长链非编码RNA调控DNA损伤修复参与乳腺癌化疗耐药的机制研究

Anthracyclines are some of the most widely used chemotherapeutic drugs for breast cancer treatment, functioning by promoting DNA double strand breaks and ultimately cell death. Enhanced DNA damage repair may render tumor cells resistant to anthracyclines and reduce the therapeutic effect. Long noncoding RNAs (lncRNAs) play crucial roles in diverse cell biological processes and are associated with cancer progression. However, few studies have looked into the roles of lncRNAs in regulating tumor resistance to anthracyclines by interfering DNA damage repair pathways. In our previous studies, we found that the novel long noncoding RNA lncER1 overexpressed in anthracycline-resistant breast cancers. Moreover, we also demonstrated that lncER1 might involve in regulating sensitivity to anthracyclines and homologous recombination DNA repair pathway. In this proposed study, we will further determine the association between lncER1 expression and resistance of breast cancer to anthracyclines，focus on the effect of dysregulation of lncER1 on DNA damage repair and sensitivity of breast cancer to anthracyclines, identify lncER1-binding protein, explore the role of lncER1-downstream binding protein regulating axis in DNA damage repair and elaborate the molecular mechanism of lncER1 mediating resistance to anthracyclines by regulating DNA damage repair. This study will complement the mechanism of the anthracycline resistance of breast cancer and provide a new point of exploring targets of reversing drug resistance and improving clinical treatment.

乳腺癌一线化疗药蒽环类药物能够诱导DNA双链断裂，促进肿瘤细胞凋亡。增强的DNA损伤修复能力能使肿瘤细胞抵抗化疗药物的杀伤。长链非编码RNA参与调控细胞多种生物学过程，与肿瘤发生发展密切相关。然而，目前长链非编码RNA调控DNA损伤修复介导乳腺癌蒽环类耐药还鲜有研究。前期我们发现长链非编码RNA-lncER1在乳腺癌蒽环类药物表阿霉素耐药肿瘤中高表达，并且能调控乳腺癌细胞对表阿霉素的敏感性和同源重组修复效率。本项目将进一步明确lncER1的表达与乳腺癌蒽环类耐药的相关性，重点研究lncER1对DNA损伤修复和乳腺癌蒽环类药物敏感性的调控作用，鉴定lncER1结合蛋白，探讨lncER1-下游结合蛋白的调节通路在DNA损伤修复中的功能，进而解析lncER1调控DNA损伤修复介导乳腺癌化疗耐药的分子机制，为深入阐明乳腺癌蒽环类药物耐药机理，发现逆转耐药新靶点、改善临床治疗效果提供新思路。

多数化疗药物和放射治疗方案能够通过造成肿瘤细胞DNA断裂损伤，诱发细胞凋亡，进而杀伤肿瘤。然而，原发性和获得性耐药的产生常常限制了化疗效果，降低患者临床获益。因此发现逆转化疗耐药新靶标，提高肿瘤化疗疗效是临床研究亟待解决的问题。前期工作中我们对乳腺癌表阿霉素敏感和耐药的肿瘤组织进行转录组测序，鉴定了一条在耐药组织中高表达的长链非编码RNA-lncER1。本项目对lncER1的乳腺癌化疗敏感性和DNA损伤修复通路活性的调控作用和具体作用机制进行了深入探索。我们研究发现，lncER1在乳腺癌表阿霉素耐药细胞株和耐药肿瘤组织中高表达，其表达水平与乳腺癌患者的无疾病进展和总体生存期负相关。细胞水平的毒性检测和克隆形成实验以及动物水平的移植瘤模型结果表明敲低lncER1能显著增加乳腺癌细胞对化疗药物表阿霉素的敏感性。利用同源重组DNA损伤修复报告系统，以及通过检测表阿霉素诱导的γH2AX水平、含有微核的细胞比例、同源重组修复关键因子RPA2和RAD51招募比例，我们明确了lncER1能够调控肿瘤细胞DNA损伤同源重组修复通路活性。研究发现，lncER1能够通过调控E3泛素连接酶接头蛋白KLHL15介导的CtIP泛素化降解过程来维持DNA末端剪切关键因子CtIP的蛋白水平，进而调控DNA同源重组修复过程。另外，我们发现敲低lncER1后DNA损伤诱导由PARP1催化生成的PAR链水平显著降低，进而影响DNA同源重组修复通路。此外，研究结果显示敲低lncER1显著抑制肿瘤细胞生长、增加细胞对放疗、PARP靶向抑制剂的敏感性，表明了靶向lncER1的临床应用潜能。本项目的研究工作为阐明乳腺癌化疗耐药机理、开发临床辅助治疗新靶标、改善临床治疗效果奠定了坚实的科学基础。