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) PDF全文下载地址:
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