Yao Jinni, Yang Huaicheng, Yuan Meng, Wang Congyu, Liao Heqiang, Song Rui, Xu Zhe, Zeng Xiangrui, Zhang Zheng
Department of Hepatobiliary and Pancreatic Surgery, The First Affiliated Hospital of Anhui University of Science and Technology, Huainan 232007, China; Graduate School of Anhui University of Science and Technology, Department of Hepatobiliary and Pancreatic Surgery, First Affiliated Hospital of Anhui University of Science and Technology, Huainan 232007, China.
Department of Hepatobiliary and Pancreatic Surgery, The First Affiliated Hospital of Anhui University of Science and Technology, Huainan 232007, China; First Clinical College of Anhui University of Science and Technology, Huainan 232007, China.
Cell Signal. 2025 Jul;131:111742. doi: 10.1016/j.cellsig.2025.111742. Epub 2025 Mar 11.
BACKGROUND: GINS4 has been identified as a regulator associated with multiple types of cancers. However, the effects of GINS4 on hepatocellular carcinoma (HCC) have not been reported. METHODS: GINS4 expression in HCC was evaluated utilizing UALCAN database. The relationship between the expression of GINS4 and the survival probability of HCC patients was analyzed using Kaplan-Meier Plotter. Cell viability was evaluated by CCK8 assay and EDU assay. qRT-PCR and western blot were performed to examine GINS4 expression. The level of cell cycle was measured by flow cytometry and western blot. Fe level and ferroptosis-related proteins were measured by corresponding kits and western blot. Lipid peroxidation was explored by C11 BODIPY 581/591 probe. STRING database and HDOCK database were performed to predict the binding of GINS4 to POLE2. Immunofluorescence and western blotting was adopted for assessing cell autophagy and mTOR signaling pathway. Ki67 and GPX4 levels were measured by immunohistochemistry. The expression levels of POLE2/PI3K/AKT were assessed by western blot. RESULTS: The data indicated that GINS4 expression was upregulated in HCC. Knockdown of GINS4 alleviated the proliferation and cycle and promoted ferroptosis of HuH7 cells. GINS4 was proved to bind to POLE2 and the silencing of GINS4 inhibited the expression of POLE2. GINS4 knockdown accelerated ferroptosis in HuH7 cells. POLE2 overexpression reversed the influences of GINS4 silencing on proliferation and cycle, and also ferroptosis. In addition, interference with GINS4 suppressed the activation of PI3K/AKT signaling via POLE2. In vivo experiments illustrated that GINS4 deletion suppressed HCC tumor growth, increased the GPX4 expression and restrained the Ki67 level, as well as reducing POLE2/PI3K/AKT signaling. CONCLUSION: GINS4 silencing suppressed proliferation and cycle while promoted ferroptosis in HCC cells by regulating PI3K/AKT signaling via binding to POLE2.
背景：GINS4已被确定为与多种癌症相关的调节因子。然而，GINS4对肝细胞癌（HCC）的影响尚未见报道。 方法：利用UALCAN数据库评估HCC中GINS4的表达。使用Kaplan-Meier Plotter分析GINS4表达与HCC患者生存概率之间的关系。通过CCK8法和EDU法评估细胞活力。进行qRT-PCR和蛋白质印迹法检测GINS4表达。通过流式细胞术和蛋白质印迹法测量细胞周期水平。用相应试剂盒和蛋白质印迹法测量铁水平和铁死亡相关蛋白。用C11 BODIPY 581/591探针探索脂质过氧化。利用STRING数据库和HDOCK数据库预测GINS4与POLE2的结合。采用免疫荧光和蛋白质印迹法评估细胞自噬和mTOR信号通路。通过免疫组织化学测量Ki67和GPX4水平。通过蛋白质印迹法评估POLE2/PI3K/AKT的表达水平。 结果：数据表明，HCC中GINS4表达上调。敲低GINS4可减轻HuH7细胞的增殖和细胞周期进程，并促进其铁死亡。证实GINS4与POLE2结合，GINS4沉默可抑制POLE2的表达。敲低GINS4可加速HuH7细胞的铁死亡。POLE2过表达可逆转GINS4沉默对增殖、细胞周期以及铁死亡的影响。此外，干扰GINS4可通过POLE2抑制PI3K/AKT信号的激活。体内实验表明，缺失GINS4可抑制HCC肿瘤生长，增加GPX4表达，抑制Ki67水平，并降低POLE2/PI3K/AKT信号。 结论：GINS4沉默通过与POLE2结合调节PI3K/AKT信号通路，从而抑制HCC细胞的增殖和细胞周期进程，同时促进铁死亡。
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