南方医科大学学报

南方医科大学学报 ›› 2024, Vol. 44 ›› Issue (3): 428-436.doi: 10.12122/j.issn.1673-4254.2024.03.03

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二甲双胍阻断乳腺癌细胞-间质细胞的交互作用：基于抑制肿瘤相关成纤维细胞缺氧诱导因子-1α的表达

邵 珊，白薇超，邹鹏程，罗敏娜，赵新汉，雷建军   

  1. 西安交通大学第一附属医院肿瘤内科，血液内科，肝胆外科，陕西 西安 710061
  • 出版日期:2024-03-20 发布日期:2024-04-03

Metformin suppresses hypoxia-inducible factor-1α expression in cancer-associated fibroblasts to block tumor-stromal cross-talk in breast cancer

SHAO Shan, BAI Weichao, ZHOU Pengcheng, LUO Minna, ZHAO Xinhan, LEI Jianjun   

  1. Department of Oncology, Department of Hematology, Department of Hepatobiliary Surgery, First Affiliated Hospital of Xi'an Jiaotong University, Xi'an 710061, China
  • Online:2024-03-20 Published:2024-04-03

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摘要： 目的 探讨二甲双胍（Met）对乳腺癌肿瘤-间质细胞交互作用的影响及机制。方法 将肿瘤相关成纤维细胞（CAFs）与乳腺癌细胞共培养，运用二甲双胍进行干预，分为对照组和Met干预组，ELISA及RT-qPCR检测Met对CAFs中HIF-1α、p-AMPK、基质衍生因子-1（SDF-1）和白细胞介素-8（IL-8）等因子的表达变化以及Transwell检测肿瘤细胞侵袭能力的变化。运用外源性SDF-1、IL-8干预后，Transwell检测肿瘤细胞侵袭能力的变化。运用缺氧诱导因子-1α（HIF-1α）shRNA或过表达质粒调节CAFs-HIF-1α的表达，以及AMPK-shRNA抑制AMPK的表达，并运用OG和2-OXO调节脯氨酸羟化酶的表达，及运用外源性TGF-β1干预后，Western blot及RT-qPCR检测CAFs中p-AMPK、HIF-1α、SDF-1、IL-8的表达，Transwell检测肿瘤细胞侵袭能力的变化。结果 相较于对照组，Met干预组中CAFs的p-AMPK、SDF-1和IL-8的表达水平升高（P<0.05），HIF-1α表达水平下降（P<0.05），AMPK的表达水平差异无统计学意义（P>0.05），Met组中乳腺癌细胞侵袭能力下降（P<0.05）。外源性SDF-1、IL-8干预可降低Met对乳腺癌细胞侵袭的抑制作用，增加乳腺癌细胞的侵袭能力（P<0.05）。过表达HIF-1α及运用脯氨酸羟化酶抑制剂OG提高HIF-1α的表达后，可降低Met对CAFs中HIF-1α、SDF-1及IL-8表达的抑制作用，并可降低Met对乳腺癌细胞侵袭的抑制作用（P<0.05）；运用HIF-1α-shRNA及运用脯氨酸羟化酶激活剂2-OXO抑制HIF-1α的表达后，降低乳腺癌细胞的侵袭能力（P<0.05）；运用AMPK-shRNA抑制p-AMPK的表达后，可降低Met对CAFs中HIF-1α表达的抑制作用，并可降低Met对乳腺癌细胞侵袭的抑制作用（P<0.05）；加入外源性TGF-β1后，可部分降低Met对CAFs中HIF-1α表达的抑制作用，并可部分降低Met对乳腺癌细胞侵袭的抑制作用（P<0.05）。结论 Met通过抑制CAFs-HIF-1α的表达进而发挥阻断乳腺癌细胞-间质细胞交互作用。

关键词: 肿瘤相关成纤维细胞；缺氧诱导因子-1α；二甲双胍；磷酸化-AMPK；肿瘤微环境

Abstract: Objective To investigate the mechanism of metformin for regulating tumor-stromal cell cross-talk in breast cancer. Methods Tumor associated fibroblasts (CAFs) co-cultured with breast cancer cells were treated with metformin, and the changes in expressions of hypoxia-inducible factor-1α (HIF-1α), p-AMPK, stroma-derived factor-1 (SDF-1) and interleukin-8 (IL-8) in the CAFs were detected using ELISA, RT-qPCR or Western blotting; Transwell assay was used to evaluate the invasiveness of the tumor cells and its changes following treatment with exogenous SDF-1, IL-8 and TGF-β1. The effects of HIF-1α shRNA or overexpression plasmid, AMPK shRNA, and treatment with OG (a proline hydroxylase inhibitor) or 2-OXO (a proline hydroxylase activator) were examined on p-AMPK, HIF-1α, SDF-1 and IL-8 expressions and invasiveness of the CAFs. Results Metformin treatment significantly increased the expression levels of p-AMPK, SDF-1 and IL-8 (P<0.05) and decreased HIF-1α expression (P<0.05) without affecting AMPK expression level (P>0.05) in the CAFs. The invasion ability of metformin-treated breast cancer cells was significantly decreased (P<0.05). Exogenous SDF-1 and IL-8, HIF-1α overexpression, and OG-induced upregulation of HIF-1α all significantly attenuated the inhibitory effects of metformin on breast cancer cell invasion (P<0.05) and HIF-1α, SDF-1 and IL-8 expressions in CAFs (P<0.05). Transfection with HIF-1α shRNA or treatment with 2-OXO significantly decreased the invasiveness of breast cancer cells (P<0.05). P-AMPK knockdown significantly suppressed the inhibitory effect of metformin on HIF-1α expression in CAFs and on invasion of breast cancer cells (P<0.05). Treatment with TGF-β1 partially decreased the inhibitory effect of metformin on HIF-1α expression in CAFs and invasiveness of the breast cancer cells (P<0.05). Conclusion Metformin suppresses HIF-1α expression in CAFs to block tumor-stromal cross talk in breast cancer.

Key words: cancer-associated fibroblasts; hypoxia-inducible factor-1α; metformin; phospho-AMPK; tumor microenvironment