UNASSIGNED:骨肉瘤(OS)是一种恶性骨肿瘤,主要影响儿童和青少年。顺铂是OS的一线化疗药物,然而,耐药性严重限制了OS的生存。然而,顺铂对OS耐药的细胞因子仍然不清楚。在这项研究中,探讨了p53在OS细胞顺铂吸收中的作用和潜在机制。
UNASSIGNED:进行CRISPR-Cas9基因编辑技术以获得p53基因敲除U2OS细胞。通过慢病毒介导的病毒感染建立了p53过表达的143B细胞系。此外,用电感耦合等离子体质谱法(ICP-MS)通过CTR1过表达和敲低评估p53和CTR1在顺铂吸收中的功能.Further,通过双荧光素酶测定法和染色质免疫沉淀(ChIP)测定法确定SP1对CTR1基因启动子的DNA结合活性。通过核质分离法和电泳迁移率变化法(EMSA)研究了p53对SP1的功能调控。通过免疫共沉淀试验验证了p53和SP1之间的相互作用。
未经批准:顺铂治疗,p53敲除促进CTR1表达和顺铂摄取,而p53过表达抑制CTR1表达和顺铂摄取。此外,p53不是通过直接与CTR1启动子结合,而是通过抑制转录因子特异性蛋白1(SP1)的核易位来调节CTR1水平。证实SP1与CTR1启动子直接结合。SP1过表达刺激CTR1表达,和SP1敲低减弱CTR1表达。
未经证实:p53可能在CTR1介导的顺铂吸收中起负调节因子的作用,p53-SP1-CTR1轴是顺铂耐药的靶点。
UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: 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.