上海交通大学学报（医学版）

上海交通大学学报（医学版） >

2021 , Vol. 41 >Issue 4: 434 - 441

DOI: https://doi.org/10.3969/j.issn.1674-8115.2021.04.004

论著·基础研究

USP47通过促进抗凋亡蛋白表达介导头颈鳞癌细胞顺铂耐药

  • 童桐 ,
  • 秦星 ,
  • 谢非 ,
  • 蒋英英 ,
  • 石剑波 ,
  • 张建军
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  • 上海交通大学医学院附属第九人民医院·口腔医学院口腔颌面头颈-肿瘤科，国家口腔疾病临床医学研究中心，上海市口腔医学重点实验室，上海市口腔医学研究所，上海 200011
童 桐（1996—），女，硕士生；电子信箱：1605049815@qq.com。

收稿日期: 2020-05-12

  网络出版日期: 2021-05-14

基金资助

国家自然科学基金(81972573);上海市科学技术委员会科研计划项目(18JC1413700);上海交通大学医学院高水平地方高校创新团队(SSMU-ZLCX20180502)

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USP47 induces cisplatin resistance in head and neck squamous cell carcinoma by up-regulation of anti-apoptotic proteins

  • Tong TONG ,
  • Xing QIN ,
  • Fei XIE ,
  • Ying-ying JIANG ,
  • Jian-bo SHI ,
  • Jian-jun ZHANG
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  • Department of Oral and Maxillofacial-Head & Neck Oncology, Shanghai Ninth People's Hospital, College of Stomatology, Shanghai Jiao Tong University School of Medicine; National Clinical Research Center for Oral Diseases; Shanghai Key Laboratory of Stomatology & Shanghai Research Institute of Stomatology, Shanghai 200011, China

Received date: 2020-05-12

  Online published: 2021-05-14

Supported by

National Natural Science Foundation of China(81972573);Project of Science and Technology Commission of Shanghai Municipality(18JC1413700);Innovative Research Team of High-Level Local Universities in Shanghai(SSMU-ZLCX20180502)

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摘要

目的·探讨去泛素化酶47（ubiquitin carboxyl-terminal hydrolase 47，USP47）对头颈鳞癌细胞顺铂耐药的影响及潜在机制。方法·通过梯度增加顺铂浓度，构建头颈鳞癌顺铂耐药细胞株；通过实时定量PCR和蛋白免疫印迹实验检测耐药株中USP47的表达情况；利用慢病毒载体构建USP47基因过表达或敲除的稳转细胞株，MTT法检测USP47表达升高或降低对头颈鳞癌细胞顺铂耐药性的影响；并检测USP47对癌细胞增殖、克隆形成和凋亡等生物学行为的影响；通过免疫印迹实验检测USP47对抗凋亡蛋白X-连锁凋亡抑制蛋白（X-linked inhibitor of apoptosis protein，XIAP）和重组人B细胞淋巴瘤因子2 xL（recombinant human B-cell leukemia/lymphoma 2 xL，Bcl-xL）的表达及泛素化水平的影响。结果·USP47在头颈鳞癌顺铂耐药细胞株中显著高表达；过表达USP47后，顺铂对头颈鳞癌细胞的半数抑制浓度（half maximal inhibitory concentration，IC50）明显升高，而敲除USP47可以降低头颈鳞癌顺铂耐药细胞株的IC50；过表达USP47的头颈鳞癌细胞，其增殖和克隆形成能力均被抑制，而抗凋亡能力明显增强。USP47基因过表达显著提高了抗凋亡蛋白XIAP和Bcl-xL的表达量。泛素化测定证实了USP47通过降低XIAP和Bcl-xL蛋白的泛素化水平从而导致其表达量升高，而敲低XIAP和Bcl-xL基因表达水平可以阻断USP47基因过表达诱导的顺铂耐药性。结论·USP47通过降低XIAP和Bcl-xL蛋白的泛素化修饰水平，提高两者的蛋白表达水平，从而介导头颈鳞癌细胞顺铂耐药；抑制USP47的表达对降低头颈鳞癌细胞顺铂耐药具有潜在应用价值。

关键词： 去泛素化酶47; 顺铂耐药; 头颈鳞癌; X-连锁凋亡抑制蛋白; 重组人B细胞淋巴瘤因子2 xL

本文引用格式

童桐 , 秦星 , 谢非 , 蒋英英 , 石剑波 , 张建军 . USP47通过促进抗凋亡蛋白表达介导头颈鳞癌细胞顺铂耐药[J]. 上海交通大学学报（医学版）, 2021 , 41(4) : 434 -441 . DOI: 10.3969/j.issn.1674-8115.2021.04.004

Abstract

Objective

· To investigate the effects and underlying mechanism of ubiquitin carboxyl-terminal hydrolase 47 (USP47) on cisplatin resistance in head and neck squamous cells carcinoma (HNSCC).

Methods

· The cisplatin-resistant cell lines of HNSCC were constructed by gradient increase of cisplatin concentration. The expression of USP47 in cisplatin-resistant cell was analyzed by real-time PCR and Western blotting. Stable USP47 over-expressed and silenced USP47 HNSCCs cell lines were generated by lentivirus vector. The influence of the increased or silenced USP47 expression on cisplation resistance were tested by MTT. The influence of USP47 on cell proliferation, colony formation, and apoptosis were examined. Western blotting was performed to detect the expression and ubiquitination levels of X-linked inhibitor of apoptosis protein (XIAP) and recombinant human B-cell lymphoma factor 2 xL (Bcl-xL).

Results

· USP47 expression was significantly increased in cisplatin-resistant cancer cells. Over-expression of USP47 significantly increased the half maximal inhibitory concentration (IC50), while knockout of USP47 gene increased the sensitivity to cisplatin. In addition, the data showed that USP47 expression was negatively related to the proliferation and colony formation abilities of HNSCC cell lines, but it remarkably enhanced the anti-apoptotic effect of HNSCC cells. The protein levels of XIAP and Bcl-xL were significantly elevated in stable USP47 over-expressed cell lines and ubiquitination assay proved that USP47 reduced the ubiquitination levels of XIAP and Bcl-xL proteins. Knockdown of the XIAP and Bcl-xL gene expression blocked cisplatin resistance induced by USP47 gene overexpression.

Conclusion

· USP47 can increase the protein expressions of XIAP and Bcl-xL by inhibiting the ubiquitination modification of them, thereby mediate the cisplatin resistance of HNSCC cells. Inhibition of USP47 expression provides potential application value to reduce cisplatin-resistance during the treatment on HNSCC.

Key words： ubiquitin carboxyl-terminal hydrolase 47 (USP47); cisplatin resistance; head and neck squamous cells carcinoma (HNSCC); X-linked inhibitor of apoptosis protein (XIAP); recombinant human B-cell lymphoma factor 2 xL (Bcl-xL)

参考文献

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