LncRNA CRNDE调控SRSF6通过对自噬相关蛋白Beclin1选择性剪切介导 胃癌化疗增敏的机制研究

Chemotherapy is an important treatment for gastric cancer (GC), but primary drug resistance and secondary drug resistance of GC result in poor chemotherapy effect. At present, the mechanism underlying chemoresistance still remains unclear. In our study, we firstly established oxaliplatin-resistance GC cell line and 5-FU-resistance GC cell line, in which we found a relatively low expression of CRNDE. Subsequently, we confirmed that CRNDE interacted with splicing factor SPSF6 and alternatively spliced autophagy-related protein Beclin1, thus inhibiting autophagy and inducing chemosensitivity. Based on these findings, we put forward an assumption that transcription factor E2F6 is overexpressed in oxaliplatin-resistance GC cell line and 5-FU-resistance GC cell line, and transcriptionally regulates CRNDE expression. By directly combining with SPSF6, CRNDE inhibits the phosphorylation of SPSF6 and interferes with its function of alternative splicing of exon 5 and exon 10 in BECN1, thus inhibiting autophagy and inducing chemosensitivity. Our study aims to use molecular pathology and molecular biology methods, combined in-vivo and in-vitro experiments, to fully elucidate the function of CRNDE in inducing drug resistance and provide direct evidence for CRNDE-mediated splicing function of SRSF6. These findings support that the CRNDE/SRSF6 axis might be a promising therapeutic target for GC patients frustrated with drug resistance.

化疗是胃癌的重要治疗手段，但是肿瘤对化疗药物的原发性耐药和继发性耐药严重影响化疗效果，目前肿瘤的耐药机制尚未完全阐明。我们前期构建胃癌奥沙利铂和5-Fu耐药株发现CRNDE低表达，并能通过剪切蛋白SRSF6的相互作用对自噬相关蛋白Beclin1选择性剪切，从而抑制自噬，介导化疗增敏。据此我们提出假设：胃癌奥沙利铂和5-Fu耐药株通过高表达转录因子E2F6，转录水平抑制CRNDE的表达，后者通过直接与剪切因子SRSF6结合，降低SRSF6磷酸化水平，干预SRSF6剪切功能，抑制其对BECN1的5号、10号外显子选择性剪切，从而抑制自噬，介导化疗增敏。本项目拟通过分子病理学和分子生物学手段，结合体内外研究，全方位阐明CRNDE在介导化疗增敏中的作用，提供CRNDE调控SRSF6剪切发挥生物学功能的直接证据，为确立CRNDE/SRSF6轴作为胃癌患者化疗耐药的治疗靶点提供新的科学依据。

胃癌严重威胁全人类生存和健康，亚洲是胃癌的高发地域之一，而我国的发病人数居亚洲之首。化疗是胃癌的重要治疗手段，在减少复发、延长生存、改善患者生活质量方面均有着举足轻重的地位。但是，肿瘤对化疗药物的原发性耐药和继发性耐药严重影响化疗效果，目前肿瘤的耐药机制尚未完全阐明，如何有效逆转化疗耐药仍是世界性难题。该研究发现长链非编码RNA CRNDE的表达与临床样本中胃癌标本的化疗敏感性有关。敲低CRNDE的表达可以促进胃癌细胞的自噬流的发生。进一步探讨CRNDE调节自噬参与胃癌化疗耐药的机制，发现CRNDE可以通过与剪切蛋白SRSF6直接结合，降低其蛋白稳定性，从而调控选择性剪接(AS)事件。二代测序联合TCGA分析确定SRSF6调控PICALM 14号外显子跳跃，导致PICALMS亚型向PICALML亚型的转变增加。总的来说，该研究揭示了CRNDE在调节自噬从而影响胃癌化疗效果中的关键作用。