USP47通过促进抗凋亡蛋白表达介导头颈鳞癌细胞顺铂耐药

doi:10.3969/j.issn.1674-8115.2021.04.004

上海交通大学学报(医学版) ›› 2021, Vol. 41 ›› Issue (4): 434-441.doi: 10.3969/j.issn.1674-8115.2021.04.004

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()

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

收稿日期:2020-05-12 出版日期:2021-04-28 发布日期:2021-05-14
通讯作者: 张建军 E-mail:1605049815@qq.com;zjjshuobo@163.com
作者简介:童桐（1996—），女，硕士生；电子信箱：1605049815@qq.com。
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)

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()

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:2020-05-12 Online:2021-04-28 Published:2021-05-14
Contact: Jian-jun ZHANG E-mail:1605049815@qq.com;zjjshuobo@163.com
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)

摘要/Abstract

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

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)

中图分类号:

R739.8

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.

Tong TONG, Xing QIN, Fei XIE, Ying-ying JIANG, Jian-bo SHI, Jian-jun ZHANG. USP47 induces cisplatin resistance in head and neck squamous cell carcinoma by up-regulation of anti-apoptotic proteins[J]. JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE), 2021, 41(4): 434-441.

图/表 9

表1 XIAP和Bcl-xL基因特异的siRNA序列

Tab 1 siRNA sequences specific for XIAP and Bcl-xL genes

Gene name

siRNA sequence (5′→3′)

XIAP

Forward：GGGCCGGAATCTTAATATTdTdT

Reverse：AATATTAAGATTCCGGCCCdTdT

Bcl-xL

Forward：AGUGCAGUUCAGUAAUAAACUdTdT

Reverse：AGTTTATTACTGAACTGCACTdTdT

Forward：UUCUCCGAACGUGUCACGUdTdT Reverse：ACGTGACACGTTCGGAGAAdTdT

表2 RT-PCR引物序列

Tab 2 RT-PCR primer sequences

Gene name

Primer sequence (5′→3′)

Human USP47

Forward：CTCGACGCTAATTTTGAGCCA

Reverse: CTCTTGGAAGCGGACCTATAAAC

Human β-actin

Forward：CACCCACACTGTGCCCATCTACGA

Reverse: CAGCGGAACCGCTCATTGCCAATGG

图1 DDP耐药HNSCC细胞株的构建Note:A.The IC50 of DDP on HN4 and HN30. B.The IC50 of HN4/DDP （left) and HN30/DDP (right) after removing DDP stimulation.

Fig 1 Construction of DDP-resistant strains of HNSCC

图2 DDP耐药细胞株HN4/DDP和HN30/DDP中USP47蛋白的表达Note:Western blotting analysis of USP47 protein levels in HNSCC cells with different DDP tolerance.

Fig 2 USP47 protein expressions in DDP-resistant HN4/DDP and HN30/DDP cell lines

图3 USP47基因过表达增强HNSCC细胞对DDP的抵抗能力Note:A.Western blotting analysis of USP47 protein levels in overexpressing stable cell lines. B.USP47 mRNA expression in overexpressing stable cell lines detected by RT-PCR. C.The IC50 of DDP on USP47 overexpression stable cell lines checked by MTT. ① P=0.000, compared with the Vector group.

Fig 3 Overexpression USP47 gene increases cell′s resistance to DDP

图4 敲除USP47基因逆转HNSCC细胞对DDP的耐药Note:A. Western blotting analysis of USP47 protein levels in USP47 knocking out cell lines. B.USP47 mRNA expression in USP47 knocking out cell lines detected by RT-PCR. C.IC50 of DDP on USP47 knocking out cell lines checked by MTT. ①P=0.000, compared with the KO NC group.

Fig 4 Knocking out USP47 gene reverses cell′s resistance to DDP

图5 过表达USP47对HNSCC细胞的增殖、克隆形成和凋亡的影响Note:A.Overexpression of USP47 in HN4 and HN30 cells inhibited the proliferation of tumor cells. B. Overexpression of USP47 significantly inhibited the colony formation of tumor cells. C.Flow cytometry analysis of the effect of USP47 overexpression on apoptosis of HN4 and HN30 cells. D.Flow cytometry analysis of the resistance of HN4 and HN30 to DDP-induced apoptosis after overexpression of USP47. ①P=0.000, ②P=0.005, ③P=0.006, ④P=0.003, ⑤P=0.015, compared with the Vector group.

Fig 5 Effect of USP47 gene overexpression on cell proliferation, colony formation and apoptosis

图6 免疫印迹实验检测USP47过表达对抗凋亡蛋白表达的影响Note:A. XIAP and Bcl-xL protein levels in USP47 overexpression stable cell lines. B. XIAP and Bcl-xL protein levels in knocking out USP47 cell lines. C.Ubiquitination level of XIAP and Bcl-xL protein levels in knocking out USP47 cell lines. D.XIAP and Bcl-xL protein levels in knocking out USP47 cell lines after inhibiting proteasome.

Fig 6 Effect of USP47 overexpression on expression of anti-apoptotic proteins by Western blotting

图7 抗凋亡蛋白表达下调阻断USP47基因过表达诱导的DDP耐药性

Fig 7 Down-regulation of anti-apoptotic protein blocks DDP resistance induced by overexpression of USP47 gene

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USP47通过促进抗凋亡蛋白表达介导头颈鳞癌细胞顺铂耐药

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

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