Numb PRR 亚型选择性剪接在HCC中的作用及其相关调控机制的研究

The cell fate determinant Numb is aberrantly expressed in cancer. Numb is alternatively spliced, with one isoform containing a long proline-rich region (PRRL) compared to the other with a short PRR (PRRS). However, the importance of Numb alternative splicing in hepatocellular carcinoma (HCC) remains unexplored. We have reported that Numb PRRL expression is increased in HCC and associated with early recurrence and reduced overall survival after surgery. In a panel of HCC cell lines, PRRL generally promotes and PRRS suppresses proliferation, migration, invasion, and colony formation. In the cell models explored in this study, alternative splicing of Numb PRR isoforms is coordinately regulated by the splicing factor RNA-binding Fox domain containing 2 (RbFox2) and the kinase serine/arginine protein–specific kinase 2 (SRPK2). Knockdown of the former causes accumulation of PRRL, while SRPK2 knockdown causes accumulation of PRRS. The subcellular location of SRPK2 is regulated by the molecular chaperone heat shock protein 90, and heat shock protein 90 inhibition or knockdown phenocopies SRPK2 knockdown in promoting accumulation of Numb PRRS. Finally, HCC cell lines that predominantly express PRRL are differentially sensitive to heat shock protein 90 inhibition. .Based on the findings above, we will further establish Numb PRRL/S over expression and knock down stable cell lines, tumor-bearing mice models and Numb PRRL high expression patients derived tumor xenograft mice model(PDX) to verify the relationship between Numb PRRL/S isoforms and HCC developing and progression, elucidate the potential mechanisms involved. Clinical derived sample testing will be performed to confirm the molecular variation and biomarkers of Numb PRR alternative splicing modulated HCC progressing. Then, we will explore the potential application and effect of HSP90 inhibitor in HCC with Numb PRRL high expression..Our present study is aiming to further elucidate the role of Numb PRR alternative splicing in the development of HCC and the detailed molecular mechanisms behind it which will be verified in large-scale HCC clinical samples. Alternative splicing of Numb may provide a useful prognostic biomarker in HCC and is pharmacologically tractable. We believe that our study will greatly contribute to the understanding of role of Numb PRR isoforms in HCC development, and thus provide a new and feasible therapeutic target for the treatment of liver cancer.

细胞命运决定子Numb选择性剪接产生PRRL、PRRS两个亚型，参与调控多种细胞生物学行为。我们研究发现，Numb PRRL在肝癌组织中表达显著升高，能促进肝癌细胞增殖、侵袭、迁徙，并与患者不良预后相关，而PRRS的作用则相反；热休克蛋白90（HSP90）可以通过影响剪接因子SRPK2的亚细胞定位而间接调控Numb PRRL/S的选择性剪接，改变肝癌细胞中Numb PRRL/PRRS的表达比例，最终影响肝癌细胞生物学行为。本研究拟在此基础上建立Numb PRRL/S稳定过表达/敲低的肝癌细胞系及荷瘤小鼠、Numb PRRL高表达的人源性肿瘤异种移植（PDX）模型，进一步阐明Numb PRR选择性剪接在HCC发生、发展中的作用及其相关分子机制；利用大规模HCC临床标本验证上述机制；此外，我们还将重点探索HSP90抑制剂通过调节Numb PRRL/S选择性剪接作为HCC新的靶向治疗药物的可行性

细胞命运决定子Numb选择性剪接产生PRRL、PRRS两个亚型，参与调控多种细胞生物学行为。我们研究发现，Numb PRRL在肝癌组织中表达显著升高，能促进肝癌细胞增殖、侵袭、迁徙，并与患者不良预后相关，而PRRS的作用则相反；热休克蛋白90（HSP90）可以通过影响剪接因子SRPK2的亚细胞定位而间接调控Numb PRRL/S的选择性剪接，改变肝癌细胞中Numb PRRL/PRRS的表达比例，最终影响肝癌细胞生物学行为。本研究拟在此基础上建立Numb PRRL/S稳定过表达/敲低的肝癌细胞系及荷瘤小鼠、Numb PRRL高表达的人源性肿瘤异种移植（PDX）模型，进一步阐明Numb PRR选择性剪接在HCC发生、发展中的作用及其相关分子机制；利用大规模HCC临床标本验证上述机制；此外，我们还重点探索了HSP90抑制剂通过影响DDX5的表达及功能调节Numb PRRL/S选择性剪接，并筛选验证确认DDX3X是调控Numb PRRL/S选择性剪接的效应蛋白之一，这为探索开发新型HCC靶向治疗药物提供了理论基础。