PCBP1介导的CD44剪接变异体对前列腺癌细胞干性调节的机制研究

Cell membrane receptor (CD44) is usually used as a surface marker of tumor stem cells, and is also involved in mediating the effects of a variety of extracellular signals on cells. CD44 can be involved in the regulation of cell function through splicing variation, including the downregulation of epithelial subtypes in EMT cells. Our previous study found that PCBP1 played an important negative regulatory role in enrichment, proliferation and tumorigenicity test in prostate cancer stem cells (LNCaP and DU145 strain). And the splice variant of CD44 mRNA in this process presented great differences. At the same time, PCBP1 could be used to mediate the CD44 pre-mRNA variation. We assume that PCBP1 mediated splicing variants of CD44 involved in the regulation of cell self-renewal, differentiation, tumorigenicity. This study compared different CD44 splicing variants and PCBP1 function in different cell conditions. With the help of fusion protein, we try to verify the relationship between CD44 and PCBP1. To explore the key function of CD44 variant splicing and corresponding protein in prostate cancer stem cell.

细胞膜受体CD44分子通常作为肿瘤干细胞分选表面标记，也参与介导多种细胞外信号对细胞的作用。CD44能通过剪接变异来参与细胞功能调节，包括其在肿瘤细胞EMT过程中下调其上皮亚型。我们的前期研究发现PCBP1对前列腺癌干细胞(LNCaP和DU145株)的富集、增殖和成瘤试验中起关键的负调节作用，且发现在此过程中CD44的剪接变异体mRNA产生巨大差异。同时，基于PCBP1可介导CD44 pre-mRNA变异剪接的证据。我们假设PCBP1参与了前列腺癌干细胞中CD44变异剪接，进而参与调节细胞自我更新、多向分化、致瘤性；本研究通过比较不同细胞状态下CD44变异剪接体、PCBP1功能和细胞功能的联系，进一步借助融合蛋白验证CD44的关键作用和PCBP1调节CD44与细胞功能关联，探索CD44变异剪接和相应蛋白在前列腺癌干细胞中的功能。

本研究基于CD44分子变异剪切产物与前列腺癌细胞干性特点之间的联系，试图揭示不同细胞状态时变异剪切产物与细胞功能的关系，基于该关系探索变异剪切产物mRNA、蛋白质和细胞干性特点(细胞周期、悬浮生长、EMT、迁移、侵袭、肿瘤启动)之间的直接联系。.我们检测了抑制CD44表达，在前列腺癌细胞（TGF-β处理）中各类表型的变化，证实了CD44的表达（特别是CD44s）是前列腺癌干细胞上皮间质转化（EMT）过程中重要的因素。再次的rescue实验证实了，它是保持前列腺癌干细胞干性特点的重要标志。.进一步实验，证实了PCBP-1和SMAD3是影响CD44变异剪接表达的关键。敲除了PCBP-1，直接影响了前列腺癌细胞（TGF-β处理）中CD44变异剪接的转化，并影响细胞EMT和干细胞特性。进一步实验，提示了TGF-β1通过蛋白磷酸化和PCBP1泛素化减少PCBP-1的蛋白水平表达，并影响到CD44的变异剪接转化。.为了测试CD44s在促进肿瘤进展中的作用，通过敲除CD44或过表达CD44s.进行细胞移植、增殖、迁移和侵袭分析。细胞和动物实验，证实了CD44s是前列腺癌干细胞干性特性重要的促进因素，以上成果揭示了前列腺癌细胞干细胞特性的新通路，将来作为药物靶向研究的重要参考。