Yong Lei, Shi Yan, Wu Hai-Long, Dong Qi-Yuan, Guo Jing, Hu Li-Sheng, Wang Wen-Hao, Guan Zhi-Ping, Yu Bin-Sheng
Shenzhen Key Laboratory of Spine Surgery, Department of Spine Surgery, Peking University Shenzhen Hospital, Shenzhen, China.
Shenzhen Engineering Laboratory of Orthopaedic Regenerative Technologies, National & Local Joint Engineering Research Center of Orthopaedic Biomaterials, Peking University Shenzhen Hospital, Shenzhen, China.
Front Oncol. 2023 Jan 26;12:1047194. doi: 10.3389/fonc.2022.1047194. eCollection 2022.
Osteosarcoma (OS) is a malignant bone tumor mainly affecting children and young adolescents. Cisplatin is a first-line chemotherapy drug for OS, however, drug resistance severely limits the survival of OS. Nevertheless, cellular factors in cisplatin resistance for OS remain obscure. In this study, the function and potential mechanism of p53 in cisplatin absorption were explored in OS cells.
The CRISPR-Cas9 gene editing technology was performed to obtain p53 gene knock-out U2OS cells. The p53 over-expression 143B cell line was established by lentivirus-mediated virus infection. Moreover, the functions of p53 and CTR1 in cisplatin absorption were assessed by inductively coupled plasma mass spectrometry (ICP-MS) through CTR1 over-expression and knock-down. Further, the DNA binding activity of SP1 on CTR1 gene promoter was determined by dual-luciferase assay and chromatin immunoprecipitation (ChIP) assay. The functional regulation of p53 on SP1 was studied by nucleocytoplasmic separation assay and electrophoretic mobility shift assay (EMSA). The interaction between p53 and SP1 was verified by Co-Immunoprecipitation assay.
Under cisplatin treatment, p53 knock-out promoted CTR1 expression and cisplatin uptake, while p53 overexpression inhibited CTR1 expression and cisplatin uptake. Moreover, p53 regulated CTR1 level not by binding to CTR1 promoter directly but by suppressing the nuclear translocation of transcription factor specificity protein 1 (SP1). It was verified that SP1 is directly bound with CTR1 promoter. SP1 overexpression stimulated CTR1 expression, and SP1 knock-down attenuated CTR1 expression.
The p53 might function as a negative regulator in CTR1 mediated cisplatin absorption, and the p53-SP1-CTR1 axis is a target for cisplatin resistance.
骨肉瘤（OS）是一种主要影响儿童和青少年的恶性骨肿瘤。顺铂是骨肉瘤的一线化疗药物，然而，耐药性严重限制了骨肉瘤患者的生存期。尽管如此，骨肉瘤顺铂耐药中的细胞因子仍不清楚。在本研究中，我们探讨了p53在骨肉瘤细胞顺铂摄取中的作用及潜在机制。
采用CRISPR-Cas9基因编辑技术获得p53基因敲除的U2OS细胞。通过慢病毒介导的病毒感染建立p53过表达的143B细胞系。此外，通过过表达和敲低铜转运蛋白1（CTR1），利用电感耦合等离子体质谱（ICP-MS）评估p53和CTR1在顺铂摄取中的作用。通过双荧光素酶报告基因检测和染色质免疫沉淀（ChIP）检测确定特异性蛋白1（SP1）对CTR1基因启动子的DNA结合活性。通过核质分离实验和电泳迁移率变动分析（EMSA）研究p53对SP1的功能调控。通过免疫共沉淀实验验证p53与SP1之间的相互作用。
在顺铂处理下，p53基因敲除促进CTR1表达及顺铂摄取，而p53过表达则抑制CTR1表达及顺铂摄取。此外，p53并非直接结合CTR1启动子来调节CTR1水平，而是通过抑制转录因子SP1的核转位来实现。已证实SP1直接与CTR1启动子结合。SP1过表达刺激CTR1表达，而SP1敲低则减弱CTR1表达。
p53可能在CTR1介导的顺铂摄取中起负调节作用，p53-SP1-CTR1轴是顺铂耐药的一个靶点。
