Institute for Transplantation Diagnostics and Cell Therapeutics, University Hospital Düsseldorf, Heinrich Heine University, D-40225 Düsseldorf, Germany.
Department of Otorhinolaryngology & Head/Neck Surgery, University Hospital Düsseldorf, Heinrich Heine University, D-40225 Düsseldorf, Germany.
Hum Mol Genet. 2022 Dec 16;31(24):4241-4254. doi: 10.1093/hmg/ddac177.
The Fanconi anemia (FA) and homologous recombination (HR) pathways, which partially overlap and include RAD51 and its paralogs, are key for the repair of different types of DNA damage, such as DNA interstrand crosslinks. First, to broadly assess the impact of microRNA-mediated regulation, we examined microRNA expression profiles in five isogenic fibroblast cell pairs, either deficient in DNA repair due to germline mutations in FANCA, FANCB, FANCC, FANCI or BRIP1/FANCJ or proficient due to correction with retroviral vectors. In each pair, we observed lower abundance of specific microRNAs in the FA-deficient cells. From the list of microRNAs, we experimentally confirmed the effects of miR-141-3p and miR-369-3p targeting RAD51B and miR-15a-5p, miR-494-3p as well as miR-544a targeting RAD51D. However, by western blotting, only RAD51D protein was reduced by a mixture of its regulating microRNAs. Gene ontology analyses and identification of additional FA/HR factors as targets of miR-15a-5p, miR-494-3p and miR-544a strongly suggested the widespread influence of these microRNAs on HR. Interestingly, only miR-494-3p directly reduced RAD51 foci formation, while a mixture of miR-15a-5p, miR-494-3p and miR-544a strongly reduced HR activity in green fluorescent protein (GFP) repair assays. In summary, by successfully employing this novel loss- and gain-of-function strategy, we have identified new microRNAs strongly inhibiting HR in mammalian cells. Understanding and modulating such miRNA regulation of DNA repair genes/pathways might help to overcome the reduced repair capacity of FA patients with biallelic hypomorphic mutations or help to engineer synthetic lethality strategies for patients with mutations in cancer-associated FA/HR genes.
范可尼贫血 (FA) 和同源重组 (HR) 途径部分重叠，包括 RAD51 及其同源物，是修复不同类型 DNA 损伤（如 DNA 链间交联）的关键。首先，为了广泛评估 microRNA 介导的调控的影响，我们检查了五个同源成纤维细胞对的 microRNA 表达谱，这些细胞要么由于 FANCA、FANCB、FANCC、FANCI 或 BRIP1/FANCJ 种系突变导致 DNA 修复缺陷，要么由于逆转录病毒载体校正而具有功能。在每一对中，我们观察到 FA 缺陷细胞中特定 microRNA 的丰度较低。在 microRNA 列表中，我们通过实验证实了 miR-141-3p 和 miR-369-3p 靶向 RAD51B 以及 miR-15a-5p、miR-494-3p 和 miR-544a 靶向 RAD51D 的作用。然而，通过 Western blot，只有 RAD51D 蛋白被其调节 microRNA 的混合物减少。基因本体分析和鉴定更多的 FA/HR 因子作为 miR-15a-5p、miR-494-3p 和 miR-544a 的靶标强烈表明这些 microRNA 对 HR 有广泛的影响。有趣的是，只有 miR-494-3p 直接减少 RAD51 焦点形成，而 miR-15a-5p、miR-494-3p 和 miR-544a 的混合物强烈降低 GFP 修复测定中的 HR 活性。总之，通过成功采用这种新的缺失和获得功能策略，我们鉴定了新的 microRNA 强烈抑制哺乳动物细胞中的 HR。了解和调节这种 miRNA 对 DNA 修复基因/途径的调控可能有助于克服具有双等位基因低功能突变的 FA 患者的修复能力降低，或有助于为具有癌症相关 FA/HR 基因突变的患者设计合成致死性策略。
