TGF-β通过miR-23a-3p/IRF1轴下调主要组织相容性复合体I类表达促进肝癌免疫逃逸

doi:10.12122/j.issn.1673-4254.2025.07.06

摘要/Abstract

摘要：

目的探讨肝癌细胞中转化生长因子-β（TGF-β）调控主要组织相容性复合体I类（MHC-I）分子的机制及其在肿瘤免疫逃逸中的作用。方法设置对照组、TGF-β处理组及TGF-β联合TGF-β Ⅰ型受体抑制剂SB-431542干预组，通过Western blotting和RT-qPCR评估TGF-β对肝癌细胞MHC-I表达水平的影响；通过RNA转染及其干扰技术、RT-qPCR和Western blotting实验探究TGF-β调控肝癌细胞MHC-I表达水平的分子机制。将各组肝癌细胞与健康人外周血单个核细胞（PBMC）共培养，采用CCK-8、克隆形成实验检测肿瘤增殖能力；通过乳酸脱氢酶（LDH）释放实验、线粒体膜电位检测量化T细胞杀伤效率，并利用流式细胞术及ELISA分析T细胞活化水平。结果 TGF-β抑制肝癌细胞MHC-I表达（P<0.01），且T细胞活化水平降低，导致共培养体系中肿瘤增殖率上升且死亡率下降；TGF-β通过特异性上调miR-23a-3p（P<0.001，P<0.01），负向调控其靶基因IRF1（P<0.05），进而抑制MHC-I转录（P<0.01）；过表达miR-23a-3p可重现IRF1/MHC-I表达抑制效应（P<0.05）。结论 TGF-β通过miR-23a-3p/IRF1信号轴下调MHC-I表达，削弱T细胞介导的抗肿瘤免疫应答，揭示了肝癌免疫逃逸的新机制。

关键词: 肝癌细胞, 转化生长因子-β, 主要组织相容性复合体I类, 肿瘤免疫逃逸

Abstract:

Objective To investigate the mechanism by which transforming growth factor‑β (TGF‑β) regulates major histocompatibility complex class I (MHC-I) expression in hepatocellular carcinoma (HCC) cells and its role in immune evasion of HCC. Methods HCC cells treated with TGF‑β alone or in combination with SB-431542 (a TGF-β type I receptor inhibitor) were examined for changes in MHC-I expression using RT-qPCR and Western blotting. A RNA interference experiment was used to explore the role of miR-23a-3p/IRF1 signaling in TGF‑β‑mediated regulation of MHC-I. HCC cells with different treatments were co-cultured with human peripheral blood mononuclear cells (PBMCs), and the changes in HCC cell proliferation was assessed using CCK-8 and colony formation assays. T-cell cytotoxicity in the co-culture systems was assessed with lactate dehydrogenase (LDH) release and JC-1 mitochondrial membrane potential assays, and T-cell activation was evaluated by flow cytometric analysis of CD69 cells and ELISA for TNF-α secretion. Results TGF‑β treatment significantly suppressed MHC-I expression in HCC cells and reduced T-cell activation, leading to increased tumor cell proliferation and decreased HCC cell death in the co-culture systems. Mechanistically, TGF-β upregulated miR-23a-3p, which directly targeted IRF1 to inhibit MHC-I transcription. Overexpression of miR-23a-3p phenocopied TGF‑β‑induced suppression of IRF1 and MHC-I. Conclusion We reveal a novel immune escape mechanism of HCC, in which TGF‑β attenuates T cell-mediated antitumor immunity by suppressing MHC-I expression through the miR-23a-3p/IRF1 signaling axis.

Key words: liver cancer cells, transforming growth factor-β, major histocompatibility complex class I, tumor immune escape

于滢, 涂丽, 刘洋, 宋雪翼, 邵倩倩, 唐小龙. TGF-β通过miR-23a-3p/IRF1轴下调主要组织相容性复合体I类表达促进肝癌免疫逃逸[J]. 南方医科大学学报, 2025, 45(7): 1397-1408.

Ying YU, Li TU, Yang LIU, Xueyi SONG, Qianqian SHAO, Xiaolong TANG. The TGF‑β/miR-23a-3p/IRF1 axis mediates immune escape of hepatocellular carcinoma by inhibiting major histocompatibility complex class I[J]. Journal of Southern Medical University, 2025, 45(7): 1397-1408.

图/表 9

表1 引物序列

Tab.1 Sequences of primers for RT-qPCR

Genes	Primer (5′-3′)
HLA-A	F:ACCCTCGTCCTGCTACTCTC
HLA-A	R:CTGTCTCCTCGTCCCAATACT
U6	F:AGAGAAGATTAGCATGGCCCCTG
U6	R:ATCCAGTGCAGGGTC CGAGG
GAPDH	F:CAGGAGGCATTGCTGATGAT
GAPDH	R:GAAGGCTGGGGCTCATTT
miR-23a-3p	F:AGCGCCTATCACATTGCCAGG
miR-23a-3p	R:ATCCAGTGCAGGGTCCGAGG
miR-106b-5p	F: TGCGGCAACACCAGTCGATGG
miR-106b-5p	R: CCAGTGCAGGGTCCGAGGT
miR-17-5p	F: GCGCAAAGTGCTTACAGTGC
miR-17-5p	R:AGTGCAGGGTCCGAGGTATT
miR-24-2-5p	F: GCGGTGCCTACTGAGCTGA
miR-24-2-5p	R:AGTGCAGGGTCCGAGGTATT

图1 TGF-β下调肝癌细胞株中MHC-I分子的表达

Fig.1 TGF-β down-regulates the expression of MHC-I molecules in hepatocellular carcinoma (HCC) cell lines. A, B: Western blotting of MHC-I protein expression in 4 human HCC cell lines. C: RT-PCR analysis of HLA-A mRNA expression in 4 human HCC cell lines. D, E: Western blotting of MHC-I protein expression in TGF-β-treated HCC cells. F: RT-PCR analysis of HLA-A mRNA expression in TGF-β-treated HCC cells. SB: SB-431542. *P<0.05, **P<0.01.

图2 TGF-β影响PBMC活化并降低其对肿瘤细胞的毒性和杀伤作用

Fig.2 TGF-β affects PBMC activation and reduces its toxicity and killing effect against HCC cells. A: CCK-8 assay for assessing HCC cell viability in the co-culture models with different treatments. B, C: Clonogenic assay for quantifying colony formation capacity of HCC cells in the co-culture models in different treatment groups. D: LDH release assay for evaluating cytotoxicity in the co-culture models with different treatments. E, F: JC-1 staining for assessing mitochondrial membrane potential of HCC cells in the co-culture models. G: ELISA for detecting TNF-α secretion by activated T cells in the co-culture systems in different groups. H: Flow cytometry for analyzing CD8+ T cell percentage with high expression of CD69 in the co-culture models in different groups. *P<0.05, **P<0.01, ***P<0.001.

图3 TGF-β降低IRF1的表达水平

Fig.3 TGF‑β decreases the expression level of IRF1. A, B: Western blotting for detecting the expression level of IRF1 in TGF-β treated cells. *P<0.05.

图4 IRF1的表达水平影响MHC-I的表达

Fig.4 Expression level of IRF1 affects the expression of MHC-I. A-C: Western blotting for detecting expression levels of IRF1 and MHC-I in HCC cells after IRF1 knockdown. *P<0.05, **P<0.01.

图5 IRF1的表达影响PBMC活化并降低其对肿瘤细胞的毒性和杀伤作用

Fig.5 Expression of IRF1 affects PBMC activation and reduces its toxicity and killing effect on HCC cells. A: CCK-8 assay for assessing viability of HCC cells with different treatments in the co-culture models. B: LDH release assay for evaluating tumor cell cytotoxicity in the co-culture systems. C,D: JC-1 staining assessing mitochondrial membrane potential of HCC cells in the co-culture models with different treatments. E: ELISA of TNF-α secretion by activated T cells in the co-culture systems. F: Flow cytometric analysis of percentage of CD8+ T cells with high CD69 expression in the co-culture models. *P<0.05, **P<0.01, ***P<0.001.

图6 Targetscan数据库、miRDB数据库和miRWalk数据库预测靶向IRF1的microRNA

Fig.6 Prediction of microRNAs targeting IRF1 using TargetScan, miRDB and miRWalk databases.

图7 TGF-β通过miR-23a-3p/IRF1下调MHC-I的表达

Fig.7 TGF-β down-regulates MHC-I expression via miR-23a-3p/IRF1. A, B: RT-PCR for analyzing expression levels of miR-23a-3p, miR-106b-5p, miR-17-5p, and miR-24-2-5p in HCC cells after TGF-β treatment. C: RT-qPCR analysis of miR-23a-3p expression in TGF-β-treated cells. D-F: Western blotting of IRF1 and MHC-I protein expressions in HCC cells following modulation of miR-23a-3p expression level. *P<0.05, **P<0.01, ***P<0.001.

图8 MiR-23a-3p影响PBMC活化并降低其对肿瘤细胞的毒性和杀伤作用

Fig.8 MiR-23a-3p affects PBMC activation and reduces its cytotoxicity and killing effect on HCC cells. A: CCK-8 assay of viability of HCC cells with different treatments in the co-culture models. B: LDH release assay for evaluating tumor cell cytotoxicity in the co-culture systems with different treatments. C, D: JC-1 staining for assessing mitochondrial membrane potential of the tumor cells in the co-culture models. E: ELISA quantification of TNF-α secretion by activated T cells in the co-culture systems. F: Flow cytometric analysis of the proportion of CD8+ T cells with high CD69 expression in the co-culture models. *P<0.05, **P<0.01, ***P<0.001.

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