肿瘤BRCA1亚细胞定位对细胞放射线及PARP抑制剂敏感性的影响

发布时间：2018-05-03 01:29

本文选题：BRCA亚细胞定位 + 同源重组修复　；参考：《临床与实验病理学杂志》2017年03期

【摘要】：目的探讨肿瘤BRCA1细胞内定位对放射线和PARP抑制剂敏感性的影响。方法采用siRNA干扰抑制乳腺癌细胞株MCF7内源性BRCA1表达,转染BRCA1细胞内定位不同的载体:p CMV-3x Flag-WT-BRCA1、p CMV-3x Flag-NES-BRCA1、p CMV-3x Flag-NLS-BRCA1。采用免疫荧光法检测BRCA1的细胞定位及细胞核γ-H2AX和Rad51核焦点形成,应用流式细胞技术检测细胞凋亡,克隆形成实验检测体外细胞存活。结果转染WT-BRCA1有47%细胞核表达,23%细胞质表达,30%细胞质和细胞核均表达,NES-BRCA1 mutant表达主要定位于细胞核(87%);NLS-BRCA1 mutant定位于细胞质(82%)。WTBRCA1、NES-BRCA1 mutant和NLS-BRCA1 mutant三种载体转染的细胞4 Gy放射处理后2 h,Rad51核焦点阳性细胞数分别为87%、84%及13%;放射后24 h,γ-H2AX核焦点阳性细胞分别为22%、25%及59%。NLS-BRCA1 mutant转染细胞较WTBRCA1和NES-BRCA1 mutant转染细胞ABT-888和放射处理后诱导的凋亡细胞增加,克隆存活减少。结论 BRCA1的细胞内定位影响DNA双链断裂同源重组修复,并可预测肿瘤对放射和PARP抑制剂的敏感性。
[Abstract]:Objective to investigate the effects of tumor BRCA1 cell localization on radiosensitivity and PARP inhibitor sensitivity. Methods siRNA interference was used to inhibit the expression of endogenous BRCA1 in breast cancer cell line MCF7, and transfected into BRCA1 cells with different vectors: P CMV-3x Flag-WT-BRCA1P CMV-3x Flag-NES-BRCA1P CMV-3x Flag-NLS-BRCA1. The cellular localization of BRCA1 and nuclear focus formation of 纬 -H2AX and Rad51 were detected by immunofluorescence assay, apoptosis was detected by flow cytometry, and cell survival in vitro was detected by clone formation assay. Results 47% of the cells transfected with WT-BRCA1 expressed 30% of the cytoplasm and 30% of the cytoplasm. The expression of NES-BRCA1 mutant was mainly located in the NLS-BRCA1 mutant of the cells transfected with NLS-BRCA1. WTBRCA1, NES-BRCA1, NES-BRCA1 mutant and NLS-BRCA1 mutant were transfected with 4 Gy radiation treatment. The number of focal point positive cells was 87% and 13% in the latter 2 h, 22% and 25% in 纬 -H2AX cells at 24 h after irradiation, respectively, and the number of 59%.NLS-BRCA1 mutant transfected cells was higher than that of ABT-888 cells transfected with WTBRCA1 and NES-BRCA1 mutant cells and apoptosis cells induced by radiation treatment. Clone survival reduced. Conclusion intracellular localization of BRCA1 affects the repair of DNA double strand break homologous recombination and can predict the sensitivity of tumor to radiation and PARP inhibitors.
【作者单位】：广州医科大学附属第一医院呼吸疾病国家重点实验室/广州呼吸疾病研究所;中山大学附属第一医院病理科;
【分类号】：R737.9

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