ELF3 通过 PKMYT1/CDK1 信号通路促进胆囊癌对吉西他滨的耐药性。
ELF3 promotes gemcitabine resistance through PKMYT1/CDK1 signaling pathway in gallbladder cancer.
机构信息
Department of Biliary-Pancreatic Surgery, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200011, China.
Shanghai Key Laboratory of Biliary Tract Disease, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200011, China.
出版信息
Cell Oncol (Dordr). 2023 Aug;46(4):1085-1095. doi: 10.1007/s13402-023-00799-5. Epub 2023 Mar 29.
BACKGROUND
Gemcitabine is the standard treatment for gallbladder cancer (GBC) patients, and the development of resistance frequently limits its efficacy. However, the molecular features and mechanisms of gemcitabine resistance (Gem-R) in GBC cells remain unknown. Herein, we aimed to explore the role of ELF3 in Gem-R of GBC, including the underlying mechanisms.
METHODS
RNA sequencing was used to screen the essential genes related to the generation of Gem-R in GBC tissues. The correlation between Gem-R and ELF3 expression was identified in GDSC, GEO database, GBC tissues, and 3 GBC cell lines. Immunohistochemical staining, quantitative real-time polymerase chain reaction, and western blot were used to examine the expression of ELF3, PKMYT1, and CDK1. Luciferase reporter assays were used to identify the binding site of ELF3 in the PKMYT1 promoter region. CCK-8 assay and clonogenic survival assays were used to evaluate the sensitivity of gemcitabine in GBC cells. A GBC xenograft model was used to evaluate the influence of ELF3 on the therapeutic effect of gemcitabine.
RESULTS
A consistently positive correlation between ELF3 expression and Gem-R, both in newly generated GBC RNA-seq data and in the datasets from GDSC and GEO. Gem-R in GBC cells was facilitated by ELF3 overexpression, whereas ELF3 knockdown had the opposite effect. In vivo experiments further proved that reducing ELF3 expression promoted the gemcitabine sensitivity of GBC cells and extended the survival time of mice that received orthotopic xenografted tumors. Mechanistically, ELF3 upregulated PKMYT1 expression by interacting with the DNA binding region of PKMYT1 in GBC cells, thereby promoting the phosphorylation of CDK1 and inducing Gem-R. Treatment with a combination of the PKMYT1 shRNA and gemcitabine significantly reduced the growth of GBC cells induced by overexpression of ELF3 in vitro and in vivo.
CONCLUSIONS
ELF3/PKMYT1/CDK1 axis significantly regulates Gem-R to GBC cells and may represent a promising drug target for treating GBC patients.
背景
吉西他滨是胆囊癌（GBC）患者的标准治疗药物，但耐药的发展常常限制了其疗效。然而，GBC 细胞中吉西他滨耐药（Gem-R）的分子特征和机制尚不清楚。在此，我们旨在探索 ELF3 在 GBC 细胞 Gem-R 中的作用，包括潜在的机制。
方法
使用 RNA 测序筛选与 GBC 组织中 Gem-R 产生相关的必需基因。在 GDSC、GEO 数据库、GBC 组织和 3 种 GBC 细胞系中鉴定 Gem-R 与 ELF3 表达的相关性。免疫组织化学染色、实时定量聚合酶链反应和蛋白质印迹法用于检测 ELF3、PKMYT1 和 CDK1 的表达。荧光素酶报告基因检测用于鉴定 ELF3 在 PKMYT1 启动子区域的结合位点。CCK-8 检测和集落形成生存检测用于评估 GBC 细胞中吉西他滨的敏感性。GBC 异种移植模型用于评估 ELF3 对吉西他滨治疗效果的影响。
结果
ELF3 表达与 Gem-R 之间存在一致的正相关，无论是在新生成的 GBC RNA 测序数据中，还是在 GDSC 和 GEO 的数据集。ELF3 过表达促进了 GBC 细胞的 Gem-R，而 ELF3 敲低则产生相反的效果。体内实验进一步证明，降低 ELF3 表达可促进 GBC 细胞对吉西他滨的敏感性，并延长接受原位异种移植肿瘤的小鼠的生存时间。机制上，ELF3 通过与 GBC 细胞中 PKMYT1 的 DNA 结合区域相互作用，上调 PKMYT1 的表达，从而促进 CDK1 的磷酸化，诱导 Gem-R。体外和体内实验中，用 PKMYT1 shRNA 和吉西他滨联合治疗显著降低了 ELF3 过表达诱导的 GBC 细胞生长。
结论
ELF3/PKMYT1/CDK1 轴显著调节 GBC 细胞的 Gem-R，可能成为治疗 GBC 患者的有前途的药物靶点。
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