过表达己糖激酶2通过激活JAK/STAT途径促进结直肠癌细胞的增殖、迁移和侵袭并调节肿瘤免疫微环境

doi:10.12122/j.issn.1673-4254.2025.03.12

摘要/Abstract

摘要：

目的探究己糖激酶2（HK2）在结直肠癌组织中的表达，阐明其生物学功能及机制和对免疫微环境的影响。方法使用生物信息学方法分析HK2在结直肠癌组织中的表达水平、预后和对免疫微环境的影响。收集本院8例结直肠癌患者的肿瘤组织及配对癌旁组织，利用免疫组化、Western blotting和RT-qPCR实验验证HK2在结直肠癌中的表达水平。筛选出HK2表达量低的结直肠癌细胞系CT26及HCT116进行慢病毒转染过表达HK2，分为空白对照组和HK2过表达组；使用HK2抑制剂3-BP处理HK2表达量高的结直肠癌细胞系MC38及CACO2；使用JAK/STAT3通路抑制剂处理结直肠癌细胞HK2过表达组。采用CCK-8、平板克隆形成实验、Transwell和小鼠皮下荷瘤实验探究HK2对结直肠癌细胞的增殖、迁移和侵袭能力的影响。通过Western blotting检测HK2过表达对JAK/STAT信号通路蛋白表达的影响。采用MCPcounter和Timer分析HK2表达与肿瘤免疫细胞浸润水平的相关性，TCGA、GEO数据库分析HK2表达与免疫检查点的相关性。结果癌症公共数据库显示，HK2在结直肠癌组织中的表达水平高于癌旁组织（P<0.001），并且高表达HK2的结直肠癌患者预后更差（P=0.09）。免疫组化、Western blotting和RT-qPCR结果显示结直肠癌组织的HK2表达水平高于癌旁组织（P<0.01）。相较于其他结直肠癌细胞，CT26和HCT116的HK2表达水平最低（P<0.05），HK2过表达组相较于空白对照组的HK2表达水平上调（P<0.01），且过表达组细胞的增殖、迁移和侵袭能力显著提高（P<0.001）。HK2被抑制后肿瘤细胞增殖、迁移和侵袭能力明显被抑制。HK2可促进JAK/STAT信号通路中STAT3磷酸化蛋白的表达，使用JAK/STAT3通路抑制剂能够有效抑制由HK2过表达介导的肿瘤细胞增殖、迁移和侵袭能力的增强。Timer和MCP counter分析显示HK2表达与多种免疫细胞具有相关性，TCGA和GEO数据库分析显示HK2的表达水平与PDCD1等免疫检查点显著正相关（P<0.05）。结论 HK2在结直肠癌中表达上调，可能通过激活JAK-STAT信号通路促进肿瘤细胞的增殖、迁移和侵袭，并调节肿瘤免疫微环境。

关键词: 结直肠癌, 己糖激酶2, JAK-STAT信号通路, 增殖、迁移和侵袭, 免疫微环境

Abstract:

Objective To explore the expression of hexokinase 2 (HK2) in colorectal cancer (CRC) and its possible mechanisms for regulating tumor cell behaviors and tumor immune microenvironment. Methods We analyzed HK2 expression in CRC and its impact on patient prognosis and tumor immune microenvironment using public databases. HK2 expression was also examined in 8 CRC and paired adjacent tissues using immunohistochemistry, Western blotting and RT-qPCR. In cultured CRC cell lines CT26 and HCT116 with low HK2 expression, the effects of lentivirus-mediated HK2 overexpression and JAK/STAT3 inhibitors on cell proliferation, migration, and invasion were assessed using CCK-8 assay, colony formation assay and Transwell assay and in a subcutaneous tumor-bearing mouse model; the changes were also observed in MC38 and CACO2 cells with high HK2 expressions following treatment with HK2 inhibitor 3-BP. Western blotting was performed to verify the relationship between HK2 and JAK/STAT signaling pathway protein expressions. Results Informatics analyses suggested that HK2 expression was significantly higher in CRC tissues than in adjacent tissues (P<0.001), and patients with high HK2 expressions had worse prognosis (P=0.09). In the 8 clinical CRC tissues, HK2 expressions were significantly higher in the tumor tissues than in the adjacent tissues (P<0.01). In CT26 and HCT116 cells, HK2 overexpression significantly enhanced cell proliferation, migration and invasion, while in HK2-overexpressing MC38 and CACO2 cells, inhibiting HK2 with 3-BP strongly suppressed these changes. HK2 overexpression promoted STAT3 phosphorylation, and JAK/STAT3 inhibitors effectively suppressed tumor cell proliferation, migration and invasion. TIMER and MCPcounter analyses indicated correlations between HK2 and immune cells, and TCGA and GEO analyses suggested significant positive correlations between HK2 and the immune checkpoints including PDCD1. Conclusion HK2 is upregulated in CRC to promote tumor cell proliferation, migration and invasion possibly by activating the JAK-STAT signaling pathway and modulating tumor immune microenvironment.

Key words: colorectal cancer, hexokinase 2, JAK-STAT signaling pathway, proliferation, migration and invasion, immune microenvironment

庆顺杰, 沈智勇. 过表达己糖激酶2通过激活JAK/STAT途径促进结直肠癌细胞的增殖、迁移和侵袭并调节肿瘤免疫微环境[J]. 南方医科大学学报, 2025, 45(3): 542-553.

Shunjie QING, Zhiyong SHEN. High expression of hexokinase 2 promotes proliferation, migration and invasion of colorectal cancer cells by activating the JAK/STAT pathway and regulating tumor immune microenvironment[J]. Journal of Southern Medical University, 2025, 45(3): 542-553.

图/表 13

表1 引物序列

Tab.1 Primer sequence for RT-qPCR

Gene	Species	Forward primer	Reverse primer
HK2	Human	GAGCCACCACTCACCCTACT	CCAGGCATTCGGCAATGTG
Hk2	Mouse	ATGATCGCCTGCTTATTCACG	CGCCTAGAAATCTCCAGAAGGG
GAPDH	Human	GGAGCGAGATCCCTCCAAAAT	GGCTGTTGTCATACTTCTCATGG
Gapdh	Mouse	AGGTCGGTGTGAACGGATTTG	GGGGTCGTTGATGGCAACA

图1 HK2在结直肠癌中的表达及与预后的关系

Fig.1 Expression of HK2 in colorectal cancer and its association with patient prognosis. A: Expression levels of HK2 in colorectal cancer from TCGA databases. B: Kaplan-Meier survival curves of overall survival (OS) for CRC patients stratified by HK2 expression level. ****P<0.0001.

图2 HK2在临床样本组织中的表达

Fig.2 Expression of HK2 in clinical samples of CRC and adjacent tissues. A: Immunohistochemical staining for HK2 expression. B, C: HK2 protein and mRNA expression levels in cancer tissues and adjacent tissues. **P<0.01. T: Tumor; N: Normal.

图3 HK2过表达CRC细胞的构建与验证

Fig.3 Construction and validation of HK2-overexpressing CRC cells. A, B: HK2 mRNA expression in CRC cells. C-F: Protein and mRNA expression of HK2 in CT26 and HCT116 cells after lentivirus-mediated transfection. *P<0.05, **P<0.01, ****P<0.0001.

图4 HK2过表达对HCT116和CT26细胞增殖能力的影响

Fig.4 Effect of HK2 overexpression on proliferation ability of CRC cells in vitro. A-B: CCK-8. C-D: Colony-forming assay. ***P<0.001, ****P<0.0001.

图5 HK2过表达对HCT116和CT26细胞迁移和侵袭能力的影响

Fig.5 Effect of HK2 overexpression on migration and invasion of CT26 cells (A, B) and HCT116 cells (C, D) (Original magnification: ×40). **P<0.01.

图6 HK2功能抑制对体外CRC细胞增殖、迁移和侵袭能力的影响

Fig.6 Effect of HK2 functional inhibition on the proliferation, migration, and invasion of CRC cells in vitroA-B, F: MC38. C-E: CACO2 (Original magnification: ×40). **P<0.01, ***P<0.001, ****P<0.0001.

图7 HK2对体内鼠源性CRC细胞增殖能力的影响

Fig.7 Effect of HK2 overexpression on the proliferation of CRC cells in vivo. A-D: Effect of HK2 overexpression in CT26 cells on subcutaneous tumor proliferation. E: Ki-67 proliferative index of subcutaneous tumors (×40). F, G: Number of CD8+ T cells in subcutaneous tumors. **P<0.01, ***P<0.001.

图8 HK2对体内人源性CRC细胞增殖能力的影响

Fig.8 Effect of HK2 overexpression on proliferation of HCT116 cells in nude mice. A-C: Effect of HK2 on subcutaneous tumor proliferation. *P<0.05.

图9 HK2调控JAK/STAT信号通路

Fig.9 HK2 regulates the JAK/STAT signaling pathway. A: GSEA confirms that the JAK/STAT signaling pathway is significantly enriched in CRC with high HK2 expression based on CRC data from TCGA. B-E: Western blotting showing expression levels of STAT and p-STAT after HK2 overexpression. ***P<0.001, ****P<0.0001.

图10 HK2通过JAK/STAT影响结直肠癌细胞生物学行为

Fig.10 HK2 affects biological behaviors of CRC cells through the JAK/STAT pathway. A, B: Transwell assay showing migration and invasion of CT26 (A) and HCT116 (B) cells in the NC, OE and OE+GA groups (×40). C, D: Quantification of numbers of migrating and invading cells in CT26 (C) and HCT116 (D) from the Transwell assays. E, F: Cell proliferation assessed by CCK-8 assay in CT26 (E) and HCT116 (F) cells in different groups. *P<0.05, **P<0.01, ****P<0.0001.

图11 HK2与肿瘤免疫细胞浸润的相关性

Fig.11 Correlation between HK2 expression and immune infiltration analyzed using MCPcounter and TIMER. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

图12 HK2与免疫检查点的相关性

Fig.12 Correlation between HK2 and immune checkpoints analyzed using TCGA (A) and GEO (B) databases. **P<0.01, ***P<0.001.

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