由于异常的血管生成，食管癌（EC）中的肿瘤干细胞（CSCs）具有缺氧微环境的特性。然而，它们可以抵抗缺氧引起的细胞凋亡。目前尚不清楚食管CSCs对缺氧引起的细胞凋亡抵抗的分子机制。因此，本研究将基于CHOP介导的内质网应激对分子机制进行研究。采用荧光激活细胞排序（FACS）筛选EC9706细胞中的CD44+CD24-细胞。为了阐明食管CSCs通过哪条凋亡途径抵抗缺氧引起的细胞凋亡，使用核染色、流式细胞术和JC-1试剂检测缺氧对凋亡的影响，使用免疫印迹（western blotting，WB）和定量聚合酶链反应（quantitative polymerase chain reaction，qPCR）检测缺氧对凋亡相关蛋白表达的影响。为了阐明CD44+CD24-细胞抵抗缺氧引起的细胞凋亡的机制是通过抑制内质网应激（ERS）通路的激活实现的，构建了EC9706细胞中CHOP和PERK基因沉默的细胞系和CD44+CD24-细胞中CHOP和PERK基因过表达的细胞系，使用流式细胞术和JC-1试剂检测缺氧对凋亡、细胞周期和线粒体膜电位的影响。采用WB和qPCR检测凋亡相关蛋白和ERS相关蛋白的表达。缺氧显著诱导EC9706细胞的凋亡和细胞周期阻滞（P<0.05），但不影响CD44+CD24-细胞的凋亡和细胞周期（P>0.05）。缺氧显著诱导EC9706细胞线粒体和ERS凋亡通路的激活（P<0.05），但不影响Fas受体凋亡通路（P>0.05）。这三条凋亡通路在CD44+CD24-细胞中不受缺氧的影响（P>0.05）。沉默CHOP和PERK基因抑制EC9706细胞的缺氧引起的凋亡（P<0.05）。CHOP和PERK过表达促进CD44+CD24-细胞的缺氧引起的凋亡（P<0.05），而线粒体膜通透性抑制剂抑制了过表达CHOP基因的CD44+CD24-细胞的缺氧引起的凋亡。EC中的CD44+CD24-肿瘤干细胞通过抑制ERS介导的线粒体凋亡途径来抵抗缺氧引起的凋亡，这表明ERS通路可以作为临床治疗中减少EC抵抗性的潜在靶点。2023《胃肠肿瘤学杂志》版权所有。

Due to the abnormal angiogenesis, cancer stem cells (CSCs) in esophageal cancer (EC) have the characteristics of a hypoxic microenvironment. However, they can resist hypoxia-induced apoptosis. the molecular mechanism underlying the resistance of esophageal CSCs to hypoxia-induced apoptosis is currently unclear. Therefore, this study will investigate the molecular mechanism based on CHOP-mediated endoplasmic reticulum stress.CD44+CD24- cells in EC9706 cells were screened by fluorescence-activated cell sorting (FACS). To clarify which apoptosis pathway esophageal CSCs resist hypoxia-induced cell apoptosis through, the effects of hypoxia on apoptosis were detected by nuclear staining, flow cytometry, and JC-1 reagent, the effects of hypoxia on the expression of apoptosis-related proteins were detected by western blotting (WB) assay and quantitative polymerase chain reaction (qPCR) assay. To clarify the mechanisms of CD44+CD24- cells resistance to hypoxia-induced apoptosis is achieved by inhibiting the activation of endoplasmic reticulum stress (ERS) pathway, silenced CHOP and PERK cell lines of EC9706 cells and overexpressed CHOP and PERK cell lines of CD44+CD24- cells were constructed, the effects of hypoxia on apoptosis, cell cycle, and mitochondrial membrane potential were detected by flow cytometry and JC-1 reagent. WB assay and qPCR assay were used to detect the expressions of apoptosis-related proteins and ERS-related proteins.Hypoxia significantly induce apoptosis and cycle arrest of EC9706 cells (P<0.05), but did not affect apoptosis and cycle of CD44+CD24- cells (P>0.05). Hypoxia considerably induced the activation of mitochondrial and ERS apoptosis pathways in EC9706 cells (P<0.05), but did not affect Fas receptor apoptosis pathways (P>0.05). The three apoptosis pathways were not affected by hypoxia in CD44+CD24- cells (P>0.05). Silencing the CHOP and PERK gene inhibited hypoxia-induced apoptosis of EC9706 cells (P<0.05). CHOP and PERK overexpression promoted hypoxia-induced apoptosis of CD44+CD24- cells (P<0.05), whereas mitochondrial membrane permeability inhibitors inhibited hypoxia-induced apoptosis of CD44+CD24- cells overexpressed CHOP gene.CD44+CD24- tumor stem cells in EC resist to hypoxia-induced apoptosis by the inhibition of ERS-mediated mitochondrial apoptosis pathway, which suggested that ERS pathway can serve as a potential target for reducing EC treatment resistance in clinical treatment.2023 Journal of Gastrointestinal Oncology. All rights reserved.