肝癌细胞通过上调巨噬细胞ANXA2和ANXA5表达诱导其向M2型极化

doi:10.12122/j.issn.1673-4254.2026.09.10

南方医科大学学报 ›› 2026, Vol. 46 ›› Issue (9): 2101-2112.doi: 10.12122/j.issn.1673-4254.2026.09.10

• • 上一篇

肝癌细胞通过上调巨噬细胞ANXA2和ANXA5表达诱导其向M2型极化

许军英¹^,²(), 刘程豪²^,³, 黄浩¹^,², 姜慧娇¹^,², 董丹¹^,², 吴向未¹^,²^,³, 陈雪玲¹^,²()

^1.石河子大学医学院基础医学系，新疆石河子 832000
^2.国家卫生健康委中亚高发病防治重点实验室，新疆石河子 832000
^3.石河子大学第一附属医院肝胆外科，新疆石河子 832000

收稿日期:2026-01-20 出版日期:2026-09-20 发布日期:2026-09-30
通讯作者: 陈雪玲 E-mail:sophiaxu4504@foxmail.com;chenxueling@shzu.edu.cn
作者简介:许军英，讲师，E-mail: sophiaxu4504@foxmail.com
基金资助:
国家自然科学基金(82573336)

Hepatocellular carcinoma cells induce M2 macrophage polarization through upregulation of ANXA2 and ANXA5 expression

Junying XU¹^,²(), Chenghao LIU²^,³, Hao HUANG¹^,², Huijiao JIANG¹^,², Dan DONG¹^,², Xiangwei WU¹^,²^,³, Xueling CHEN¹^,²()

^1.Department of Basic Medicine, School of Medicine, Shihezi 832000, China
^2.National Health Commission Key Laboratory of Prevention and Treatment for High-Incidence Diseases in Central Asia, Shihezi 832000, China
^3.Department of Hepatobiliary Surgery, First Affiliated Hospital, Shihezi University, Shihezi 832000, China

Received:2026-01-20 Online:2026-09-20 Published:2026-09-30
Contact: Xueling CHEN E-mail:sophiaxu4504@foxmail.com;chenxueling@shzu.edu.cn
Supported by:
Natural Science Foundation of China(82573336)

摘要/Abstract

摘要：

目的基于多组学数据探究膜联蛋白A2（ANXA2）、膜联蛋白A5（ANXA5）和膜联蛋白A11（ANXA11）对肝细胞癌（HCC）预后及肿瘤微环境的影响。方法收集公共数据集中HCC转录组以及临床数据、空间转录组数据，采用生物信息学方法（如生存分析、相关性分析、免疫微环境分析以及富集分析等）分析ANXA2、ANXA5与患者预后、肿瘤微环境的影响以及其可能机制；通过免疫荧光实验，检测ANXA2、ANXA5和ANXA11在肝细胞癌组织表达以及与CD206共表达情况；使用人肝细胞癌细胞系MHCC-97H培养上清（TCM）处理THP-1细胞，qRT-PCR检测THP-1细胞的IL-10、ANXA2、ANXA5和ANXA11的mRNA水平；Western blotting实验检测THP-1细胞ANXA2、ANXA5、ANXA11以及KLF4的蛋白水平变化；流式细胞术检测巨噬细胞CD206和PD-L1的表达。结果生物信息学分析显示ANXA2、ANXA5和ANXA11在肝细胞癌中表达上调，与肝细胞癌不良预后相关（P<0.05）；GO分析和GSEA富集分析显示，ANXA2、ANXA5和ANXA11与IL-10、IL-4/IL-13信号通路正向富集；免疫微环境分析显示，ANXA2、ANXA5和ANXA11转录水平与巨噬细胞浸润呈正相关（P<0.05）。实验结果显示，ANXA2、ANXA5与M2型巨噬细胞标志CD206，在人肝细胞癌组织中共表达；使用TCM诱导THP-1细胞，仅ANXA2、ANXA5以及CD206、PD-L1表达上调（P<0.01）；使用ANXA2、ANXA5蛋白处理THP-1细胞，THP-1细胞CD206表达上调（P<0.01），而PD-L1无变化；相关性分析显示，KLF4转录水平分别与ANXA2、ANXA5呈正相关，使用KLF4抑制剂Kenpaullone，可抑制THP-1细胞ANXA2和ANXA5表达（P<0.05）。结论肝癌细胞通过上调巨噬细胞ANXA2和ANXA5表达，促进巨噬细胞向M2型极化，进而参与构建肝细胞癌免疫抑制微环境。ANXA2和ANXA5可作为联合免疫治疗的分子靶标。

关键词: 肝细胞癌, 膜联蛋白A2, 膜联蛋白A5, 肿瘤相关巨噬细胞, CD206

Abstract:

Objective To explore the prognostic value of annexins A2 (ANXA2) and A5 (ANXA5) in hepatocellular carcinoma (HCC) and their impact on tumor microenvironment. Methods Public datasets of HCC transcriptome and clinical data and spatial transcriptomic data were used for survival analysis, correlation analysis, immune microenvironment analysis, and enrichment analysis to explore the effects of annexin A2 (ANXA2) and ANXA5 on patient prognosis and tumor microenvironment and the potential mechanisms. Immunofluorescence assays were performed to detect ANXA2, ANXA5, and ANXA11 expressions in HCC tissues and their co-expression with CD206. THP-1 cells were treated with culture supernatant of human HCC MHCC-97H cells (TCM), and the changes in mRNA levels of IL-10, ANXA2, ANXA5, and ANXA11 and the protein levels of ANXA2, ANXA5, ANXA11, and KLF4 were examined using qRT-PCR and Western blotting. Flow cytometry was used to detect the expression of CD206 and PD-L1 in the macrophages. Results ANXA2, ANXA5 and ANXA11 expressions were all significantly up-regulated in HCC tissues in correlation with poor patient prognosis. GO and GSEA revealed enrichment of IL-10 and IL-4/IL-13 signaling pathways in tumors overexpressing ANXA2, ANXA5 and ANXA11, whose high expressions were positively correlated with macrophage infiltration. Immunofluorescence staining demonstrated co-expressions of ANXA2 and ANXA5 with M2-macrophage markers in human HCC tissues. The conditioned medium from HCC cells significantly up-regulated the expressions of ANXA2, ANXA5, CD206 and PD-L1 in THP-1 cells, whereas treatment with the recombinant ANXA2 or ANXA5 protein alone increased CD206 expression only. Correlation analysis indicated that KLF4 was positively correlated with both ANXA2 and ANXA5, and inhibition of KLF4 caused significant reduction of ANXA2, ANXA5 and CD206 levels in THP-1 cells. Conclusion HCC promotes M2 macrophage polarization by up-regulating ANXA2 and ANXA5 expressions, which contribute to an immunosuppressive microenvironment in HCC and potentially serve as molecular targets for HCC immunotherapy.

Key words: hepatocellular carcinoma, annexin A2, annexin A5, Tumor-Associated Macrophage, CD206

许军英, 刘程豪, 黄浩, 姜慧娇, 董丹, 吴向未, 陈雪玲. 肝癌细胞通过上调巨噬细胞ANXA2和ANXA5表达诱导其向M2型极化[J]. 南方医科大学学报, 2026, 46(9): 2101-2112.

Junying XU, Chenghao LIU, Hao HUANG, Huijiao JIANG, Dan DONG, Xiangwei WU, Xueling CHEN. Hepatocellular carcinoma cells induce M2 macrophage polarization through upregulation of ANXA2 and ANXA5 expression[J]. Journal of Southern Medical University, 2026, 46(9): 2101-2112.

图/表 9

表1 引物序列

Tab.1 Primer sequence for qRT-PCR

Gene	Primer (5'-3')
ANXA2	F: ATATTGCCTTCGCCTACCAGAG R: GCGTCATACTGAGCAGGTGTCTT
ANXA5	F: GACTTCCCTGGATTTGATGAGC R: GAGGGTTTCATCAGAGCCACAA
ANXA11	F: AAAGGCTTCGGGACGGAT R: AGCCGTCTTGAAGGAAAGTAGGAT
IL-10	F: CTCCGAGATGCCTTCAGCAG R: TCACATGCGCCTTGATGTCT
β-actin	F: CACCCAGCACAATGAAGATCAAGAT R: CCAGTTTTTAAATCCTGAGTCAAGC

图1 ANXA2、ANXA5和ANXA11在肝细胞癌中表达与预后关系

Fig.1 Expression of ANXA2, ANXA5, and ANXA11 in hepatocellular carcinoma (HCC) and their association with prognosis. A, B: ANXA family expression in HCC. C, E: Analysis of the association between the ANXA family and prognosis of HCC. D: PPI network analysis of molecular functional relationships within the ANXA family. *P<0.05, **P<0.01, ***P<0.001

图2 空间转录组数据分析肝细胞癌ANXA2、ANXA5和ANXA11 的mRNA水平

Fig.2 Spatial transcriptomic analysis of ANXA2, ANXA5, and ANXA11 mRNA levels in HCC. A-H: Spatial transcriptomic analysis of ANXA2, ANXA5, and ANXA11 mRNA levels in non-malignant (nMal) and malignant (Mal) regions of HCC tissue. ***P<0.001 vs nMal.

图3 ANXA2、ANXA5和ANXA11的mRNA水平与免疫细胞浸润相关性

Fig.3 Correlation between expression levels of ANXA2, ANXA5, and ANXA11 and immune cell infiltration. A, C, E: ssGSEA analysis of ANXA2, ANXA5 and ANXA11 mRNA levels in relation to immune-cell infiltration. B, D, F: Spatial transcriptomic analysis of ANXA2, ANXA5 and ANXA11 mRNA levels in relation to immune-cell infiltration.

图4 ANXA2、ANXA5和ANXA11与免疫抑制以及黏附基因表达的相关性

Fig.4 Correlation of ANXA2, ANXA5, and ANXA11 with immunosuppressive and adhesion gene expression. A-C: Top 30 co-expressed gene profiling analysis of ANXA2, ANXA5, and ANXA11. D: PPI-based interactome analysis of ANXA2, ANXA5, and ANXA11 together with their top 30 co-expressed genes.

图5 ANXA2、ANXA5和ANXA11与IL-4/IL-13及PD-1信号通路相关

Fig.5 ANXA2, ANXA5, and ANXA11 are associated with IL-4/IL-13 and PD-1 signaling pathways. A, B: GO functional annotation and GSEA enrichment analysis of ANXA2, ANXA5, and ANXA11.

图6 ANXA2、ANXA5与ANXA 11 在肝细胞癌中表达

Fig.6 Expression of ANXA2, ANXA5, and ANXA11 in HCC tissues. A-C: Immunofluorescence staining of the co-expression of ANXA2, ANXA5, and ANXA11 with CD206 in HCC tissues. D, E: mRNA and protein levels of ANXA2, ANXA5, and ANXA11 (n=3). CON: Control; TCM: Tumor conditioned medium. **P<0.01, ***P<0.001 vs CON group.

图7 ANXA2与ANXA5诱导巨噬细胞M2型标志CD206表达

Fig.7 ANXA2 and ANXA5 induce the expression of the M2 macrophage marker CD206. A-C: flow cytometric analysis of CD206 (n=5), PD-L1 (n=5) expression and IL-10 (n=3) mRNA levels in THP-1 cells after treatment with tumor-conditioned medium (TCM). D-F: CD206, PD-L1 expression and IL-10 mRNA level in THP-1 cells induced by ANXA2 and ANXA5 (n=3). ***P<0.001 vs CON group.

图8 肝癌细胞通过KLF4上调THP-1细胞ANXA2和ANXA5表达

Fig.8 TCM of MHCC-97H cells promotes ANXA2 and ANXA5 expression in THP-1 cells by upregulating KLF4. A: Correlation analysis of ANXA2, ANXA5, and KLF4 mRNA levels using TCGA-LIHC transcriptome data (n=3). B, C: Protein levels of KLF4, ANXA2, and ANXA5 of THP-1 cells treated by TCM and Kenpaullone (n=3). D, E: Flow cytometric analysis of CD206 and PD-L1 expression in THP-1 cells (n=4). Ken: Kenpaullone. *P<0.05, ***P<0.001 vs CON group; ##P<0.01, #P<0.05 vs TCM group.

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肝癌细胞通过上调巨噬细胞ANXA2和ANXA5表达诱导其向M2型极化

Hepatocellular carcinoma cells induce M2 macrophage polarization through upregulation of ANXA2 and ANXA5 expression

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