HIF-1α介导的PUF60高表达通过pre-mRNA可变剪接调控卵巢癌发生发展的机制研究

Ovarian cancer is the most lethal in malignancies gynecology, and its pathogenesis is not very clear. Hypoxia microenvironment is one of the most common conditions for the initiation of tumor, which has a closely relationship with the occurrence of ovarian cancer. Abnormal alternative splicing involves almost all of the biological process of tumor cells, including proliferation, invasion, metabolism and apoptosis and so on. In our previous studies, we found for the first time that the expression of splicing factor PUF60 was significantly increased in ovarian cancer cells after being induced with hypoxia. Moreover, the expression level of PUF60 was significantly correlated with pathological type, clinical stage and patient survival rate. The growth of ovarian cancer cells was significantly inhibited when PUF60 interfered by ShRNA in vitro and in vivo. Moreover, by analyzing data of gene sequence and transcriptome we found that the change of alternative splicing in WNT pathway was most significant. In this study, we will go further to reveal how the PUF60 expression is modulated by HIF-1α in hypoxic microenvironment.The study is expected to clarify the molecular mechanisms of PUF60 which will promote the development of ovarian cancer through regulating the WNT pathway and indicate a potential therapeutic strategy for comprehensive treatment of ovarian cancer.

卵巢癌是死亡率最高的妇科恶性肿瘤，其发病机制尚不清楚。低氧微环境是驱动肿瘤形成的最常见条件之一，与卵巢癌发生关系密切。异常可变剪接几乎涉及肿瘤细胞所有的生物学行为，包括增殖、侵袭、代谢和凋亡等过程。我们在前期研究中首次发现，卵巢癌细胞经低氧诱导后剪接因子PUF60表达显著升高，且其表达水平与卵巢癌的病理类型、临床分期和患者总生存率显著相关。通过shRNA干扰PUF60的表达，能显著抑制卵巢癌细胞在体内外的生长，进一步通过转录组基因测序和数据分析，发现WNT通路的可变剪接变化最为显著。本课题将深入研究低氧微环境中HIF-1α介导PUF60高表达的调控关系，并进一步阐明PUF60通过调控WNT通路可变剪接促进卵巢癌发展的具体分子机制。为卵巢癌的治疗提供新的思路。

卵巢癌是全球妇科肿瘤相关死亡的第二大主要原因，病因及发病机制不清。本研究旨在通过分析剪接因子PUF60在卵巢癌中表达及功能进一步探究其在卵巢癌发生发展的可能分子机制，筛选出靶向PUF60的特异性小分子抑制剂，以期为卵巢癌的治疗提供新靶点和新思路。多个数据库及免疫组化的结果显示，高表达PUF60的卵巢癌患者预后差，与病理分型和临床分期密切相关，高表达PUF60具有促进卵巢癌细胞增殖和迁移能力。RIP-seq、RNA-pull own及Western blot实验显示PUF60优先结合发生m6A的通用序列“GGACU”序，发生m6A的RNA探针与PUF60的亲和力明显高于未发生者。综合分析RIP-seq、RNA-seq、m6A-seq实验发现氧化磷酸化途径为PUF60靶基因富集最明显的信号通路；Seahorse和mRNA稳定测定实验显示PUF60具有降低卵巢癌细胞氧化磷酸化、增加糖酵解和促进氧化磷酸化相关靶基因的mRNA降解的能力；CO-IP及IF实验显示PUF60与PABPC1有相互作用关系，两者明显共定位于P小体以促进卵巢癌细胞中P小体的形成；靶向PUF60蛋白的小分子化合物初步虚拟筛选出GW9508和ZK 756326为PUF60的潜在小分子抑制剂。在基于GW9508和ZK 756326分子结构的进一步优化筛选分析出20个类似物中，发现小分子抑制剂QL-448B对PUF60的蛋白表达的影响具有浓度依赖性，其与PUF60蛋白的KD为0.38μM；细胞克隆形成实验和mRNA稳定性测定表明QL-448B可以降低卵巢细胞的克隆形成并抑制PUF60对靶基因mRNA稳定性。裸鼠皮下瘤模型显示，QL-448B可显著降低瘤体的大小和重量，其中50mg/kg组对移植瘤的体积和重量的抑制力更强，具有较好的浓度依赖性。.综上所述，PUF60具有作为卵巢癌不良预后分子标志物、促进卵巢癌细胞的增殖迁移功能和被鉴定为新的m6A结合蛋白的潜力；PUF60作为m6A结合蛋白可调节氧化磷酸化和糖酵解水平来满足卵巢癌细胞对能量的需求；PUF60与PABPC1相互作用促进P小体的形成，进而促进氧化磷酸化相关靶基因mRNA降解；靶向PUF60的小分子抑制剂QL-448B在卵巢细胞中抑制能力强、特异性高、亲和力好，具有良好的转化价值。