可变剪切基因REST调控SRRM3的表达在前列腺癌神经内分泌分化中的作用机制研究

Androgen depletion treatment related neuroendocrine differentiated prostate cancer is recently recognized as one pivotal mechanism for castration resistant prostate cancer. Accumulated evidence had demonstrated that RE1-silencing transcription factor (REST) was closely with neuroendocrine differentiation. In neuroendocrine prostate cancer, the expression of REST was downregulated and alternative splicing of REST was observed. However, the mechanism is unclear and further investigations are urgent. Our previous work found that downregulation of REST in prostate cancer cell line LNCaP could contribute to an elevated level of serine/arginine repeat matrix 3(SRRM3). Meanwhile, the expression of alternative splicing variant REST4 was also higher than ever. Based on these results, we hypothesize that there may be a reciprocal loop between REST and SRRM3. Consequently, we will investigate the role of SRRM3 after knocking down REST, exploring the impact on biogenesis of neuroendocrine differentiation in prostate cancer. Furthermore, we will regulate the expression of SRRM3 in the cells to explore its influence on the biological behavior and the role of SRRM3 in the reciprocal feedback loop on REST, resulting alternative splicing. Finally, we would like to screen effectively prompted neuroendocrine prostate cancer indicators, providing the laboratory basis for further investigations of therapeutic targets and feasible clinical neuroendocrine prostate biomarkers.

前列腺癌雄激素剥夺治疗后神经内分泌分化的发生近期被认为是去势抵抗发生机制之一。近年来发现可变剪切基因RE-1沉默转录因子REST与神经内分泌分化关系密切，在神经内分泌分化的前列腺癌中表达下降，但具体功能机制有待深入发掘。我们前期发现下调前列腺癌细胞LNCaP中REST的表达可导致剪切调节基因SRRM3表达增高，REST发生可变剪切，推论REST和SRRM3可能存在相互调节，REST能够调控SRRM3的表达，而SRRM3可影响REST可变剪切。基于此假说，我们拟下调前列腺癌细胞中REST水平来影响SRRM3表达，探索对REST可变剪切的作用和肿瘤神经内分泌分化的影响；诱导前列腺癌细胞株的神经内分泌分化，调节SRRM3的表达来改变REST剪切状态，探讨二者相互作用，对肿瘤细胞生物学行为的影响；筛选出有效地提示神经内分泌分化的指标，为临床进一步探索可行治疗靶点和预后指标提供新的实验室依据。

前列腺癌细胞神经内分泌分化近期被认为可能导致前列腺癌去势抵抗发生。可变剪切基因RE-1沉默转录因子REST与细胞神经内分泌分化关系密切，在神经内分泌分化的前列腺癌中表达下降。通过质粒下调前列腺癌细胞中REST及SRRM3表达，realtime-PCR检测mRNA表达，western-blot和免疫组化检测蛋白表达水平，细胞增殖速率体外采用CCK8法、体内试验建立裸鼠移植瘤模型测定，细胞周期及凋亡使用流式细胞仪进行分析，迁移和侵袭实验采用transwell检测，REST和SRRM3结合状态通过ChIP检测。研究结果显示下调前列腺癌细胞LNCaP、PC-3和DU145中REST的表达可导致剪切调节基因SRRM3表达增高，REST发生可变剪切，前列腺癌细胞增殖加快，S期细胞增多，神经内分泌指标CgA、CgB表达改变。REST间接调节SRRM3发挥作用。诱导前列腺癌细胞株神经内分泌分化筛选出neuro-LNCaP、neuro-PC-3细胞株，体内和体外实验均显示细胞增殖速率快于对照组，S期细胞增多，神经内分泌指标CgA、CgB、NSE表达增高。因此，下调REST水平能影响SRRM3表达，导致前列腺癌细胞株的神经内分泌分化，促进细胞恶性增殖，为临床进一步探索前列腺癌治疗靶点和预后指标提供新的实验室依据。

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