抑制USP9x表达增强食管癌Ec9706-R细胞的放射敏感性

doi:10.19401/j.cnki.1007-3639.2019.07.002

中国癌症杂志 ›› 2019, Vol. 29 ›› Issue (7): 486-493.doi: 10.19401/j.cnki.1007-3639.2019.07.002

抑制USP9x表达增强食管癌Ec9706-R细胞的放射敏感性

晋瑞¹ ，金迎迎² ，王敏聪² ，惠文涛³ ，李毅² ，李芳² ，贾辉² ，潘继元² ，马红兵²

1. 西安交通大学第二附属医院医学影像科，陕西西安 710004 ；
2. 西安交通大学第二附属医院肿瘤放疗科，陕西西安 710004 ；
3. 西安交通大学第二附属医院肿瘤科，陕西西安 710004

出版日期:2019-07-30 发布日期:2019-07-12
通信作者: 金迎迎 E-mail: yingyingjin717@sina.com
基金资助:
陕西省重点研发计划（2017SF-063）。

Inhibition of USP9x expression enhances the radiosensitivity of esophageal cancer Ec9706-R cells51

JIN Rui ¹, JIN Yingying² , WANG Mincong ² , XI Wentao ³ , LI Yi ² , LI Fang ², JIA Hui ² , PAN Jiyuan ² , MA Hongbing ²

1. Department of Medical Imaging, the Second Affiliated Hospital of Xi’an Jiaotong University, Xi’an 710004, Shaanxi Province, China; 2. Department of Oncology Radiotherapy, the Second Affiliated Hospital of Xi’an Jiaotong University, Xi’an 710004, Shaanxi Province, China; 3. Department of Oncology, the Second Affiliated Hospital of Xi’an Jiaotong University, Xi’an 710004, Shaanxi Province, China

Published:2019-07-30 Online:2019-07-12
Contact: JIN Yingying E-mail: yingyingjin717@sina.com

摘要/Abstract

摘要： 背景与目的：泛素特异性蛋白酶9x（ubiquitin-specific protease 9x，USP9x）与多种肿瘤的发生、发展以及肿瘤细胞的放射抗拒相关。研究发现，USP9x的表达与食管鳞状细胞癌的浸润深度和淋巴结转移相关，但其食管癌细胞放射抗拒作用尚未见报道。探究USP9x对放射抗拒食管癌Ec9706-R细胞放射敏感性的作用及其机制。方法：首先通过实时荧光定量聚合酶链反应（real-time fluorescent quantitative polymerase chain reaction，RTFQ-PCR）和蛋白质印迹法（Western blot）检测放射线照射后Ec9706-R细胞及其亲本细胞Ec-9706中USP9x和抗髓样细胞白血病-1（myeloid cell leukemia-1，Mcl-1）mRNA表达和蛋白水平。然后将Ec9706-R细胞随机分成3组：放射（irradiation，IR）组、IR+对照siRNA组（IR+si-NC组，转染Control siRNA）和IR+USP9x siRNA组（IR+si-USP9x组，转染USP9x siRNA），各组细胞均使用一定量的6 MV-X射线照射。噻唑蓝（methyl thiazolyl tetrazolium，MTT）比色法检测不同剂量（0、2、4、6和8 Gy）6 MV-X射线照射下各组细胞的活力。Transwell、流式细胞术、RTFQ-PCR和Western blot分别检测3组细胞在6 Gy照射下的细胞迁移、凋亡、Mcl-1 mRNA表达和蛋白水平以及增殖细胞核抗原（proliferating cell nuclear antigen，PCNA）和DNA损伤修复相关基因核苷酸切除修复交叉互补基因1（excision repair cross-complementing gene 1，ERCC1）蛋白水平。结果：放射后Ec9706-R和Ec-9706细胞中USP9x和Mcl-1 mRNA表达和蛋白水平均增加，Ec9706-R细胞尤为显著（P<0.05）。与IR组相比，IR+si-USP9x组中细胞活力、迁移细胞数目、PCNA、ERCC1和Mcl-1的表达均降低，细胞凋亡增加（P<0.05）。但是与IR组相比，IR+si-NC组中上述指标均无显著变化（P>0.05）。结论：抑制USP9x表达能够增强放射抗拒食管癌Ec9706-R细胞的放射敏感性，这可能是通过下调Mcl-1的表达发挥作用的。

关键词: 泛素特异性蛋白酶9x, 放射抗拒食管癌细胞, 放射敏感性, 抗髓样细胞白血病-1

Abstract: Background and purpose: Ubiquitin-specific protease 9x (USP9x) is related to the development and radioresistance of multiple cancers. Currently, it is reported that the USP9x expression is associated with invasive depth and lymphatic metastasis of esophageal squamous cancer. However, the effect of U5P9x on the radioresistance of esophageal cancer remains unknown. This study aimed to investigate the role of USP9x in the radiosensitivity of radioresistant esophageal cancer Ec9706-R cells and its mechanism. Methods: The mRNA expression and protein level of USP9x and myeloid cell leukemia-1 (Mcl-1) were detected by real-time fluorescent quantitative polymerase chain reaction (RTFQ-PCR) and Western blot in Ec9706-R cells and the parental Ec9706 cells after irradiation. Then, Ec9706-R cells were randomly divided into 3 groups: irradiation (IR) group, IR+control siRNA group (IR+si-NC, transfected with control siRNA), and IR+USP9x siRNA group (IR+si-USP9x, transfected with USP9x siRNA). All cells were exposed to designated dose of 6 MV-X irradiation. MTT analysis was used to determine the viability of cells following 6 MV-X irradiation with different doses (0, 2, 4, 6 and 8 Gy). The migration, apoptosis and expression levels of Mcl-1, proliferating cell nuclear antigen (PCNA) and excision repair cross-complementing gene 1 (ERCC1) of Ec9706-R cells under 6 Gy irradiation were analyzed using Transwell, flow cytometry, real-time PCR and Western blot, respectively. Results: The mRNA expression and protein level of USP9x and Mcl-1 were elevated in both Ec9706-R and Ec9706 cells after irradiation, and their expression levels were more obvious in Ec9706-R cells (P<0.05). Compared with the IR group, cell viability, migrated cell numbers and the expression levels of PCNA, Mcl-1 and ERCC1 were decreased, whereas cell apoptosis was increased in the IR+si-USP9x group (P<0.05). However, compared with the IR group, these indexes were not changed in the IR+si-NC group (P>0.05). Conclusion: Inhibition of USP9x enhances the radiosensitivity of esophageal cancer Ec9706-R cells, which may be dependent on the downregulation of Mcl-1.

Key words: Ubiquitin-specific protease 9x, Radioresistant esophageal cancer cells, Radiosensitivity, Myeloid cell leukemia-1

晋瑞,金迎迎,王敏聪,惠文涛,李毅,李芳,贾辉,潘继元,马红兵 . 抑制USP9x表达增强食管癌Ec9706-R细胞的放射敏感性[J]. 中国癌症杂志, 2019, 29(7): 486-493.

JIN Rui,JIN Yingying,WANG Mincong,XI Wentao,LI Yi,LI Fang,JIA Hui,PAN Jiyuan,MA Hongbing . Inhibition of USP9x expression enhances the radiosensitivity of esophageal cancer Ec9706-R cells51[J]. China Oncology, 2019, 29(7): 486-493.

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抑制USP9x表达增强食管癌Ec9706-R细胞的放射敏感性

Inhibition of USP9x expression enhances the radiosensitivity of esophageal cancer Ec9706-R cells51

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