剪接蛋白SRSF9在胶质瘤中的功能及作用机制研究

Glioma is the most common and highly lethal type of primary brain cancer. Previous studies have documented that GRβ (glucocorticoid receptor β) serves as a tumor promoting factor in glioma. In the present study, we found alternative splicing factor SRSF9 may regulate alternative splicing of GR mRNA and enhance GRβ expression. Aberrant alternative splicing may be a significant but yet under-explored contributor to heterogeneous pathological characteristics of malignant tumor. We found that SRSF9 was upregulated in human gliomas, and there was a highly significant correlation between SRSF9 and overall survival of patients. In vitro assays demonstrated that SRSF9 is a potential glioma cancer promoting factor, and GRβ was regulated by SRSF9. Further study showed that SRSF9 mediates the anti-apoptotic effect by regulating the alternative splicing of another target gene BCL-XL. According to these results, we proposed that alternative splicing mediated by SRSF9 may play an important role in the development of glioma. The present study will investigate the function and mechanisms of SRSF9 in glioma, by using glioma tissues, cellular biology techniques and animal models, combined with high throughput transcriptome sequencing and RIP-sequence. We hope this study will enrich our knowledge of glioma pathogenesis at the splicing regulation level and provide novel strategies and potential therapeutic targets for malignant glioma treatment.

胶质瘤是最常见的颅内恶性肿瘤。糖皮质激素受体β（GRβ）是我们新鉴定的胶质瘤促癌因子，但其高表达机制尚未明确。我们前期工作提示这可能与RNA剪接蛋白SRSF9介导的GR基因mRNA的选择性剪接有关。RNA的异常剪接在肿瘤组织中广泛存在，我们前期研究发现SRSF9在胶质瘤中高表达，且高表达患者生存期更短，体外功能实验提示其是胶质瘤的一个潜在促癌因子。初步的机制研究显示SRSF9不仅调控GRβ表达，而且介导凋亡相关分子BCL-XL的选择性剪接，发挥抗凋亡作用。据此我们推测SRSF9介导的RNA选择性剪接在胶质瘤发生发展中发挥重要作用。本课题将通过组织、细胞和动物水平的研究，结合高通量转录组测序及RIP-seq技术，解析SRSF9在胶质瘤中的作用及分子机制，以期从RNA剪接调控水平上探讨胶质瘤的发病机理，为胶质瘤的防治提供新靶点和新思路。

胶质母细胞瘤（Glioblastoma, GBM）是人颅内恶性度最高的原发肿瘤，平均生存期仅为15个月。目前胶质瘤的治疗选择有限，从关键基因及通路研究胶质瘤发生发展机制可能为胶质瘤的诊疗提供新的线索。本项目在发现RNA剪接蛋白SRSF9调控糖皮质激素受体β（GRβ）表达的基础上系统研究了SRSF9基因在胶质瘤中的功能，并进一步探讨其调控机理。主要有如下发现：1）通过大数据分析及病人组织样本验证SRSF9在胶质瘤组织中高表达，高表达SRSF9的病人生存期更短；2）通过过表达和shRNA敲减SRSF9等手段，在体内外证实SRSF9在胶质瘤中发挥促癌功能；3）通过CRISPR/Cas9技术，我们获得完全敲除SRSF9的U87单克隆细胞株，通过突变技术成功在单克隆细胞株中回补SRSF9，发现可以挽救SRSF9缺失所致的细胞增殖和干细胞成球能力降低的表型，进一步明确了SRSF9在胶质瘤中的促癌功能。免疫荧光和WB方法验证了外源表达的SRSF9蛋白定位于细胞核，这与SRSF9作为剪接因子执行选择性剪接的功能相符合;4）证实SRSF9通过选择性剪接BCL2L1发挥抗凋亡作用; 5）对敲降SRSF9后的U87成神经干细胞球及对照细胞做全转录组测序，并分析结果，为更全面研究SRSF9促胶质瘤发生发展的机制提供实验依据。