Radiosensitization of esophageal carcinoma cells by knockdown of HMGB1 expression
Radiosensitization of esophageal carcinoma cells by knockdown of HMGB1 expression
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敲低 HMGB1 表达对食管癌细胞的放射增敏作用
DOI：
10.3892/or.2018.6923
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发表时间：
2019-03-01
期刊：
影响因子：
4.2
通讯作者：
Zou, Naiyi
中科院分区：
文献类型：
--
作者：
Zhang, Xueyuan;Yang, Xingxiao;Zou, Naiyi
放射治疗（RT）是食管癌的传统治疗方法，与手术、化疗并列沿着。高迁移率族蛋白1（HMGB 1）在抑制放射治疗后的癌细胞凋亡中起着重要作用。本研究旨在分析HMGB 1在食管癌发生发展中的作用，并探讨HMGB 1对食管癌细胞放射敏感性的影响。在本研究中，HMGB 1的免疫组化评价77活检，结果显示，HMGB 1的过度表达与肿瘤的总体积（GTV），肿瘤淋巴结转移（TNM）阶段，T分类，远处转移，复发呈正相关，与患者的生存率呈负相关，提示HMGB 1作为一个关键因素在食管癌的发展。设计HMGB 1靶向shRNA，在ECA 109和TE 13细胞中敲低HMGB 1，并采用实时定量逆转录聚合酶链反应和western blot分析评估shRNA的转染效率。采用CCK-8法和克隆形成法分别检测HMGB 1对食管癌细胞增殖和放射敏感性的影响。分别采用创伤愈合实验、Transwell实验和流式细胞术检测HMGB 1对辐射诱导的肿瘤细胞迁移、侵袭、细胞周期和凋亡的影响。此外，还构建了异种移植瘤模型，观察HMGB 1对体内肿瘤生长的影响。体外实验结果显示，照射后HMGB 1-shRNA组的增殖能力下降，放射治疗使异种移植瘤模型的肿瘤体积缩小，HMGB 1-shRNA组更为明显。此外，HMGB 1参与了照射后H2 AX的磷酸化，HMGB 1敲低可使细胞周期阻滞于G（0）/G（1）期，并增加细胞凋亡。HMGB 1缺陷还与p16、Bax和caspase-9的上调以及MMP-2、MMP-9、cyclin D1、CDK 4、H2 AX和Bcl-2的下调相关。这些数据表明，治疗前HMGB 1的过表达与食管癌的不良临床结局相关，并且在人食管癌细胞系中敲低HMGB 1表达通过允许诱导细胞凋亡和暴露于辐射后的G（0）/G（1）停滞而增加其辐射敏感性。
Radiotherapy (RT) is a traditional and important treatment for carcinoma of the esophagus along with surgery and chemotherapy. High mobility group box 1 (HMGB1) plays a crucial part in inhibiting the apoptosis of cancer cells after irradiation treatment. The present study, was designed to analyze the function of HMGB1 in esophageal cancer progression and elucidate the effects of HMGB1 on the radiosensitivity of human esophageal cancer cell lines. In the present study, an immunohistochemical evaluation of HMGB1 was performed on 77 biopsies, and the results revealed that HMGB1 overexpression was positively correlated with gross tumor volume (GTV), tumor-node-metastasis (TNM) stage, T classification, distant metastasis, and relapse and negatively correlated with patient survival rates, suggesting that HMGB1 acts as a key factor in the development of esophageal cancer. An shRNA targeting HMGB1 was designed for the knockdown of HMGB1 in ECA109 and TE13 cells, and the transfection efficiency of the shRNA was assessed using quantitative real-time reverse transcription polymerase chain reaction and western blot analysis. CCK-8 and clonogenic assays were used to analyze the effect of HMGB1 on the proliferation and radiosensitivity, respectively, of esophageal cancer cells in vitro. The influence of HMGB1 on radiation-induced changes in the migration, invasion, and cell cycle as well as apoptosis of tumor cells was examined by wound-healing and Transwell assays and flow cytometry, respectively. In addition, xenograft tumor models were constructed to observe the effect of HMGB1 on tumor growth in vivo. The results of the study in vitro revealed that the proliferation of the HMGB1-shRNA group decreased after irradiation, and the radiation treatment reduced the tumor volume of the xenograft model which was more marked in HMGB1-shRNA group. Moreover, HMGB1 was involved in the phosphorylation of H2AX after irradiation, and HMGB1 knockdown blocked the cell cycle in the G(0)/G(1) phase and increased apoptosis. HMGB1 deficiency was also correlated with the upregulation of p16, Bax and caspase-9 and the downregulation of MMP-2, MMP-9, cyclin D1, CDK4, H2AX and Bcl-2. These data indicated that the overexpression of HMGB1 prior to treatment was correlated with poor clinical outcome in esophageal carcinoma and that knockdown HMGB1 expression in human esophageal cancer cell lines increased their radiosensitivity by allowing the induction of apoptosis and G(0)/G(1) arrest after exposure to radiation.