Department of Obstetrics and Gynecology, Shengjing Hospital of China Medical University, Shenyang, China.
Technol Cancer Res Treat. 2020 Jan-Dec;19:1533033820983289. doi: 10.1177/1533033820983289.
We have previously reported that heterozygous variation presented in platinum-resistant patients. This study aimed to further investigate the mechanism of mutation in the development of platinum resistance in ovarian cancer.
The was synthesized and used to exchange 1 wildtype allele followed by sequencing to confirm the mutant allele sequence. Plasmids were constructed and transfected into the OVCAR-3 cells after lentiviral packaging. mRNA was detected by qPCR. BRCA2 protein was assessed by immunoblotting. Binding of the BRCA2 to Rad51 was detected by immunofluorescence staining. Sensitivity of the cells to cisplatin treatment was assessed with CCK-8 assay.
It was found that expression of BRCA2 protein in ovarian cancer cells transfected with (2.177 ± 0.003) was significantly increased compared to that of the cells transfected with lenti-EGFP only (1.227 ± 0.003, < 0.001). Binding of the BRCA2 and Rad51 proteins was significantly increased in the cells with mutation (3.542 ± 0.24) than that in the cells transfected with lenti-EGFP (1.29 ± 0.32) or empty cells (1.363 ± 0.32, < 0.001). Cell viability significantly increased in the cells transfected with mutant gene. The IC50 value was significantly higher in the cells transfected with mutant gene (1.963 ± 0.04) than that of the cells transfected with lenti-EGFP (0.955 ± 0.03, < 0.01) or empty cells (1.043 ± 0.007, < 0.01).
Over expression of mRNA and protein of BRCA2 was detected in the cells with mutation but not in the lentivirus negative control (lenti-EGFP) or the cells without transfection (empty cells), which may lead to resistance to platinum-based drugs in ovarian cancer cells through homologous recombination repair pathway.
我们之前曾报道过，铂耐药患者存在杂合性变异。本研究旨在进一步探讨 突变在卵巢癌铂耐药发展中的机制。
合成 并用于交换 1 个野生型等位基因，然后测序以确认突变等位基因序列。构建质粒，通过慢病毒包装转染至 OVCAR-3 细胞。通过 qPCR 检测 mRNA。通过免疫印迹法评估 BRCA2 蛋白。通过免疫荧光染色检测 BRCA2 与 Rad51 的结合。通过 CCK-8 assay 评估细胞对顺铂治疗的敏感性。
发现转染 （2.177 ± 0.003）的卵巢癌细胞中 BRCA2 蛋白表达明显高于仅转染 lenti-EGFP（1.227 ± 0.003， < 0.001）的细胞。突变细胞中 BRCA2 和 Rad51 蛋白的结合明显增加（3.542 ± 0.24），明显高于转染 lenti-EGFP（1.29 ± 0.32）或空载细胞（1.363 ± 0.32， < 0.001）的细胞。转染 突变基因的细胞活力明显增加。转染 突变基因的细胞 IC50 值明显高于转染 lenti-EGFP 的细胞（1.963 ± 0.04）和空载细胞（1.043 ± 0.007， < 0.01）。
在携带 突变的细胞中检测到 BRCA2 mRNA 和蛋白的过表达，但在阴性对照慢病毒（lenti-EGFP）或未转染的细胞（空载细胞）中未检测到，这可能通过同源重组修复途径导致卵巢癌细胞对铂类药物产生耐药性。
