USP28在ECA109细胞中不同表达状态对照射后凋亡及相关蛋白表达影响

doi:10.3760/cma.j.issn.1004-4221.2019.10.011

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摘要目的采用基因转染技术观察USP28不同表达状态对ECA109细胞放射敏感性的影响,为食管癌的综合治疗提供理论依据。方法 qRT-PCR检测USP28和c-Myc在食管上皮细胞Het-1A、食管癌细胞ECA109和放射抗拒细胞ECA109R中的表达。针对USP28基因和c-Myc基因设计特异性siRNA序列,构建pcDNA-USP28和pcDNA-c-Myc质粒,转染食管癌细胞ECA109,观察转染效果及相关蛋白表达情况。6Gy X射线照射细胞,流式细胞仪检测各组的细胞凋亡情况。克隆形成实验评价放射敏感性。结果 USP28和c-Myc在ECA109中的表达高于正常的食管上皮细胞Het-1A (P<0.05),在ECA109R中的表达高于ECA109(P<0.05)。成功构建pcDNA-USP28和pcDNA-c-Myc重组质粒,转染结果显示与阴性对照组比较无论mRNA水平和蛋白水平,si-USP28组中USP28的表达明显下降,而在pcDNA-USP28组中USP28的表达明显上升。针对c-Myc的研究得到类似结果。与对照组比较,pcDNA-USP28组c-Myc在蛋白水平表达明显升高,si-USP28中c-Myc表达下降(P<0.05)。6Gy照射ECA109后pcDNA-USP28组细胞凋亡率下降,放射敏感性下降;ECA109R中si-USP28组是细胞凋亡率增加,放射敏感性增强。结论 USP28的蛋白表达与食管癌细胞的放射敏感性密切相关,其机制可能与USP28对c-Myc的表达调控有关。

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	张魏丽
	刘志坤
	张钧
	牛玉杰
	宋波

关键词 ： USP28基因, c-Myc基因, 食管癌细胞系, 放射敏感性

Abstract：Objective To observe the effect of different expression levels of USP28 on the radiosensitivity of ECA109 cells by gene transfection method,aiming to provide theoretical basis for comprehensive treatment of esophageal cancer. Methods The expression levels of USP28 and c-Myc in the esophageal epithelial cells Het-1A,ECA109 and ECA109R were quantitatively measured by qRT-PCR. The specific siRNA sequences were designed according to the USP28 and c-Myc genes. The pcDNA-USP28 and pcDNA-c-Myc plasmids were constructed. The esophageal cancer cell ECA109 was transfected with Lipofectamine 2000 to observe the transfection effect and related protein expression. ECA109 and ECA109R cells were exposed to 6Gy X-ray radiation. The cell apoptosis in each group was detected by flow cytometry. The radiosensitivity was evaluated by clone formation assay. Results The expression levels of USP28 and c-Myc in ECA109 were significantly higher than those in Het-1A (both P<0.05),and the expression levels of USP28 and c-Myc in ECA109R were remarkably higher than those in ECA109(both P<0.05). The pcDNA-USP28 and pcDNA-c-Myc recombinant plasmids were successfully constructed. Compared with the negative control group, the expression of USP28 at the protein and mRNA levels in the si-USP28 group was significantly down-regulated, whereas those in the pcDNA-USP28 group were remarkably up-regulated. Similar results were obtained in terms of c-Myc. Compared with the control group, the expression level of c-Myc protein was significantly up-regulated in the pcDNA-USP28 group, whereas considerably down-regulated in the si-USP28 group. After 6Gy irradiation, the apoptosis rate and radiosensitivity of ECA109 cells were significantly declined. The apoptosis rate and radiosensitivity of ECA109R cells were increased in the si-USP28 group. Conclusions The expression of USP28 protein is closely correlated with the radiosensitivity of esophageal cancer cells. The underlying mechanism may be related to the regulation of c-Myc expression by USP28.

Key words： USP28 gene c-Myc gene Esophageal carcinoma cell line Radiosensitivity

收稿日期: 2018-05-18

通讯作者: 刘志坤,Email:zhikunliu1978@163.com

引用本文:

张魏丽,刘志坤,张钧等. USP28在ECA109细胞中不同表达状态对照射后凋亡及相关蛋白表达影响[J]. 中华放射肿瘤学杂志, 2019, 28(10): 767-767.

Zhang Weili,Liu Zhikun,Zhang Jun et al. Effect of different expression levels of USP28 on apoptosis and related protein expression in esophageal cancer cell ECA109 after irradiation[J]. Chinese Journal of Radiation Oncology, 2019, 28(10): 767-767.

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http://journal12.magtechjournal.com/Jweb_fszlx/CN/10.3760/cma.j.issn.1004-4221.2019.10.011 或 http://journal12.magtechjournal.com/Jweb_fszlx/CN/Y2019/V28/I10/767

[1] Popovic D,Vucic D,Dikic I. Ubiquitination in disease pathogenesis and treatment. Nat Med,2014,20(11):1242-1253. DOI:10.1038/nm.3739.
[2] Buneeva OA,Medvedev AE. Ubiquitin-independent protein degradation in proteasomes[J]. Biomed Khim,2018,64(2):134-148. DOI:10.18097/PBMC 2018 6402134.
[3] 于娟, 陈玮琳. 泛素羧基末端水解酶及其在肿瘤发生发展中的作用研究进展[J]. 浙江大学学报(医学版), 2015,44(2):217-222. DOI:10.3785/j.issn.1008-9292.2015.03.016.
Yu J,Chen WL. Research advances on ubiquitin C-terminal hydrolase in oncogenesis and progression[J]. J Zhejiang Univ (Med Sci),2015,44(2):217-222. DOI:10.3785/j.issn.1008-9292.2015.03.016.
[4] Upadhyay A,Joshi V,Amanullah A,et al. E3 ubiquitin ligases neu-robiological mechanisms:development to degeneration[J]. Front Mol Neurosci,2017,10(2):151. DOI:10.3389/fnmol.2017.00151.
[5] Ristic G,Tsou WL,Todi SV. An optimal ubiquitin-proteasome pathway in the nervous system:the role of deubiquitinating enzymes[J]. Front Mol Neurosci,2014,7(1):72. DOI:10.3389/fnmol.2014.00072.
[6] Zhang L,Xu B,Qiang Y,et al. Overexpression of deubiquitinating enzyme USP28 promoted non-small cell lung cancer growth[J]. J Cell Mol Med,2015,19(4):799-805. DOI:10.1111/jcmm.12426.
[7] Diefenbacher ME,Chakraborty A,Blake SM,et al. USP28 counteracts Fbw7 in intestinal homeostasis and cancer[J]. Cancer Res,2015,75(7):1181-1186. DOI:10.1158/0008-5472. CAN-14-1726.
[8] Guo G,Xu Y,Gong M,et al. USP28 is a potential prognostic marker for bladder cancer[J]. Tumour Biol,2014,35(5):4017-4022. DOI:10.1007/s13277-013-1525-1.
[9] Chang L,Graham P,Hao J,et al. Proteomics discovery of radioresistant cancer biomarkers for radiotherapy[J]. Cancer Lett,2015,369(2):289-297. DOI:10.1016/j.canlet.2015.09.013.
[10] Murtaza M,Jolly LA,Gecz J,et al. La FAM fatale:USP9X in development and disease[J]. Cell Mol Life Sci,2015,72(11):2075-2089. DOI:10.1007/s00018-015-1851-0.
[11] Lee JK,Chang N,Yoon Y,et al. USP1 targeting impedes GBM growth by inhibiting stem cell maintenance and radioresistance[J]. Neuro Oncol,2016,18(1):37-47. DOI:10.1093/neuonc/nov091.
[12] Li J,Liang Y,Lyu H,et al.miR-26a and miR-26b inhibit esophageal squamous cancer cell proliferation through suppression of c-Myc pathway[J]. Gene,2017,625(1):1-9. DOI:10.1016/j.gene.2017.05.001.
[13] Xie Y,Tu W,Zhang J,et al. SirT1 knockdown potentiates radiation-induced bystander effect through promoting c-Myc activity and thus facilitating ROS accumulation[J]. Mutat Res,2015,772(1):23-29. DOI:10.1016/j.mrfmmm.2014.12.010.
[14] Gravina GL,Festuccia C,Popov VM,et al.c-Myc sustains transformed phenotype and promotes radioresistance of embryonal rhabdomyo-sarcoma cell lines[J]. Radiat Res,2016,185(4):411-422. DOI:10.1667/RR14237.1.
[15] Wang WJ,Wu SP,Liu JB,et al. MYC regulation of CHK1 and CHK2 promotes radioresistance in a stem cell-like population of nasopharyngeal carcinoma cells[J]. Cancer Res,2013,73(3):1219-1231. DOI:10.1158/0008-5472. CAN-12-1408.
[16] Cui F,Fan R,Chen Q,et al. The involvement of c-Myc in the DNA double-strand break repair via regulating radiation-induced phosphorylation of ATM and DNA-PKcs activity[J]. Mol Cell Biochem,2015,406(1-2):43-51. DOI:10.1007/s11010-015-2422-2.
[17] Schülein-Vlk C,Wolf E,Zhu J,et al. Dual regulation of Fbw7 function and oncogenic transformation by Usp28[J]. Cell Rep,2014,9(3):1099-1109. DOI:10.1016/j.celrep.2014.09.057.
[18] Wang Z,Song Q,Xue J,et al. Ubiquitin-specific protease 28 is overexpressed in human glioblastomas and contributes to glioma tumorigenicity by regulating MYC expression[J]. Exp Biol Med,2016,241(1):255-264. DOI:10.1177/1535370215595468.
[19] Diefenbacher ME,Popov N,Blake SM,et al. The deubiquitinase USP28 controls intestinal homeostasis and promotes colorectal cancer[J]. J Clin Invest,2014,124(8)8:3407-3418. DOI:10.1172/JCI73733.