干扰HMGB1基因表达对食管鳞癌细胞侵袭迁移及放射敏感性的影响

doi:10.3969/j.issn.1004-616x.2021.05.002

癌变·畸变·突变 ›› 2021, Vol. 33 ›› Issue (5): 327-333.doi: 10.3969/j.issn.1004-616x.2021.05.002

干扰HMGB1基因表达对食管鳞癌细胞侵袭迁移及放射敏感性的影响

杨兴肖¹, 张雪原², 邹乃祎², 单保恩³, 马鸣⁴, 祝淑钗²

1. 河北医科大学第四医院感染科, 河北石家庄 050011;
2. 河北医科大学第四医院放疗科, 河北石家庄 050011;
3. 河北医科大学第四医院科研中心, 河北石家庄 050011;
4. 河北医科大学第四医院检验科, 河北石家庄 050011

收稿日期:2020-05-11 修回日期:2021-09-02 出版日期:2021-09-30 发布日期:2021-10-09
通讯作者: 祝淑钗,E-mail:sczhu1965@163.com E-mail:sczhu1965@163.com
作者简介:杨兴肖,E-mail:yangxingxiao2007@sina.com。
基金资助:
国家自然科学基金（81872456，81903118）；河北省自然科学基金（H2020206292）；河北省医学科学研究所资助项目（20180483）

Effects of HMGB1 silencing on migration, invasion and radiosensitivity of esophageal squamous cell carcinoma cells

YANG Xingxiao¹, ZHANG Xueyuan², ZOU Naiyi², SHAN Bao'en³, MA Ming⁴, ZHU Shuchai²

1. Department of Infection, the Fourth Affiliated Hospital of Hebei Medical University, Shijiazhuang 050011;
2. Department of Radiation Oncology, the Fourth Affiliated Hospital of Hebei Medical University, Shijiazhuang 050011;
3. Research Centre, the Fourth Affiliated Hospital of Hebei Medical University, Shijiazhuang 050011;
4. Department of Laboratory, the Fourth Affiliated Hospital of Hebei Medical University, Shijiazhuang 050011, Hebei, China

Received:2020-05-11 Revised:2021-09-02 Online:2021-09-30 Published:2021-10-09

摘要/Abstract

摘要： 目的：探讨干扰HMGB1基因对X线照射后食管鳞癌细胞增殖活性、存活能力及侵袭、迁移能力的影响及其机制。方法：构建含有HMGB1 shRNA的慢病毒载体并转染人食管鳞癌EC9706细胞（HMGB1 shRNA组），并以转染阴性序列的细胞为对照组，两组均设置X射线照射和未照射2个亚组。采用Western blot法评估HMGB1基因的干扰效果；MTS法和集落形成实验分别检测食管鳞癌EC9706细胞的增殖活性和存活能力；划痕实验和Transwell小室实验分别检测干扰HMGB1基因对EC9706细胞迁移和侵袭能力的影响；流式细胞术和Western blot分别检测照射后各组的细胞凋亡率及上皮间质转化（EMT）相关蛋白的表达。结果：与阴性对照组比较，Western blot结果显示干扰HMGB1基因明显降低了HMGB1蛋白的表达（P < 0.01），MTS法检测结果显示X射线照射后干扰HMGB1显著抑制了食管鳞癌细胞的增殖水平（P < 0.05），集落形成实验结果显示HMGB1 shRNA组细胞的放射敏感性明显增加（P < 0.01）。划痕实验结果显示，X射线照射后HMGB1 shRNA组细胞24 h的划痕愈合率为（20.78±4.38）%，低于阴性对照组的（39.02±3.25）%（P < 0.01）。Transwell实验结果显示X射线照射后HMGB1 shRNA组细胞在3.5 h的迁移细胞数为（67.00±16.56）个，低于阴性对照组的（194.00±19.74）个（P < 0.01）。流式细胞术结果显示，X射线照射后阴性对照组细胞凋亡率为（13.64±1.24）%，HMGB1shRNA组细胞凋亡率为（20.67±1.38）%；与阴性对照组比较，X射线照射后HMGB1 shRNA组细胞E-cadherin表达显著增加，而Ncadherin、Vimentin、MMP-2、MMP-9的表达明显降低（均为P < 0.01）。结论：干扰食管鳞癌细胞中HMGB1基因的表达后经X射线照射，降低了肿瘤细胞的增殖、侵袭、迁移水平和存活能力，诱导了细胞凋亡，增加了放射敏感性，并调控EMT相关蛋白的表达。

关键词: 食管鳞癌, HMGB1基因, 照射, 侵袭, 迁移, 放射敏感性

Abstract: OBJECTIVE: To examine the effect of HMGB1 silencing on proliferation, survival ability, migration and invasion of human esophageal squamous cell carcinoma after X-ray radiation. METHODS: Using siRNA technique, sequences of HMGB1 mRNA were synthesized and transfected into cultured EC9706 cells as the HMGB1 shRNA group. A negative sequence was synthesized and used as negative control (NC). Both groups were irradiated or not with Xrays. Protein levels of HMGB1 and metastasis-related molecules were determined using Western blot. MTS method, clone formation, and wound healing, Transwell assay were employed to examine the proliferation, survival ability, migration and invasion of the cells. Flow cytometry assay was used to analyze cell apoptosis rates. Western blot was used to examine the expression of cell. RESULTS: Western blot results demonstrated that silencing HMGB1 gene significantly reduced the HMGB1 protein expression compared with NC group (P < 0.01). MTS data demonstrated that,after irradiation, HMGB1 silencing significantly inhibited the proliferation of esophageal squamous cell carcinoma cells compared with NC group (P < 0.01). The data from the clone formation assay revealed that the radiosensitivity was increased after down-regulation of HMGB1 expression compared with NC group (P < 0.01). The apoptosis rate of tumor cells in HMGB1 shRNA group after irradiation was markedly increased (P < 0.01). Wound-healing assays show that Wound-healing rate of HMGB1 silencing cells was significantly lower than that of NC group[(20.78±4.38)% and (39.02 ±3.25)%, respectively, P < 0.01]. Transwell assays with matrigel show the invasion capability of HMGB1 silencing cells at 3.5 h was deeply suppressed compared with NC group (67.00±16.56 and 194.00±19.74, respectively, P < 0.01). Our data show that apoptosis rate of HMGB1 silencing cells was significantly higher than that of NC group[(20.67 ±1.38)% and (13.64 ±1.24)%, respectively, P < 0.01]. In addition, expressions of N-cadherin,Vimentin,MMP-2 and MMP-9 protein were downregulated,and expressions of Ecadherin were upregulated after HMGB1 silencing compared with NC group (P < 0.01). CONCLUSION: siRNA interference inhibited expression of the HMGB1 gene, reduced the proliferation, migration and invasion of cells,induced cell apoptosis,and increased radiosensitivity,followed by regulating the expression of metastasisrelated molecules in oesophageal squamous cell carcinoma cells.

Key words: esophageal squamous cell carcinoma, HMGB1, irradiation, migration, invasion, radiosensitivity

中图分类号:

R735.1

杨兴肖, 张雪原, 邹乃祎, 单保恩, 马鸣, 祝淑钗. 干扰HMGB1基因表达对食管鳞癌细胞侵袭迁移及放射敏感性的影响[J]. 癌变·畸变·突变, 2021, 33(5): 327-333.

YANG Xingxiao, ZHANG Xueyuan, ZOU Naiyi, SHAN Bao'en, MA Ming, ZHU Shuchai. Effects of HMGB1 silencing on migration, invasion and radiosensitivity of esophageal squamous cell carcinoma cells[J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2021, 33(5): 327-333.

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干扰HMGB1基因表达对食管鳞癌细胞侵袭迁移及放射敏感性的影响

Effects of HMGB1 silencing on migration, invasion and radiosensitivity of esophageal squamous cell carcinoma cells

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