| 黄安中,张大伟,赵文生,等.AQP9通过调控细胞炎症及氧化应激抑制肝癌细胞转移[J].肿瘤学杂志,2025,31(6):488-497. |
| AQP9通过调控细胞炎症及氧化应激抑制肝癌细胞转移 |
| AQP9 Inhibits Metastasis of Hepatocellular Carcinoma Through Regulating Cellular Inflammation and Oxidative Stress |
| 投稿时间：2024-08-14 |
| DOI：10.11735/j.issn.1671-170X.2025.06.B004 |
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| 中文关键词: 肝细胞癌 水通道蛋白 细胞系 炎症 氧化应激 干性维持 |
| 英文关键词:hepatocellular carcinoma aquaporin cell line inflammation oxidative stress stemness maintenance |
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| 中文摘要: |
| 摘 要：［目的］ 探讨水通道蛋白AQP9对肝细胞癌（hepatocellular carcinoma，HCC）转移的作用及其分子机制。［方法］ 通过Western blot和qPCR检测不同临床分期肝癌组织（Ⅰ期、Ⅱ/Ⅲ期、Ⅳ期，各6例）及癌旁组织中AQP9的蛋白和mRNA表达水平，结合ELISA检测促炎因子［白细胞介素（interleukin，IL）-6、IL-1β、肿瘤坏死因子（tumor necrosis factor α，TNF-α）］的表达。在低表达AQP9的HepG2和SMMC-7721细胞系中过表达AQP9，评估其对细胞迁移（划痕实验、Transwell）、增殖（克隆形成）、炎症因子（ELISA）、氧化应激指标［活性氧（reactive oxygen species，ROS）、丙二醛（malondialdehyde，MDA）、超氧化物歧化酶（superoxide dismutase，SOD）、谷胱甘肽（glutathione，GSH）、谷胱甘肽过氧化物酶（glutathione peroxidase，GSH-Px）］及干性相关蛋白（CD133、Nanog、Oct4、Bmi1）的影响。通过免疫共沉淀法验证AQP9调控β-catenin/TCF4/FOX3a信号通路的相互作用。［结果］ 临床样本结果显示，随着肝癌临床分期增加，AQP9在蛋白和mRNA水平显著下调（Ⅳ期较正常组织分别降至21%±3%和22%±4%，P<0.001），而IL-6、IL-1β、TNF-α表达升高（P<0.05）。体外实验中，AQP9过表达显著抑制肝癌细胞迁移（HepG2伤口愈合率由52.20%±9.34%降至29.20%±3.18%，P=0.020 6）和增殖（SMMC-7721克隆数减少61%，P<0.001），并降低促炎因子（SMMC-7721中TNF-α降至23%±3%，P<0.001）及氧化应激标志物（ROS和MDA均显著降低，P<0.01）水平。机制研究表明，AQP9通过增强β-catenin与FOX3a的结合，竞争性抑制β-catenin/TCF4复合体形成，从而下调干性相关基因（CD133、Nanog）。［结论］ AQP9通过抑制炎症反应、缓解氧化应激及调控β-catenin/TCF4/FOX3a信号通路，降低HCC干性，进而抑制其迁移和侵袭，为靶向AQP9的肝癌治疗策略提供了理论依据。 |
| 英文摘要: |
| Abstract： ［Objective］ To investigate the effect of AQP9 on metastasis of hepatocellular carcinoma (HCC) and its molecular mechanism. ［Methods］ Specimens of liver cancer tissue and pericancerous liver tissues were collected from 24 HCC patients with stage Ⅰ (n=6)， stage Ⅱ/Ⅲ (n=12) and stage Ⅳ (n=6). The protein and mRNA expression levels of AQP9 in HCC tissues and pericancerous tissues were detected by Western blot (WB) and qPCR， respectively. The expression of pro-inflammatory factors interleukin(IL)-6， IL-1β， tumor necrosis factor α(TNF-α) was detected by ELISA. Human HCC HepG2 and SMMC-7721 cells were transfected with AQP9 gene to overexpress AQP9， cell migration was determined with scratch test and Transwell test， cell proliferation was determined with clonogenesis test， inflammatory factor levels were measured with ELISA， oxidative stress indexes reactive oxygen species (ROS)， malondialdehyde (MDA)， superoxide dismutase (SOD)， glutathione (GSH) and glutathione peroxidase (GSH-Px) and stemness cancer cells related proteins CD133， Nanog， Oct4， Bmi1 were also measured. The interaction of AQP9 regulating β-catenin/TCF4/FOX3a signaling pathway was verified by Co-immunoprecipitation method. ［Results］ The protein and mRNA expression levels of AQP9 were significantly down-regulated in late stage HCC (21%±3% and 22%±4% in stage Ⅳ compared with normal tissues， P<0.001)， while the expressions of IL-6， IL-1β and TNF-α were increased (P<0.005). In vitro study showed that overexpression of AQP9 significantly inhibited hepatoma cell migration (HepG2 wound healing rate decreased from 52.20%±9.34% to 29.20%±3.18%， P=0.020 6) and proliferation (SMMC-7721 clone number decreased by 61%， P<0.001)， and the level of pro-inflammatory factors (TNF-α decreased to 23%±3% in SMMC-7721， P<0.001) and markers of oxidative stress decreased (ROS and MDA were significantly decreased， P<0.01). Mechanism studies showed that AQP9 competitively inhibited the formation of the β-catenin/TCF4 complex by enhancing the binding of β-catenin to FOX3a， downregulating tumor stemness related genes (CD133， Nanog). ［Conclusion］ AQP9 can reduce the stemness of HCC cells by inhibiting inflammatory response， relieving oxidative stress and regulating β-catenin/TCF4/FOX3a signaling pathway， and then inhibit its migration and invasion. |
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