肺腺癌放射敏感性受泛素结合酶2T抑制作用的研究

Study on the inhibitory effect of UBE2T on radiosensitivity of lung adenocarcinoma

目的探讨泛素结合酶2T(UBE2T)对肺腺癌放射敏感性的影响及其可能的机制。方法收集2019年3月至2021年12月在遵义医科大学第二附属医院经病理证实, 且经单纯放射治疗的各期肺腺癌患者45例, 按实体肿瘤疗效评价(RECIST)1.1标准进行疗效评价, 分为放疗敏感组(25例)、放疗抵抗组(20例), 前者为完全缓解+部分缓解, 后者为疾病稳定+疾病进展。免疫组化(IHC)检测通过染色强度和阳性细胞数计分, 结合卡方检验分析患者石蜡标本UBE2T表达水平与放射敏感性间的相关性。利用人肺腺癌细胞, 构建慢病毒UBE2T干扰(UBE2Tsh)的A549细胞和UBE2T过表达的SPC-A-1细胞, 行放射及克隆形成实验检测细胞的存活分数, 多靶单击模型拟合生存曲线。免疫荧光技术检测DNA双链损伤(DSB)标记物γH2AX聚焦点水平(γH2AX聚焦点数/单个细胞)。蛋白质印迹法检测UBE2T、γH2AX、Rad51蛋白表达。流式细胞术检测细胞周期变化和凋亡率。二分类变量资料统计采用Fisher’’s精确概率法, 计量资料采用t检验。结果 UBE2T高表达同肺腺癌患者放疗抵抗相关(P<0.05)。慢病毒UBE2T干扰提高A549细胞的放射敏感性, 放射增敏比(SER)为1.795, UBE2T过表达降低SPC-A-1细胞的放射敏感性, SER为0.293。UBE2Tsh A549细胞照射后细胞核内γH2AX聚焦点数显著增加(P<0.01), γH2AX蛋白表达量增加(P<0.01), Rad51蛋白表达量降低(P<0.001), UBE2T过表达SPC-A-1细胞γH2AX蛋白表达量降低(P<0.05), Rad51蛋白表达量增加(P<0.001)。UBE2Tsh A549细胞照射后G2期细胞占比减少(P<0.01), 细胞凋亡增加(P<0.001)。结论 UBE2T可能通过增强肺腺癌细胞放疗所致DNA双链损伤修复, 诱导细胞周期G2期阻滞, 减少细胞凋亡介导放疗抵抗。

Objective To investigate the effect of ubiquitin binding enzyme 2T(UBE2T)on the radiosensitivity of lung adenocarcinoma and unravel its possible mechanism.Methods A total of 45 patients pathologically diagnosed with different stages of lung adenocarcinoma and treated with radiotherapy in the Second Affiliated Hospital of Zunyi Medical University from March,2019 to December,2021 were enrolled,and the efficacy was evaluated according to response evaluation criteria in solid tumors(RECIST1.1).All patients were divided into radiosensitive group(n=25)and radioresistant group(n=20).Radiosensitive group was complete remission(CR)+partial remission(PR),and radioresistant group was stable disease(SD)+progression disease(PD).Immunohistochemistry(IHC)was used to calculate the score based on the staining intensity and the number of positive cells.Chi-square test was combined to analyze the correlation between the expression level of UBE2T in paraffin specimens of lung adenocarcinoma patients and the radiosensitivity of patients.Lentivirus UBE2T-interfered(UBE2Tsh)A549 and UBE2T-overexpressed SPC-A-1 lung adenocarcinoma cells and their respective controls were constructed for irradiation and colony formation assay.The survivor fraction curve was fitted by single-hit multi-target model.The DNA double-strand break(DSB)markerγH2AX foci were detected by immunofluorescence(IF).The expression levels of UBE2T,γH2AX and Rad51 proteins were detected by Western blot.Cell cycle and apoptosis rate of A549 were determined by flow cytometry.Binary variables were statistically analyzed by Fisher's exact probability method and measurement data were assessed by t-test.Results High-expression level of UBE2T was correlated with the radiosensitivity of lung adenocarcinoma patients(P<0.05).UBE2Tsh improved the radiosensitivity of A549 lung adenocarcinoma cells,and the sensitizing enhancement ratio(SER)was 1.795.UBE2T overexpression decreased the radiosensitivity of SPC-A-1 lung adenocarcinoma cells with an SER of 0.293.γH2AX foci number per cell were significantly increased in UBE2Tsh A549 cells after irradiation(P<0.01).Compared with the control group,the expression level ofγH2AX protein was up-regulated(P<0.01)and that of Rad51 protein was down-regulated in UBE2Tsh A549 cells after radiation(P<0.001).Compared with the control group,the expression level ofγH2AX protein was down-regulated(P<0.05)and that of Rad51 protein was up-regulated in UBE2T overexpressed SPC-A-1 cells(P<0.001).The proportion of UBE2Tsh A549 cells in G2 phase was decreased(P<0.01)and cell apoptosis was increased(P<0.001).Conclusions UBE2T might promote the radioresistance of lung adenocarcinoma cells by enhancing DNA DSB repair induced by radiotherapy,inducing cell cycle G2 phase arrest,and reducing cell apoptosis.