Department of Gastroenterology, Shanxi Province Cancer Hospital/Shanxi Hospital Affiliated to Cancer Hospital, Chinese Academy of Medical Sciences/Cancer Hospital Affiliated to Shanxi Medical University, Taiyuan, China.
Department of Scientific Research, Shanxi Province Cancer Hospital/Shanxi Hospital Affiliated to Cancer Hospital, Chinese Academy of Medical Sciences/Cancer Hospital Affiliated to Shanxi Medical University, Taiyuan, China.
J Biochem Mol Toxicol. 2024 Sep;38(9):e23778. doi: 10.1002/jbt.23778.
Hepatocellular carcinoma (HCC) is a lethal form of liver cancer, and the tumor microenvironment, particularly cancer-associated fibroblasts (CAFs), plays a critical role in its progression. This study aimed to elucidate the mechanism by which CAF-derived exosomes regulate the development of HCC. The study employed quantitative real-time polymerase chain reaction for mRNA expression analysis and western blot analysis for protein expression detection. Chromatin immunoprecipitation assay and dual-luciferase reporter assay were performed to investigate the relationship between zinc finger protein 250 (ZNF250) and programmed cell death 1 ligand 1 (PD-L1). Transmission electron microscopy and western blot analysis were used to characterize the isolated exosomes. The transferability of CAF-derived exosomes and normal fibroblasts (NFs)-derived exosomes into HCC cells was analyzed using a green fluorescent labeling dye PKH67. Cell proliferation was assessed via a 5-Ethynyl-2'-deoxyuridine assay, while Transwell assays were conducted to evaluate cell migration and invasion. Flow cytometry was performed to measure cell apoptosis, while enzyme-linked immunosorbent assays were used to assess the levels of tumor necrosis factor-α and perforin. Finally, a xenograft mouse model was constructed to examine the effects of exosomes derived from ZNF250-deficient CAFs on the tumor properties of HCC cells. The study revealed increased expression of ZNF250 in HCC tissues and cells, with ZNF250 transcriptionally activating PD-L1 in HCC cells. ZNF250 expression was associated with HbsAg, clinical stage and tumor size of HCC patients. CAF-derived exosomal ZNF250 can regulate PD-L1 expression in HCC cells. Furthermore, exosomes derived from ZNF250-deficient CAFs inhibited the proliferation, migration, invasion, and immune escape of HCC cells by downregulating PD-L1 expression. Moreover, CAF-derived exosomal ZNF250 promoted tumor formation in vivo. These findings provide insights into the role of CAF-derived exosomes in the suppression of HCC development, highlighting the significance of ZNF250 and PD-L1 regulation in tumor progression.
肝细胞癌 (HCC) 是一种致命形式的肝癌，肿瘤微环境，特别是癌相关成纤维细胞 (CAF)，在其进展中起着关键作用。本研究旨在阐明 CAF 衍生的外泌体调节 HCC 发展的机制。该研究采用定量实时聚合酶链反应进行 mRNA 表达分析，采用蛋白质印迹分析进行蛋白质表达检测。染色质免疫沉淀测定和双荧光素酶报告基因测定用于研究锌指蛋白 250 (ZNF250) 与程序性细胞死亡 1 配体 1 (PD-L1) 之间的关系。透射电子显微镜和蛋白质印迹分析用于表征分离的外泌体。使用绿色荧光标记染料 PKH67 分析 CAF 衍生的外泌体和正常成纤维细胞 (NF) 衍生的外泌体向 HCC 细胞的转移能力。通过 5-乙炔基-2'-脱氧尿苷测定评估细胞增殖，通过 Transwell 测定评估细胞迁移和侵袭。流式细胞术用于测量细胞凋亡，酶联免疫吸附测定用于评估肿瘤坏死因子-α和穿孔素的水平。最后，构建异种移植小鼠模型，以研究 ZNF250 缺陷型 CAF 衍生的外泌体对 HCC 细胞肿瘤特性的影响。研究表明，ZNF250 在 HCC 组织和细胞中表达增加，ZNF250 在 HCC 细胞中转录激活 PD-L1。ZNF250 的表达与 HCC 患者的 HbsAg、临床分期和肿瘤大小有关。CAF 衍生的外泌体 ZNF250 可调节 HCC 细胞中 PD-L1 的表达。此外，ZNF250 缺陷型 CAF 衍生的外泌体通过下调 PD-L1 表达抑制 HCC 细胞的增殖、迁移、侵袭和免疫逃避。此外，CAF 衍生的外泌体 ZNF250 促进体内肿瘤形成。这些发现提供了关于 CAF 衍生的外泌体在抑制 HCC 发展中的作用的见解，强调了 ZNF250 和 PD-L1 调节在肿瘤进展中的重要性。
