Department of Pathology, The First People's Hospital of Yunnan Province, Kunming, Yunnan, China.
Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.
Cancer Biother Radiopharm. 2024 Aug;39(6):406-424. doi: 10.1089/cbr.2021.0346. Epub 2022 Feb 18.
To investigate the relationship between breast cancer susceptibility gene-1 () gene methylation and the radiosensitivity of breast cancer. The authors studied three breast cancer cell lines: MDA-MB-435, MDA-MB-231, and MCF-7 cells. They constructed five short hairpin RNAs (shRNAs) and five small interfering RNAs to target selected promoter loci and initiate sequence-specific methylation in breast cancer cells. Pyrosequencing was used to analyze the state of DNA methylation. Quantitative real-time polymerase chain reaction was used to detect mRNA expression and RNA-directed DNA methylation (RdDM)-related gene expression. Western blotting was performed to analyze BRCA1 protein expression. Colony formation assays and γ-histone H2A foci formation assays were conducted to assess the surviving fraction (SF) and double-strand break (DSB) repair ability of cells after irradiation. The authors constructed five strains of lentivirus vectors and five plasmid vectors targeting promoter region. In MDA-MB-435 cells, lentivirus-mediated RNA interference targeting Site 1 of BRCA1 increased the methylation levels of and reduced mRNA and protein expression. The SF and the ability to repair DNA DSBs were reduced in the combined LV-BRCA1RNAi-Site 1 infection and irradiation group. The authors' findings suggest that the shRNA suppressed the expression levels of the gene and reduced the SF and DNA repair ability of cells after irradiation through RdDM. In summary, the radiosensitivity of breast cancer cells may correlate with methylation. The authors first utilized a lentivirus-based shRNA-mediated specific-sequence DNA methylation of the gene mediated by RdDM.
为了研究乳腺癌易感基因 1（）基因甲基化与乳腺癌放射敏感性之间的关系。作者研究了三种乳腺癌细胞系：MDA-MB-435、MDA-MB-231 和 MCF-7 细胞。他们构建了五个短发夹 RNA（shRNA）和五个小干扰 RNA，以靶向选定的启动子位点，并在乳腺癌细胞中引发序列特异性甲基化。焦磷酸测序用于分析 DNA 甲基化状态。定量实时聚合酶链反应用于检测 BRCA1 mRNA 表达和 RNA 指导的 DNA 甲基化（RdDM）相关基因表达。Western blot 用于分析 BRCA1 蛋白表达。集落形成实验和γ-组蛋白 H2A 焦点形成实验用于评估细胞照射后的存活分数（SF）和双链断裂（DSB）修复能力。作者构建了五个靶向 BRCA1 启动子区域的慢病毒载体和质粒载体。在 MDA-MB-435 细胞中，靶向 BRCA1 第 1 位的慢病毒介导的 RNA 干扰增加了和的甲基化水平，降低了 BRCA1 mRNA 和蛋白表达。在 LV-BRCA1RNAi-Site1 感染和照射联合组中，SF 和 DNA DSB 修复能力降低。作者的研究结果表明，shRNA 通过 RdDM 抑制基因表达水平，降低照射后细胞的 SF 和 DNA 修复能力。综上所述，乳腺癌细胞的放射敏感性可能与甲基化有关。作者首次利用基于慢病毒的 shRNA 介导 RdDM 特异性序列 DNA 甲基化。
