UBE2T通过调节性T细胞诱导肝细胞癌的放疗抵抗

doi:10.12122/j.issn.1673-4254.2024.06.16

摘要/Abstract

摘要：

目的探索泛素结合酶2T（UBE2T）对肝细胞癌放疗敏感性的影响及机制。方法采用空白对照载体或过表达UBE2T慢病毒载体转染小鼠Hepa1-6肝癌细胞建立对照组（LV-Control）和过表达组（LV-UBE2T），qPCR以及Western blotting检测上述细胞UBE2T表达情况；对两组细胞进行射线照射（IR）处理，克隆形成实验检测UBE2T过表达对Hepa1-6肝癌细胞放疗敏感性影响；分别在裸鼠和C57BL/6小鼠皮下注射上述细胞建立肝癌皮下荷瘤小鼠模型，对皮下瘤予IR处理，建立LV-Control组、LV-Control+IR组、LV-UBE2T组和LV-UBE2T+IR组，5~6只/组，观察皮下瘤生长速度及体积。通过CIBERSORT算法分析肝癌免疫细胞浸润情况与UBE2T表达量的关系。流式细胞术检测上述4组小鼠的肝癌中CD4⁺T细胞以及调节性T细胞（Tregs）浸润情况。比色法测定细胞培养上清液中葡萄糖及乳酸的含量；癌症和肿瘤基因图谱（TCGA）的公共数据分析肝癌UBE2T表达量与糖酵解水平和Tregs浸润关系。Western blotting检测UBE2T表达与糖酵解相关蛋白HK1、LDHA表达相关关系。体外共培养模型联合流式细胞术以及qPCR验证UBE2T过表达肝癌与Tregs关系。结果 qPCR、Western blotting结果显示过表达组中UBE2T表达显著升高（P<0.0001）。克隆形成实验、裸鼠肝癌皮下瘤实验显示UBE2T过表达导致肝细胞癌放疗抵抗（P<0.05），UBE2T导致的放疗抵抗在C57BL/6小鼠肝癌皮下瘤模型上更显著（P<0.01）。CIBERSORT分析提示UBE2T高表达组肝癌中树突状细胞（P<0.01）、滤泡辅助性T细胞（P<0.001）、M2型巨噬细胞（P<0.01）、单核细胞（P<0.05）、总体淋巴细胞（P<0.05）以及Tregs（P<0.0001）浸润比例上调。流式细胞术显示过表达UBE2T小鼠肝癌免疫微环境中Tregs数量上调（P<0.05），IR导致UBE2T组CD4⁺T细胞以及Tregs浸润增加（P<0.01或P<0.001）。与对照组细胞培养上清液相比，过表达UBE2T组的培养上清液葡萄糖浓度降低（P<0.05），乳酸浓度上调（P<0.01）。GSEA分析提示UBE2T高表达肝癌与糖酵解水平（P<0.001）、Tregs浸润水平呈正相关（P<0.001）。Western blotting显示糖酵解相关蛋白HK1、LDHA表达水平与UBE2T表达水平相关。体外共培养模型显示UBE2T过表达肝癌使Tregs细胞内乳酸含量上调（P<0.001），增殖能力增加（P<0.05）以及免疫抑制功能上调（Il-10，P<0.05；TGF-β，P<0.001）。结论 UBE2T介导的肝癌细胞放疗抵抗可能与肝癌细胞糖酵解水平提高介导的免疫微环境中Tregs富集相关。

关键词: 泛素结合酶2T, 肝细胞癌, 放疗抵抗, 肿瘤免疫微环境, 调节性T细胞

Abstract:

Objective To investigate the effect of overexpression of ubiquitin-conjugating enzyme 2T (UBE2T) on radiosensitivity of hepatocellular carcinoma (HCC). Methods Hepa1-6 cells were transfected with a UBE2T-overexpressing or a control lentiviral vector, and the changes in their radiotherapy sensitivity and concentrations of glucose and lactate in the supernatant were assessed using colony-forming assay and colorimetric assay. The transfected cells were inoculated subcutaneously in nude mice or C57BL/6 mice, and tumor growth following irradiation were recorded. The xenografts were collected for analyzing infiltration of CD4⁺ T cells and regulatory T cells (Tregs) using flow cytometry and detecting expressions of HK1 and LDHA using Western blotting. The correlations of UBE2T expression with immune cell infiltration, glycolysis and Tregs in HCC were analyzed using CIBERSORT algorithm and TCGA database, and the results were verified in a co-culture system of Hepa1-6 cells and Tregs. Results UBE2T overexpression caused radiotherapy resistance in both cultured Hepa1-6 cells and xenografts in the tumor-bearing mouse models (especially in C57BL/6 mice). CIBERSORT analysis suggested that a high expression of UBE2T was associated with increased percentages of dendritic cells, T follicular helper cells, M2 macrophages, monocytes, lymphocytes and Tregs in HCC. The UBE2T-overexpressing xenografts showed an increased percentage of Tregs and enhanced expressions of HK1 and LDHA, and irradiation increased infiltration of CD4⁺ T cells and Tregs in the tumor microenvironment. Hepa1-6 cells overexpressing UBE2T showed a decreased glucose concentration and an increased lactate concentration. GSEA analysis suggested that a high UBE2T expression was positively correlated with increased glycolysis and Tregs infiltration in HCC. In the cell co-culture system, UBE2T overexpression significantly enhanced lactate production, proliferation and immunosuppressive functions of Tregs. Conclusion A high UBE2T expression results in radiotherapy resistance of HCC possibly by enhancing glycolysis and cause enrichment of Tregs in the tumor microenvironment.

Key words: ubiquitin-conjugating enzyme 2T, hepato-cellular carcinoma, radiotherapy resistance, tumor immune microenvironment, regulatory T cells

何欣容, 熊斯丽, 朱真如, 孙景苑, 曹传辉, 王惠. UBE2T通过调节性T细胞诱导肝细胞癌的放疗抵抗[J]. 南方医科大学学报, 2024, 44(6): 1149-1158.

Xinrong HE, Sili XIONG, Zhenru ZHU, Jingyuan SUN, Chuanhui CAO, Hui WANG. Overexpression of ubiquitin-conjugating enzyme 2T induces radiotherapy resistance in hepatocellular carcinoma by enriching regulatory T cells in the tumor microenvironment[J]. Journal of Southern Medical University, 2024, 44(6): 1149-1158.

图/表 6

表1 引物序列

Tab.1 Primers used for RT-qPCR

Primer

Sequence (5'→3')

Il-10-F

Il-10-R

GTCCTTTCACTTGCCCTCATC

CAAACYGGTCACAGCTTTCGA

图1 过表达UBE2T可导致肝癌放疗抵抗

Fig.1 Overexpression of UBE2T results in radiotherapy resistance in HCC. A: Relative mRNA level of Ube2t adjusted to Actin. B: Western blotting of UBE2T protein in LV-Control and LV-UBE2T cells. C, D: Colony formation assays of Hepa 1-6 cells with stable UBE2T overexpression. E: Treatment schedules of ionizing radiation (IR) and endpoint for recording. F, G: Tumor growth curves of the nude mice (F, n=5 or 6) and the dissected xenografts from the mice (G). in each group. H-J: Tumor growth curves of C57BL/6 mice (H, n=5 or 6), the tumor-bearing mice (I), and the dissected xenografts (J). In (F) and (H), statistical analyses were performed using a mixed-effects model followed by Tukey's multiple comparison test. In (A), student's t-test was used for comparisons. Data are presented as Mean±SD. *P<0.05, **P<0.01, ****P<0.0001.

图2 CIBERSORT肝癌免疫微环境分析

Fig.2 Immunological microenvironmental analysis of HCC by CIBERSORT. A: Percentages of different cell types in the two groups. B-J: Estimated fractions of DCs (B), T follicular helper cells (C), M1 macrophages (D), M2 macrophages (E), monocytes (F), lymphocytes (G), CD8+ T cells (H), Tregs (I), and CD8+T cells/Tregs (J) in the two groups. Student's t-test was used for comparisons. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. DCs: Dendritic cells, Tregs: Regulatory T cells.

图3 过表达UBE2T肝癌中Tregs增多

Fig.3 The number of Tregs is increased in HCC overexpressing UBE2T. A: Treatment schedules of IR and endpoint for analysis. B, C: Representative contour plots (B) and quantitation (C) of CD4+/CD3+ ratios (n=6-8). D, E: Representative contour plots (D) and quantitation (E) of the number of TIL Tregs (CD25+ Foxp3+) per 104 cells (n=6-8). Student's t-test was used for comparisons. Data are presented as Mean±SD. *P<0.05, **P<0.01, ***P<0.001.

图4 UBE2T表达量对肝癌糖酵解水平的影响及其与调控肝癌免疫微环境的关系

Fig.4 Effect of UBE2T expression on glycolysis levels in HCC and its regulatory effect on immune microenvironment. A, B: Concentration of glucose (A) and lactic acid (B) in the two groups with or without IR. C-E: Results of GSEA are plotted to visualize the correlation between the expression of UBE2T and the gene signatures of glycolysis and Tregs infiltration in the TCGA cohort. Student's t-test was used for comparisons. F, G: Western blotting of HK1, LDHA, and UBE2T protein in siControl, siUNE2T, LV-Control and LV-UBE2T cells. *P<0.05, **P<0.01, ***P<0.001.

图5 UBE2T过表达肝癌分泌乳酸增多扩增激活Tregs

Fig.5 HCC overexpressing UBE2T promoted lactate secretion and activated Tregs.A: Schematic diagram of the co-culture model. B: Concentrations of Tregs in different co-culture groups (n=5). C, D: Representative contour plots (C) and quantitation (D) of Ki67+ ratios (n=6). E: Relative mRNA level of IL-10 adjusted to Actin in different co-culture groups (n=3). F, G: Representative contour plots (F) and quantitation (G) of the TGF‑β+ ratios (n=5). One-way ANOVA was used for comparisons. Data are presented as Mean±SD. *P<0.05, **P<0.01, ***P<0.001.

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