剪切因子SNRPB2通过调控Bcl-X可变剪切促进乳腺癌进展的机制研究

As one of the most prevalent mechanisms of gene regulation, alternative splicing (AS) plays a vital role in the intricate regulation of protein function, and splicing dysregulation is closely associated with human cancer. This RNA processing event is mediated by a complex interplay of splicing factors (SFs) and defined RNA sequences (splice sites) within pre-mRNA transcripts. These SFs and other RNA-binding proteins are tightly regulated and embedded in signaling pathways in the cancer cell, making AS a major modulator of cancer cell properties and functions. Using an in vivo CRISPR screen targeting RNA-binding proteins, we have previously identified Small Nuclear Ribonucleoprotein Polypeptide B2 (SNRPB2) as a SF involved in breast tumor progression. To date, there is almost no lecture to uncover the role of SNRPB2 in tumor and the underlying molecular mechanisms remain poorly defined. In this study, SNRPB2 was frequently up-regulated in breast cancer and was associated with poor clinical outcome in patients. Knockout of SNRPB2 gene by CRISPR/Cas9 significantly inhibited breast cancer cell growth and migration and caused apoptosis. RNA-seq analysis revealed that SNRPB2 ablation altered the splicing patterns of cancer-related genes in transcriptome-scale, including downstream target gene Bcl-X. Additionally, SNRPB2 was found to bind to SF RBM4. We therefore hypothesize that SNRPB2 promotes breast tumor progression by regulating Bcl-X AS and antagonizing the AS effect of RBM4 on Bcl-X. We plan to perform relevant cell and molecular biology experiments, transcriptome sequencing, animal experiments and clinical sample analysis to investigate the biological role of SNRPB2 on breast tumor progression as well as the mechanism of SNRPB2 regulating the AS of Bcl-X. Further, we are going to identify the key downstream molecules that mediate the effect of SNRPB2 on breast tumor progression and to elucidate the primary signaling pathway that is directly regulated by SNRPB2. In summary, these proposed experiments in the project are to lay a solid foundation for identifying a prognostic marker and a translatable therapeutic target for breast tumor.

剪切因子调控转录后可变剪切过程，其表达量异常与肿瘤进展密切相关。本研究前期利用CRISPR/Cas9文库技术筛选发现剪切因子SNRPB2在乳腺肿瘤进展中不可或缺。有关SNRPB2在肿瘤研究中报道甚少，相关分子机理有待研究。预实验发现：高表达SNRPB2的乳腺癌预后更差；敲除SNRPB2基因显著抑制乳腺癌细胞生长与迁移；SNRPB2基因缺失会改变靶基因Bcl-X的剪切模式而引起显著凋亡效应。我们假设SNRPB2通过调控Bcl-X剪切并拮抗RBM4对Bcl-X的剪切效应，促进乳腺肿瘤进展。本项目计划运用细胞和分子生物学实验、转录组测序技术、动物实验和临床样本分析，研究SNRPB2对靶基因Bcl-X可变剪切的直接与间接调控机制，鉴定介导SNRPB2促癌效应的关键靶基因，阐明SNRPB2直接调节的细胞信号分子通路。本研究将揭示SNRPB2与乳腺肿瘤进展的内在联系，为乳腺肿瘤治疗提供潜在新靶点。

剪切因子调控转录后可变剪切过程，其表达量异常与肿瘤进展密切相关。本研究前期利用CRISPR/Cas9文库技术筛选发现剪切因子SNRPB2在乳腺肿瘤进展中不可或缺。有关SNRPB2在肿瘤研究中报道甚少，相关分子机理有待研究。本研究利用免疫组化染色分析、公共数据库分析等方法明确了SNRPB2在乳腺癌组织中高表达，尤其在三阴性乳腺癌中高表达，且高表达SNRPB2转录本的乳腺癌患者预后更差；敲除SNRPB2基因显著抑制乳腺癌细胞增殖、迁移及动物体内成瘤能力，但不影响正常乳腺细胞表型；转录组测序、基因本体论分析、可变剪切分析及凋亡实验等表明，SNRPB2基因缺失会使靶基因Bcl-X的转录本更多地剪切为Bcl-xS而引起显著凋亡效应；RNA免疫沉淀实验、剪切报告基因、功能回复等实验显示，SNRPB2通过与5’剪切位点附近mut2位点与Bcl-X前体mRNA结合，行使可变剪切调控作用，使Bcl-xL剪切形式明显增多，由此介导抗凋亡效应，产生相应的生物学表型；RBM4蛋白能通过调控Bcl-X更多地剪切为Bcl-xS而发挥抑癌效应；而免疫沉淀实验表明SNRPB2与RBM4相结合，通过减少RBM4蛋白的表达，拮抗RBM4对Bcl-X的剪切效应，促进乳腺癌增殖、迁移、动物体内成瘤。由此总结：SNRPB2通过调控Bcl-X剪切并拮抗RBM4对Bcl-X的剪切效应，促进乳腺肿瘤进展。本研究探索了SNRPB2对靶基因Bcl-X可变剪切的直接与间接调控机制，鉴定了介导SNRPB2促癌效应的关键靶基因，阐明了SNRPB2直接调节的细胞信号分子通路。本研究揭示了SNRPB2与乳腺肿瘤进展的内在联系，为乳腺肿瘤治疗提供了潜在新靶点。