非编码RNA及其互作因子表观调控DNA损伤修复研究

Interstrand crosslinks (ICLs), induced by either endogenous metabolites or exogenous therapeutic drugs, represent a major challenge for DNA replication and transcription by preventing DNA strand separation. Cells deficient in ICL repair exhibit excessive genomic instability. Patients with deficient ICL repair are predisposed to a broad spectrum of cancers. It is known that the complicated ICL repair process involves highly regulated orchestration of multiple DNA repair pathways. However, it remains largely unknown how non-coding RNAs (ncRNAs) and their interacting factors regulate the ICL repair process. In our new preliminary studies, we found that many RNA-interacting proteins exhibit a mitomycin C (MMC)-induced enhanced chromatin association through an isobaric tag for relative and absolute quantitation (iTRAQ)-based quantitative proteomic approach. Among them, one protein, which involves in DNA damage response, is found to be recruited to laser-induced damage sites via a RNA-dependent fashion. These results suggest that some RNAs might be used as scaffolds for mediating DNA repair factors recruitment at DNA damage sites. By integrating multi-cross-disciplinary omics technologies including ncRNAs, proteomics, etc., this project aims at identifying the ICL-induced differentially enriched ncRNAs as well as the RNA-interacting factors recruited to damage sites via a RNA-dependent fashion, determining their functions in DNA damage repair and underlying mechanisms, unveiling the potential associations between these factors with breast cancer development and treatment. We believe these results would be beneficial to developing new breast cancer epigenetic biomarkers and significantly improving ICL-based chemotherapeutic drugs’ efficacy.

DNA链间交联（ICL）损伤可经内源代谢物或外源化疗药物诱导产生，严重影响DNA复制和转录，ICL修复缺陷将导致基因组不稳定性和肿瘤发生。ICL修复过程相当复杂，涉及多种修复途径的相互协调，目前关于非编码RNA（ncRNA）及其互作因子调控ICL修复的研究几乎空白。在前期研究中，申请人通过定量蛋白质组发现许多RNA互作蛋白在ICL损伤后与染色质结合发生显著改变，其中一个RNA互作蛋白能通过RNA依赖的方式被招募到损伤位点，调控DNA损伤应答，提示一些RNA分子可能作为支架介导DNA损伤修复蛋白的招募和功能发挥。本项目整合多组学(ncRNA和蛋白组)技术，鉴定ICL诱导的差异表达ncRNA以及通过RNA介导招募到损伤位点的RNA互作蛋白，解析ncRNA及其互作因子调控DNA损伤修复机理，阐明其与乳腺癌发生、发展和化疗耐药的关联，为开发早期诊疗表观标记和降低化疗耐药性提供重要理论基础。

细胞的基因组时刻受到内、外源损伤因素的威胁，诱导产生的DNA损伤如果不能及时修复将导致基因组不稳定性和肿瘤发生。另外，DNA损伤修复异常也与肿瘤细胞的治疗耐药密切相关。然而前期关于长链非编码RNA（lncRNA）和RNA结合蛋白（RBP）调控DNA损伤应答（DDR）的研究相对较少。我们通过定量蛋白质组筛选到多个参与DDR的RBP，深入探讨了其中RBM45（与肌萎缩侧索硬化症相关）和SART3（一种RNA剪切因子）等调控DDR的机理，相关成果发表在Nucleic Acids Res和Nat Commun等期刊；我们通过转录组测序筛选到一个能调控肿瘤化疗耐药的lncRNA，首次揭示该lncRNA通过编码的小肽PACMP抑制KLHL15与CtIP结合、抑制CtIP泛素化和蛋白降解。此外我们发现PACMP能以PAR依赖的方式招募到DNA损伤位点、促进PARP1催化的PAR链合成。小鼠原位成瘤实验表明敲低PACMP后肿瘤生长显著变慢，对EPI更敏感。敲低PACMP还能改善肿瘤细胞对多种肿瘤治疗方案的疗效，其中包括化疗试剂喜树碱、PARP抑制剂、ATR抑制剂、CDK4/6抑制剂、放疗等，显示了广泛的应用前景。相关成果目前被Molecular Cell接收，我们同时也已经递交了专利申请。