryonic stem cells to study the role of IPO13 in transcriptional response to stress.

Purpose: The importin (IPO) superfamily member IPO13 is able to mediate nuclear transport bidirectionally. To better understand IPO13 involvement in cellular signalling, we performed transcriptomic analysis on wild-type and IPO13-knockout mouse embryonic stem cells (mESCs), with gene ontology (GO) annotation results indicating enrichment of differentially expressed genes involved in stress responses and regulation of apoptosis. To examine IPO13's contribtion to the response to cellular stress, we employed RNA sequencing to investigate and compare the transcription profile of wild-type and IPO13-knockout ESCs in the presence and absence of oxidative stress. Methods: Wild-type and IPO13-knock out mESCs were treated with or without 125 µM H2O2 for 1 h, after which the medium was replaced and cells recovered in the absence of H2O2 for 2 h. Total RNA was isolated and used to generate sequencing libraries using Illumina TruSeq Stranded mRNA Library Prep Kit. Sequencing was then performed on an Illumina HiSeq2500 platform. qRT-PCR validation was performed using SYBR Green assays. Results: Results comparing Wild-type and IPO13-knock out cell lines indicated that IPO13 knock out results in the down-regulation of 338 genes and up-regulation of 536 genes. This included genes involved in stress response and apoptotic pathways. Comparison of specific gene subset differentially expressed in response to H2O2 treatment identifiedd 277 IPO13-dependent genes up- or down-regulated exclusively in the Wild-type cell line and not in the IPO13-knock out cell line, highlighting the importance of IPO13 in the transcriptional response to oxidative stress. Conclusion: IPO13 plays a key role in the stress response. Overall design: Examination of mRNA levels from Wild-type and IPO13-/- mouse embryonic stem cells under untreated and hydrogen peroxide treated conditions to identify genetic targets of IPO13 in cells undergoing oxiative stress.

研究目的：核转运蛋白（importin, IPO）超家族成员IPO13可介导双向核转运。为深入解析IPO13在细胞信号传导中的作用，我们对野生型及IPO13敲除小鼠胚胎干细胞（mouse embryonic stem cells, mESCs）开展了转录组学分析，基因本体（Gene Ontology, GO）注释结果显示，差异表达基因显著富集于应激反应与细胞凋亡调控通路。为探究IPO13对细胞应激应答的调控作用，我们采用RNA测序（RNA Sequencing）技术，分析并比较了氧化应激处理与未处理条件下野生型及IPO13敲除胚胎干细胞的转录谱。
实验方法：将野生型及IPO13敲除mESCs分别用125 μM H₂O₂处理1小时，随后更换培养基，在不含H₂O₂的条件下让细胞恢复培养2小时。提取总RNA，使用Illumina TruSeq Stranded mRNA Library Prep Kit构建测序文库，随后在Illumina HiSeq2500平台上完成测序。采用SYBR Green染料法开展qRT-PCR验证实验。
实验结果：对比野生型与IPO13敲除细胞系的转录组数据显示，IPO13敲除可导致338个基因表达下调、536个基因表达上调，其中包含参与应激反应与细胞凋亡通路的基因。对H₂O₂处理后差异表达的特定基因子集进行分析，共鉴定出277个IPO13依赖型基因，这些基因仅在野生型细胞系中出现表达上调或下调，而在IPO13敲除细胞系中无此变化，凸显了IPO13在细胞应对氧化应激的转录应答中的关键作用。
结论：IPO13在细胞应激应答中发挥关键调控作用。
整体实验设计：检测未处理及过氧化氢处理条件下野生型与IPO13基因敲除（IPO13-/-）小鼠胚胎干细胞的mRNA表达水平，以鉴定细胞在氧化应激状态下IPO13调控的靶基因。