| 李昂,查文章,轩福明,等.缺氧微环境下 HIF-1α通过 PD-L1调控肝癌恶性生物学行为和上皮间质转化的机制研究[J].安徽医药,2025,29(1):189-195. |
| 缺氧微环境下 HIF-1α通过 PD-L1调控肝癌恶性生物学行为和上皮间质转化的机制研究 |
| HIF-1α regulates malignant biological behavior and epithelial interstitial transformation of hepatocellular carcinoma through PD-L1 in hypoxia microenvironment |
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| DOI：10.3969/j.issn.1009-6469.2025.01.039 |
| 中文关键词: 癌，肝细胞 缺氧微环境 缺氧诱导因子 -1α 程序性死亡受体 -配体 1 上皮间质转化 |
| 英文关键词: Carcinoma, hepatocellular Hypoxia microenvironment Hypoxia-inducible factor-1α (HIF-1α) Programmed death ligand 1 (PD-L1) Epithelial-mesenchymal transformation (EMT) |
| 基金项目:江苏省卫生健康委员会科研项目面上项目( M2020100)；江苏大学医教协同创新基金项目( JDYY2023106) |
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| 中文摘要: |
| 目的探讨缺氧微环境下，缺氧诱导因子 -1α(HIF-1α)通过程序性死亡配体 1(PD-L1)调控肝癌细胞增殖、迁移、侵袭和上皮间质转化的机制。方法选取 2016年 9月至 2018年 9月在徐州医科大学附属盐城临床学院确诊治疗的 57例肝癌病人作为研究对象。免疫组织化学检测 HIF-1α、PD-L1的表达水平，统计学分析 HIF-1α、PD-L1与肝癌病人临床病理及总生存期之间的关系。通过氯化钴建立肝癌细胞缺氧模型，用细胞计数试剂盒( cell counting kit-8，CCK-8)法检测不同浓度氯化钴作用下细胞存活率；划痕实验、 Transwell实验检测缺氧细胞及亲本细胞迁移、侵袭能力的变化；蛋白质印迹法检测缺氧细胞及亲本细胞 HIF-1α、PD-L1蛋白表达水平。设计针对 HIF-1α的 siRNA(小干扰 RNA，small interfering RNA)及 PD-L1质粒，脂质体转染肝癌细胞氯化钴缺氧细胞，划痕实验、 Transwell侵袭实验检测转染前后细胞迁移、侵袭能力的变化；蛋白质印迹法检测转染前后细胞 HIF-1α、PD-L1、上皮间质转化( EMT)相关蛋白表达水平。结果与癌旁组织相比， HIF-1α、PD-L1在肝癌组织中高表达( P< 0.05)。肝细胞癌中 HIF-1α高表达组与低表达组在肿瘤长径、肿瘤分化程度和 CNLC分期差异有统计学意义( P<0.05)； PD-L1高表达组与低表达组在肿瘤数目、 AFP表达水平和 CNLC分期差异有统计学意义( P<0.05)；且 log-rank检验方法显示: HIF-1α、 PD-L1高表达组病人的总生存期较低表达组明显降低( P<0.05)。与常氧细胞相比，缺氧细胞穿透小室的细胞数显著增加［( 429.33±12.01)个比( 244.3±8.14)个， t=.22.08，P<0.001］，氯化钴缺氧肝癌细胞增殖、迁移及侵袭能力增强， HIF-1α、PD-L1表达水平明显增加(P<0.05)。与亲本细胞相比， HIF-1α siRNA作用后肝癌缺氧细胞增殖、迁移及侵袭能力明显降低(P<0.05)且 PD-L1、波形蛋白(Vimentin)及扭曲相关蛋白1(Twist1)蛋白表达明显降低，而上皮钙黏素(E-cadherin)蛋白表达明显增加(P<，0.05)； HIF-1α siRNA和 PD-L1质粒共转染后，可逆转 HIF-1α siRNA介导的侵袭、迁移能力的改变及 EMT相关指标的改变。结论 HIF-1α、PD-L1在肝癌中高表达，两者高表达预示肝癌病人预后较差。缺氧微环境下， HIF-1α可通过 PD-L1调控肝癌恶性生物学行为及上皮间质转化进程。 |
| 英文摘要: |
| Objective To investigate the mechanism of HIF-1α regulating hepatocellular carcinoma (HCC) cell proliferation, migra-tion, invasion and epithelial mesenchymal transformation through programmed death ligand 1 (PD-L1) in hypoxia microenvironment. Methods Fifty-seven patients diagnosed with liver cancer and treated at the Yancheng Clinical College of Xuzhou Medical Universityfrom September 2016 and September 2018 were selected as the study objects. The expression of HIF-1α and PD-L1 were detected by immunohistochemistry, and the relationship between HIF-1α, PD-L1 and clinicopathological and overall survival was statistically ana-lyzed. The hypoxia model of HCC was established by Cobalt Chloride (CoCl2), and the survival rate of HCC under different concentra-tions of CoCl2 was detected by Cell Counting Kit-8 (CCK-8) method. The changes of migration and invasion ability of hypoxic cells andparental cells were detected by scratch test and transwell invasion test. The expressions of HIF-1α and PD-L1 in hypoxic cells and pa-rental cells were detected by Western blot. Small interfering RNA (siRNA) targeting HIF-1α and PD-L1 plasmid were designed to trans-fect HCC cells with liposome into hypoxic cells. The changes of cell migration and invasion ability before and after transfection were de-tected by scratch assay and Transwell (migration/invasion) invasion assay. The expression levels of HIF-1α, PD-L1 and Epithelial mes-enchymal transformation (EMT)-related proteins before and after transfection were detected by Western blotting. Results HIF-1α and PD-L1 were highly expressed in HCC tissues compared with paracancellular tissues (P<0.05). There were significant differences in tu-mor size, tumor differentiation degree and China liver cancer staging (CNLC) stage between high and low PD-L1 expression groups (P< 0.05). There were significant differences in tumor number, AFP expression level, and CNLC stage between high and low PD-L1 expres-sion groups (P<0.05). Kaplan-Meier analysis showed that the survival rate of patients in HIF-1α and PD-L1 high expression groups was significantly lower than that in low expression group (P<0.05). Compared with normoxic cells, the number of cells penetrating the com-partment was significantly increased by hypoxic cells [(429.33±12.01) vs. (244.3±8.14), t=.22.08, P<0.001]. The proliferation, migra-tion and invasion ability of CoCl2 anoxic HCC cells were enhanced, and the expression levels of HIF-1α and PD-L1 were significantly increased (P<0.05). Compared with parental cells, the proliferation, migration and invasion ability of hypoxic HCC cells were signifi-cantly decreased after HIF-1α siRNA treatment (P<0.05), and the expressions of PD-L1, Vimentin and Twist1 were significantly de-creased, while the expressions of E.cadherin were significantly increased (P<0.05). After co-transfection of HIF-1α siRNA and PD-L1 plasmid, the changes of HIF-1α siRNA-mediated invasion, migration ability and EMT-related indexes could be reversed. Conclusion HIF-1α and PD-L1 are highly expressed in liver cancer. Liver cancer patients with high HIF-1α and PD-L1 expression have poor prog-nosis. In hypoxia microenvironment, HIF-1α can regulate malignant biological behavior and epithelial interstitial transformation pro-cess through PD-L1 in HCC cells. |
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