目的：通过RNA-seq分析眼表外胚层(OSE)与表面外胚层(SE)转录组表达差异，揭示OSE转录组景观及转录调控网络。

方法：人类胚胎干细胞(hES)被分化成OSE细胞和SE细胞，利用RNA-seq分析了OSE与SE之间差异表达的基因(DEGs)。基于这些差异表达基因，进行基因本体论(GO)、京都基因与基因组百科全书(KEGG)通路富集以及蛋白质-蛋白质交互作用(PPI)网络分析，并筛选出了关键的转录因子(TFs)和枢纽基因。使用NetworkAnalyst平台构建了TF-基因和TF-miRNA调控网络。

结果：OSE与SE共筛选出4 182个差异基因，OSE细胞中上调基因2 771个，下调基因1 411个。GO-BP富集分析显示OSE上调基因主要集中在离子跨膜转运的调节、轴突发育、调节化学突触传递等相关生物学进程，下调基因主要富集在核分裂、染色体分离、细胞周期相变的调控等生物学进程； KEGG富集分析显示OSE上调基因主要富集在可卡因成瘾、轴突导向、苯丙胺成瘾等通路，下调基因主要富集于癌症中的蛋白聚糖、ECM-受体相互作用、蛋白质消化和吸收、细胞因子-细胞因子受体相互作用等通路。与SE相比，OSE细胞中有204个TFs(FOS、EGR1、POU5F1、SOX2和PAX6等)上调，80个TFs(HAND2、HOXB6、HOXB5、HOXA5和HOXB8等)下调。在OSE细胞中发现了6个上调和9个下调枢纽基因，并根据这些枢纽基因构建了TF-基因和TF-miRNA调控网络。

结论：RNA-seq分析阐明了OSE和SE细胞的转录组特征，这些数据可为OSE及角结膜上皮发育调控及体外定向诱导等研究提供指导。

METHODS:OSE and SE cells were differentiated from human embryonic stem(hES)cells. Differentially expressed genes(DEGs)between OSE and SE were analyzed using RNA-seq. Based on the DEGs, we performed gene ontology(GO)analysis, Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis, and protein-protein interaction(PPI)network analysis. Transcription factors(TFs)and hub genes were screened. Subsequently, TF-gene and TF-miRNA regulatory networks were constructed using the NetworkAnalyst platform.

RESULTS:A total of 4 182 DEGs were detected between OSE and SE cells, with 2 771 up-regulated and 1 411 down-regulated genes in OSE cells. GO-BP analysis revealed that up-regulated genes in OSE were enriched in the regulation of ion transmembrane transport, axon development, and modulation of chemical synaptic transmission. Down-regulated genes were primarily involved in nuclear division, chromosome segregation, and regulation of cell cycle phase transition. KEGG analysis indicated that up-regulated genes in OSE cells were enriched in signaling pathways such as cocaine addiction, axon guidance, and amphetamine addiction, while down-regulated genes were enriched in proteoglycans in cancer, ECM-receptor interaction, protein digestion and absorption, and cytokine-cytokine receptor interaction. Additionally, compared with SE, 204 TFs(including FOS, EGR1, POU5F1, SOX2, and PAX6)were up-regulated, and 80 TFs(including HAND2, HOXB6, HOXB5, HOXA5, and HOXB8)were down-regulated in OSE cells. Furthermore, we identified 6 up-regulated and 9 down-regulated hub genes in OSE cells, and constructed TF-gene and TF-miRNA regulatory networks based on these hub genes.

CONCLUSIONS:The transcriptome characteristics of OSE and SE cells were elucidated through RNA-seq analysis. These findings may provide a novel insight for studies on the development and in vitro directed induction of OSE and corneal epithelial cells.