LncRNA RRL1在黑色素瘤中的耐药机制研究

Melanoma is an extraordinarily malignant primary tumor, which is characterized with highly metastatic potential and resistance to traditional chemotherapy and radiotherapy. Although RAS/BRAF pathway plays a key role in the malignant progression of melanoma, selection of drug resistant forms can occur during RAS/BRAF targeted drug treatment, while the underlying mechanism is still unclear. Long non-coding RNA (lncRNA) is extensively active in gene transcription regulation, and involving in drug resistance development, here, by combining our-designed microarray and Chip-Seq technologies, we found lncRNA-RRL1 was specifically highly expressing in drug resistant malignant melanoma cell lines and clinical patients. Further, extensive pathway inhibitors screening results show that RRL1 is at the downstream of RAS/BRAF, suggesting a pivotal role of RRL1 in the development of drug resistance of melanoma to RAS/BRAF targeted drugs. In this project, we will combine RNA-Seq、Pulldown、RNAIP technologies to comprehensively analyze the function of RRL1 in melanoma drug-resistance development and the underlying mechanism, providing a theoretical basis to overcome the drug resistance problem in clinical treating of malignant melanoma.

恶性黑色素瘤恶性程度高，转移时机早，且对常规放化疗不敏感。RAS/BRAF信号通路对该肿瘤的恶性进展起关键驱动作用，黑色素瘤细胞却易在短时间内对该通路的靶向治疗产生耐药，但其耐药机制尚不清楚。研究表明，长链非编码RNA(lncRNA)广泛参与肿瘤的耐药过程。前期工作中，我们利用基因芯片及RNA-Seq数据，发现lncRNA-RRL1特异高表达于恶性黑色素瘤中，且敲除RRL1后黑色素瘤肿瘤细胞药敏性增加。进一步实验表明RRL1位于RAS/BRAF信号通路下游，在长时间(3个月)使用Vemurafenib的患者体内其表达会急剧升高，提示RRL1在恶性黑色素瘤对RAS/BRAF通路抑制剂耐药过程中起重要作用。本项目中，我们将在此基础上综合运用Pulldown、RNAIP等技术，在基因调控层面上阐明RRL1的相互作用蛋白及分子网络，为设计针对RRL1的抗黑色素瘤联合治疗方案奠定理论基础。

RAS及其下游级联传递细胞信号通路的激活会导致参与细胞生长和分裂的基因转录增加。目前，受RAS信号通路调控的蛋白质编码基因相关靶点被广泛研究，但受这些过程调控的长链非编码RNA（lncRNA）研究甚少。我们以RAS信号通路为切入点，通过基因芯片技术，筛选出对RAS信号通路的激活至关重要的lncRNA RRL1。通过实验发现RAS–RAF–MEK–ERK信号途径中的调节转录因子AP1调控RRL1的表达，当RRL1的表达下调时，导致细胞周期蛋白表达增加，诱导肿瘤细胞G1–S细胞周期阻滞。通过研究发现RRL1在BRAF突变的肿瘤中高表达，如黑色素瘤。RRL1的沉默在体内外阻断了肿瘤细胞的增殖和生长，凋亡增加。综上所述，我们的结果表明RRL1是一种新的非蛋白介导RAS/RAF激活的介质，可能作为RAS/RAF驱动的癌症的治疗靶点。