First Clinical Medical College, Shaanxi University of Chinese Medicine, Xianyang, 712046, Shaanxi, China.
College of Integrated Traditional Chinese and Western Medicine, Shaanxi University of Chinese Medicine, Xianyang, 712046, Shaanxi, China.
Sci Rep. 2024 Nov 18;14(1):28539. doi: 10.1038/s41598-024-80160-4.
Gastric cancer (GC) is the fifth most common cancer and the third most common cause of cancer death globally, with high invasiveness, high recurrence rate, and poor prognosis. Multiple studies have shown that Metallothionein-1G (MT1G) is closely associated with oxidative stress, ferroptosis, and autophagy. However, the role and potential mechanisms of MT1G in GC have not been fully elucidated. This study aims to explore the biological functions and regulatory mechanisms of MT1G in GC. Perform bioinformatics analysis using the TCGA database to investigate the expression of MT1G in GC. RT-qPCR and Western blot were used to detect the expression of MT1G, ferroptosis related proteins, autophagy related proteins and ARNTL clock autophagy related proteins in Hgc27, MKN45 and AGS cell lines. Exploring the biological functions of MT1G overexpressing GC cell lines through wound healing and transwell experiments. Use specific fluorescence probes to examine mitochondrial membrane potential and Fe fluorescence intensity. Using immunoprecipitation analysis (CO-IP) to elucidate the association between GC cell lines GPX4, SQSTM and ARNTL. Use flow cytometry to detect ROS expression. Observation of autophagy related morphological changes in cells using transmission electron microscopy. Compared with gastric mucosal cell lines, the expression of MT1G is decreased in three gastric cancer cell lines (Hgc27, MKN45 and AGS). Overexpression of MT1G inhibits the proliferation, migration, and invasion functions of GC cells, reduces SOD and GSH content, increases MDA content, cause the mitochondrial membrane potential to weaken and promote the transformation of JC-1 aggregates to JC-1 monomer, increases Fe, affects ROS, and reduces GPX4 and SLC7A11 protein expression, promoting ferroptosis. Overexpression of MT1G promotes the transformation of LC3B I to LC3B II, reduces SQSTM1 protein expression, and leads to the appearance of more autophagosomes and autolysosomes at low magnification. At high magnification, mitochondrial autophagy, endoplasmic reticulum autophagy, lipid droplet autophagy, and wrinkled mitochondrial cristae are observed, promoting autophagy. Overexpression of MT1G inhibits GPX4, thereby affecting SQSTM1 as a vector to promote ARNTL autophagy and EGLN2, promoting ARNTL clock autophagy through the GPX4/SQSTM1 axis. Our research findings elucidate that overexpression of MT1G promotes iron autophagy centered around ARNTL in GC cells via the GPX4/SQSTM1 axis, thereby inhibiting GC cell function and providing a new molecular mechanism and therapeutic target for the development of GC.
胃癌（GC）是全球第五大常见癌症和第三大癌症死亡原因，具有侵袭性高、复发率高、预后差等特点。多项研究表明，金属硫蛋白 1G（MT1G）与氧化应激、铁死亡和自噬密切相关。然而，MT1G 在 GC 中的作用和潜在机制尚未完全阐明。本研究旨在探讨 MT1G 在 GC 中的生物学功能和调控机制。使用 TCGA 数据库进行生物信息学分析，以研究 MT1G 在 GC 中的表达情况。通过 RT-qPCR 和 Western blot 检测 Hgc27、MKN45 和 AGS 细胞系中 MT1G、铁死亡相关蛋白、自噬相关蛋白和 ARNTL 时钟自噬相关蛋白的表达情况。通过划痕愈合和 Transwell 实验探索过表达 MT1G 的 GC 细胞系的生物学功能。使用特异性荧光探针检测线粒体膜电位和 Fe 荧光强度。使用免疫沉淀分析（CO-IP）阐明 GC 细胞系中 GPX4、SQSTM 和 ARNTL 之间的关联。使用流式细胞术检测 ROS 表达。通过透射电子显微镜观察细胞自噬相关形态变化。与胃黏膜细胞系相比，三种胃癌细胞系（Hgc27、MKN45 和 AGS）中 MT1G 的表达降低。过表达 MT1G 抑制 GC 细胞的增殖、迁移和侵袭功能，降低 SOD 和 GSH 含量，增加 MDA 含量，导致线粒体膜电位减弱，促进 JC-1 聚集体向 JC-1 单体转化，增加 Fe，影响 ROS，并降低 GPX4 和 SLC7A11 蛋白表达，促进铁死亡。过表达 MT1G 促进 LC3B I 向 LC3B II 的转化，降低 SQSTM1 蛋白表达，导致低倍镜下自噬体和自噬溶酶体增多。高倍镜下观察到线粒体自噬、内质网自噬、脂滴自噬和皱缩的线粒体嵴，促进自噬。过表达 MT1G 抑制 GPX4，从而影响 SQSTM1 作为载体促进 ARNTL 自噬和 EGLN2，通过 GPX4/SQSTM1 轴促进 ARNTL 时钟自噬。我们的研究结果表明，过表达 MT1G 通过 GPX4/SQSTM1 轴促进 GC 细胞中以 ARNTL 为中心的铁自噬，从而抑制 GC 细胞功能，并为 GC 的发展提供了新的分子机制和治疗靶点。
