Primer sequences for RT-qPCR.

The aim of this study was to investigate how the cholangiocarcinoma cell lines RBE and HCCC-9810 responded to NCOA4 downregulation in terms of proliferation, migration and invasive.First,we analyzed the differential expression and survival prognosis of the NCOA4 gene using a bioinformatic approach,as well as validation using clinical samples.Next,cholangiocarcinoma cells were cultured and the NCOA4 gene was down-regulated with siRNA,and then NCOA4 and GPX4 expression was detected using qPCR and Western blot.Cell was measured using CCK8, cell cloning, wound healing, and transwell migration and invasion.Levels of changes in indicators related to ferroptosis were measured after induction of iron metamorphosis by Erastin. Data from TCGA showed that NCOA4 shows greater downgrade in tumor tissues than in non-tumor tissues and the overall survival (OS) of patients with low NCOA4 expression was significantly shorter than that of patients with high NCOA4 expression.The qPCR results showed that NCOA4 was expressed at low levels in cholangiocarcinoma tissue specimens; the mRNA expression of NCOA4 decreased after knocking down NCOA4 in cells. Western blot (WB) analysis showed that NCOA4 downregulation led to an increase in GPX4 expression. The cell cloning assay confirmed that downregulation of NCOA4 significantly increased cell viability. The transwell and wound healing assays demonstrated that the proliferation rate increased after downregulation of NCOA4. After NCOA4 silencing, ferroptosis indicators such as Fe2+, MDA, and ROS expression were lowered;GSH expression was increased.Our findings indicated the regulatory effects of NCOA4 on GPX4 protein and its contribution to malignant progression in CCA, which could provide a potential therapeutic target for CCA.本研究旨在探讨胆管癌细胞系RBE与HCCC-9810在核受体辅激活因子4（NCOA4）下调后，其增殖、迁移及侵袭能力的变化情况。首先，本研究通过生物信息学方法分析NCOA4基因的差异表达与生存预后，并结合临床样本进行验证。随后，培养胆管癌细胞，利用小干扰RNA（siRNA）下调NCOA4基因的表达，随后通过实时荧光定量PCR（qPCR）与蛋白质印迹（Western blot）检测NCOA4及谷胱甘肽过氧化物酶4（GPX4）的表达水平。分别采用CCK8法、细胞克隆形成实验、划痕愈合实验以及Transwell迁移与侵袭实验评估细胞的相关生物学行为。经埃拉司汀（Erastin）诱导铁死亡（ferroptosis）后，检测铁死亡相关指标的变化水平。 癌症基因组图谱（TCGA）数据显示，NCOA4在肿瘤组织中的表达水平显著低于非肿瘤组织，且NCOA4低表达患者的总生存期（OS）显著短于高表达患者。实时荧光定量PCR结果表明，胆管癌组织标本中NCOA4呈低表达状态；细胞中敲低NCOA4后，其mRNA表达水平显著下调。蛋白质印迹分析显示，NCOA4下调可导致GPX4的表达水平升高。细胞克隆形成实验证实，下调NCOA4可显著增强细胞增殖活力。Transwell实验与划痕愈合实验表明，下调NCOA4后细胞迁移能力显著提升。在NCOA4沉默后，Fe²+、丙二醛（MDA）、活性氧（ROS）等铁死亡相关指标的表达水平降低，而谷胱甘肽（GSH）的表达水平升高。 本研究结果揭示了NCOA4对GPX4蛋白的调控作用及其在胆管癌恶性进展中的贡献，可为胆管癌的临床治疗提供潜在的靶向方向。