Shen Wenlong, Liu Yi, Dai Bing, Qin Changling, Fu Yongli, Li Xi, Liu Chi
The Department of Pancreatic Surgery, General Surgery Department, Nanyang City Center Hospital, Nanyang, Henan, China.
Life Science Research Center, The First Affiliated Hospital of Xinxiang Medical University, Xinxiang, China.
PLoS One. 2025 Aug 11;20(8):e0327722. doi: 10.1371/journal.pone.0327722. eCollection 2025.
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.
本研究的目的是探讨胆管癌细胞系RBE和HCCC-9810在增殖、迁移和侵袭方面对NCOA4下调的反应。首先，我们使用生物信息学方法分析了NCOA4基因的差异表达和生存预后，并使用临床样本进行了验证。接下来，培养胆管癌细胞并用siRNA下调NCOA4基因，然后使用qPCR和蛋白质印迹法检测NCOA4和GPX4的表达。使用CCK8、细胞克隆、伤口愈合以及Transwell迁移和侵袭实验检测细胞情况。用Erastin诱导铁死亡后，测量与铁死亡相关指标的变化水平。来自TCGA的数据显示，NCOA4在肿瘤组织中的下调程度高于非肿瘤组织，NCOA4低表达患者的总生存期（OS）明显短于NCOA4高表达患者。qPCR结果显示，NCOA4在胆管癌组织标本中低表达；在细胞中敲低NCOA4后，NCOA4的mRNA表达降低。蛋白质印迹（WB）分析表明，NCOA4下调导致GPX4表达增加。细胞克隆实验证实，NCOA4下调显著提高了细胞活力。Transwell和伤口愈合实验表明，NCOA4下调后增殖率增加。NCOA4沉默后，铁死亡指标如Fe2+、MDA和ROS表达降低；GSH表达增加。我们的研究结果表明NCOA4对GPX4蛋白的调节作用及其对CCA恶性进展的影响，这可能为CCA提供一个潜在的治疗靶点。
