miR-195靶向调控CLSPN基因影响人前列腺癌进展的作用机制研究

Clinical indexes could not predict prostate cancer prognosis accurately which fails to achieve precision management and lead to serious over-treatment of prostate cancer. Thus, it is urgent to investigate the molecular mechanism underlying prostate cancer progression. Our previous results have shown that miR-195 act as a critical role in the process of prostate cancer progression by targeting RPS6KB1 (Clin Cancer Res. 2015). However, miRNAs is characterized by controlling multiple targets. In order to better understand the molecular network controlled by miR-195 in prostate cancer, we then continued to screen other targets and find that CLSPN is a novel candidate targets of miR-195 by gene microarray, target prediction bioinformatics software and human prostate cancer database. Moreover, miR-195 and CLSPN together could predict prostate cancer prognosis accurately. In addition, dual-luciferase reporter assay further revealed that the 3’ untranslated region (UTR) sequence of CLSPN was targeted by miR-195. However, the biological role of miR-195-CLSPN axis and functional interaction between them in prostate cancer is unknown. In this study, we are planning to perform series of experiments, such as cellular functional experiments in vitro, tumor metastasis model in vivo and tissue microarray, to clarify the tumor progression-related network controlled by miR-195, which may afford some critical clues for precision treatment and help us identify some novel therapeutic targets in human prostate cancer.

依赖临床指标判断前列腺癌预后导致严重过度治疗，无法满足精准医疗的要求，因而迫切需要明确前列腺癌进展的分子机制。我们的研究已证实miR-195调控RPS6KB1在前列腺癌进展中起重要作用（Clin Cancer Res. 2015）。然而，小分子RNA（miRNAs）能同时调控多个靶基因，为了揭开miR-195在前列腺癌调控的分子网络，我们用基因芯片、预测软件和临床数据库继续筛选，首次发现CLSPN为miR-195潜在的靶基因，并且miR-195-CLSPN联合与前列腺癌预后关系密切。预实验也证实CLSPN的3’端非编码区序列被miR-195特异性结合。但miR-195-CLSPN在前列腺癌的分子生物学功能、二者表达和功能上的调控关系如何？我们拟用细胞体内、体外功能实验和组织芯片等进一步明确miR-195在前列腺癌进展中的调控网络，为实现前列腺癌个体化精准治疗和发现新的治疗靶点提供科学依据。

目的 临床指标在精确判断前列腺癌的预后方面存在不足，小分子RNA-195（miR-195）已被证实是前列腺癌进展过程中一个重要的调控因子。本文进一步探索miR-195调控的靶标基因及其生物学功能。材料和方法 核苷酸瞬时转染前列腺癌细胞株DU145和LNCaP构建miR-195过表达的细胞模型，用体外细胞实验探讨miR-195过表达后对细胞增殖、血管生长和克隆形成的影响。利用RNA基因芯片、信号通路分析软件和靶标基因预测软件及GEO前列腺癌公共数据库（Taylor队列）对候选靶基因进行筛选。荧光素酶报告基因系统进一步确认miR-195调控的靶基因。最后，用裸鼠皮下移植瘤模型及组织芯片探讨靶基因在动物体内的功能及临床意义。结果 miR-195表达上调能够明显抑制DU145和LNCaP细胞增殖和克隆形成的能力，还能抑制脐静脉内皮细胞形成血管。RNA基因芯片检测发现miR-195过表达的细胞对比对照组细胞存在153个差异表达1.5倍以上的基因，包括138（90.2%）个下调基因和 15 （9.8%）个上调基因。靶基因预测软件预测下调最明显的前10个基因中有6个可能是靶基因，分别是CLSPN、ZFHX3、 PRR11、 GCC2、 RPS6KB1和ENAH。用Taylor队列进一步分析，我们发现脯氨酸富集蛋白11 （PRR11）与miR-195在前列腺癌组织的表达水平成负相关，联合二者的表达水平可作为预测前列腺癌根治术后生化复发的独立指标。荧光素酶报告基因系统证实PRR11是miR-195调控的靶基因。体外细胞功能实验表明PRR11重新表达能够拮抗miR-195对细胞增殖能力、克隆能力、血管形成能力及细胞周期的抑制效果。最后，裸鼠皮下移植瘤模型发现siRNA干扰PRR11表达后，DU145和LNCaP细胞形成的移植瘤生长明显变缓，重量也减轻。前列腺组织芯片免疫染色发现PRR11在前列腺癌中表达上调，结合Taylor队列分析得出PRR11上调与前列腺癌恶性程度高及预后差有关。结论 我们的研究确认了PRR11是miR-195新的靶基因，miR-195通过调控PRR11的表达影响肿瘤增殖和血管形成的过程。该研究首次报道了PRR11在前列腺癌是一个促癌基因。联合miR-195和PRR11的表达水平可协助临床指标判断前列腺癌的预后，从而优化治疗方案。