应激反应蛋白RBM3调节CD44可变剪接对前列腺癌进展的影响

The adaptive envolution to microenvironment play a central role in cancer progression, especially in the process of metastasis and the development of drug resistance. One of important ways taken by cancer cells to respond to the microenviromental stress is producing a variety of proteins from the same pre-mRNA through alternative splicing. We have found that the cold-inducible RNA-binding protein RBM3 is supressed by different microenvironmental stress and help prostate cancer cells to maitain a typical stem cell-like features, which is critical to the development and progression of cancer. Importantly, our previous study demonstrated that RBM3 inhibit the alternative splicing of CD44v8-10, which is a typical splicing variant of prostate cancer and contribute to the survival of cancer cells in a stressful situation like cloning formation in soft agar. These results raise a potential mechanism, in that cancer cells develop an adaptive capability to the microenvironmental stress through decreasing the expression of RBM3, which in turn trigger the alternative splicing of CD44. On the basis of this hypothesis, we propose to explore how RBM3 regulating the alternative splicing of CD44 using crosslinking-immunprecipitation (CLIP) and high-throughput sequencing together with a splicing reporter system. Further, the potential signialing pathways by which the alterantive splicing variants of CD44 especially CD44v8-10 regulate the progression of prostate cancer will be investigated through flow cytometry, DNA microarray, cytokine antibody array, etc. Thus, the successful completion of this project will likely provide a new insight into the mechanisms underling the progression of prostate cancer that will in turn lead to identification of targets for cancer therapy.

恶性肿瘤最根本的特征之一是在发生转移和接受药物治疗时对所处的应激微环境具有适应性，而以基因可变剪接为代表的表观基因调控对恶性肿瘤的适应进化性具有重要作用。我们前期研究发现前列腺癌细胞中冷激蛋白RBM3在多种微环境应激状态下表达降低，并因此增强前列腺癌干细胞特征和生存能力，而RBM3对前列腺癌干细胞相关因子CD44的可变剪接具有抑制作用。因此，我们推测在应激微环境中肿瘤细胞下调RBM3的表达促进CD44剪接变异体的生成可能是提高环境适应性的一种机制。本研究拟利用RNA交联免疫沉淀结合测序和可变剪接报告系统来揭示RBM3对CD44可变剪接的调节机制，并运用流式细胞分选、基因表达芯片和荧光素酶报告系统等在体内、体外鉴定CD44剪接变异体影响前列腺癌恶性表型的信号转导通路，从而阐明RBM3调节CD44可变剪接影响前列腺癌进展、转移的分子机制，为进一步从前列腺癌适应性进化角度筛选治疗靶点打下基础。

本研究针对RBM3对CD44基因可变剪接及前列腺癌干细胞特性的影响和作用机制加以探讨。肿瘤细胞的干性是恶性肿瘤的基本特征之一，他表现为肿瘤细胞的自我更新能力，治疗抵抗能力和微环境适应能力。正是因为癌干细胞或具有干细胞特性的癌细胞存在，才使得恶性肿瘤总是在治疗后死灰复燃，或迁徙到不同的组织微环境中生根发芽，这些都是恶性肿瘤难以治疗的重要原因。我们前期的研究发现了RBM3抑制前列腺癌细胞干性，并初步判定了RBM3对干细胞相关蛋白CD44可变剪接的影响。在此基础上，我们开展了本项研究。我们发现RBM3可以广泛地影响调控外部刺激应答等重要生理过程的基因转录，以及与细胞外基质、细胞黏附等功能相关基因的可变剪接。同时，我们还发现RBM3并没有与CD44基因直接结合的特异序列，表明RBM3可能不是直接与CD44转录调节序列结合调节可变剪接，RBM3对基因可变剪接的调节应该是在更广泛、更上游的层面上进行，这为进一步的研究指明了方向。接下来的研究，我们证实了CD44可变剪接体CD44v8-10能够增加前列腺癌细胞的干性，而这种作用是通过干扰金属蛋白水解酶对标准CD44蛋白水解作用，从而抑制CD44对周期蛋白的促转录作用而实现的。肿瘤细胞在具有选择压力的微环境中停止增殖而进入休眠状态正是干细胞的一种特征，我们的结果提示了RBM3通过抑制CD44v8-10而干扰了这种干细胞特性形成。此外，通过PTEN与RBM3双基因前列腺特异性敲除小鼠模型，我们发现了RBM3基因敲除可能促进小鼠前列腺癌的发展，这一结果也支持体外实验所显示的RBM3对前列腺癌干性的抑制作用。