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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.
CONCLUSIONS: A high UBE2T expression results in radiotherapy resistance of HCC possibly by enhancing glycolysis and cause enrichment of Tregs in the tumor microenvironment.
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.
CONCLUSIONS: A high UBE2T expression results in radiotherapy resistance of HCC possibly by enhancing glycolysis and cause enrichment of Tregs in the tumor microenvironment.
摘要：
目的：探讨泛素结合酶2T(UBE2T)过表达对肝细胞癌(HCC)放射敏感性的影响。
方法：用UBE2T过表达或对照慢病毒载体转染Hepa1-6细胞，使用集落形成试验和比色法评估其放疗敏感性以及上清液中葡萄糖和乳酸浓度的变化。转染的细胞皮下接种在裸鼠或C57BL/6小鼠中，并记录照射后的肿瘤生长情况。收集异种移植物用于使用流式细胞术分析CD4+T细胞和调节性T细胞(Tregs)的浸润并使用Western印迹检测HK1和LDHA的表达。UBE2T表达与免疫细胞浸润的相关性,使用CIBERSORT算法和TCGA数据库分析HCC中的糖酵解和Treg，结果在Hepa1-6细胞和Tregs共培养体系中得到验证。
结果：UBE2T过表达在荷瘤小鼠模型（尤其是C57BL/6小鼠）中培养的Hepa1-6细胞和异种移植物中均引起放疗抵抗。CIBERSORT分析表明，UBE2T的高表达与树突状细胞的百分比增加有关，滤泡辅助性T细胞,M2巨噬细胞，单核细胞,肝癌中的淋巴细胞和Tregs。UBE2T过表达的异种移植物显示Tregs百分比增加，HK1和LDHA表达增强，照射增加了肿瘤微环境中CD4+T细胞和Tregs的浸润。过表达UBE2T的Hepa1-6细胞显示出葡萄糖浓度降低和乳酸浓度升高。GSEA分析表明，高UBE2T表达与HCC中糖酵解和Tregs浸润增加呈正相关。在细胞共培养系统中，UBE2T过表达显著提高乳酸产量，Tregs的增殖和免疫抑制功能。
结论：UBE2T的高表达可能通过增强糖酵解并导致肿瘤微环境中Tregs的富集而导致HCC的放疗抵抗。
方法：用UBE2T过表达或对照慢病毒载体转染Hepa1-6细胞，使用集落形成试验和比色法评估其放疗敏感性以及上清液中葡萄糖和乳酸浓度的变化。转染的细胞皮下接种在裸鼠或C57BL/6小鼠中，并记录照射后的肿瘤生长情况。收集异种移植物用于使用流式细胞术分析CD4+T细胞和调节性T细胞(Tregs)的浸润并使用Western印迹检测HK1和LDHA的表达。UBE2T表达与免疫细胞浸润的相关性,使用CIBERSORT算法和TCGA数据库分析HCC中的糖酵解和Treg，结果在Hepa1-6细胞和Tregs共培养体系中得到验证。
结果：UBE2T过表达在荷瘤小鼠模型（尤其是C57BL/6小鼠）中培养的Hepa1-6细胞和异种移植物中均引起放疗抵抗。CIBERSORT分析表明，UBE2T的高表达与树突状细胞的百分比增加有关，滤泡辅助性T细胞,M2巨噬细胞，单核细胞,肝癌中的淋巴细胞和Tregs。UBE2T过表达的异种移植物显示Tregs百分比增加，HK1和LDHA表达增强，照射增加了肿瘤微环境中CD4+T细胞和Tregs的浸润。过表达UBE2T的Hepa1-6细胞显示出葡萄糖浓度降低和乳酸浓度升高。GSEA分析表明，高UBE2T表达与HCC中糖酵解和Tregs浸润增加呈正相关。在细胞共培养系统中，UBE2T过表达显著提高乳酸产量，Tregs的增殖和免疫抑制功能。
结论：UBE2T的高表达可能通过增强糖酵解并导致肿瘤微环境中Tregs的富集而导致HCC的放疗抵抗。
