The miR-96 and RARG signaling axis governs androgen signaling and prostate cancer progression V. The miR-96 and RARG signaling axis governs androgen signaling and prostate cancer progression V

Expression levels of retinoic acid receptor gamma (NR1B3/RARG, encodes RARG), are commonly reduced in prostate cancer (PCa). Therefore we sought to establish the cellular and gene regulatory consequences of reduced RARG expression, and determine RARG regulatory mechanisms. RARG shRNA approaches in non-malignant (RWPE-1 and HPr1-AR) and malignant (LNCaP) prostate models revealed that reducing RARG levels, rather than adding exogenous retinoid ligand, had the greatest impact on prostate cell viability and gene expression. ChIP-Seq defined the RARG cistrome which was significantly enriched at active enhancers associated with AR binding sites. Reflecting a significant genomic role for RARG to regulate androgen signaling, RARG knockdown in HPr1-AR cells significantly regulated the magnitude of the AR transcriptome. RARG down-regulation was explained by increased miR-96 in PCa cell and mouse models, and TCGA PCa cohorts. Biochemical approaches confirmed that miR-96 directly regulated RARG expression and function. Capture of the miR-96 targetome by biotin-miR96 identified that RARG and a number of RARG interacting co-factors including TACC1 were all targeted by miR-96, and expression of these genes were prominently altered, positively and negatively, in the TCGA-PRAD cohort. Differential gene expression analyses between tumors in the TCGA-PRAD cohort with lower quartile expression levels of RARG and TACC1 and upper quartile miR-96, compared to the reverse, identified a gene network including several RARG target genes (e.g. SOX15) that significantly associated with worse disease free survival (hazard ratio 2.23, 95% CI 1.58 to 2.88, p=0.015). In summary, miR-96 targets a RARG network to govern AR signaling, PCa progression and disease outcome. RNA-seq: HPr1-AR non-malignant, immortalized cell line +/- shRARG, +/- 10nM DHT for 0,24,96 hours Overall design: 18 samples total, 6 experimental conditions (each in triplicate); shCTL-0hr, shCTL_24hr, shCTL_96hr, shRARG-0hr, shRARG-24hr, shRARG-96hr

视黄酸受体γ（NR1B3/RARG，编码RARG）的表达水平在前列腺癌（PCa）中普遍降低。因此本研究旨在明确RARG表达下调所带来的细胞与基因调控效应，并解析RARG的调控机制。在非恶性（RWPE-1与HPr1-AR）及恶性（LNCaP）前列腺细胞模型中采用RARG短发夹RNA（shRNA）干预手段，结果显示，相较于添加外源性类维生素配体，下调RARG表达对前列腺细胞活力与基因表达的影响更为显著。染色质免疫沉淀测序（ChIP-Seq）明确了RARG的顺式调控元件组（cistrome），其在与雄激素受体（AR）结合位点相关的活性增强子区域存在显著富集。这反映出RARG在调控雄激素信号通路中具有重要的基因组学功能：在HPr1-AR细胞中敲低RARG可显著改变AR转录组的调控幅度。前列腺癌的细胞与小鼠模型、癌症基因组图谱（TCGA）前列腺癌队列中均发现，miR-96表达上调可解释RARG的下调现象。生化实验证实，miR-96可直接调控RARG的表达与功能。通过生物素标记的miR-96捕获miR-96的靶标组，结果显示RARG以及包括TACC1在内的多种RARG互作辅因子均为miR-96的靶基因；且这些基因在TCGA-PRAD队列中的表达呈现显著的正负向调控变化。对TCGA-PRAD队列中，RARG与TACC1表达处于下四分位数、miR-96处于上四分位数的肿瘤样本，与反之的样本进行差异基因表达分析，鉴定出包含若干RARG靶基因（如SOX15）的基因网络，该网络与较差的无病生存期显著相关（风险比=2.23，95%置信区间：1.58~2.88，p=0.015）。综上，miR-96通过靶向RARG调控网络，进而调控雄激素信号通路、前列腺癌进展与疾病预后。RNA测序：HPr1-AR非恶性永生化细胞系经±shRARG、±10nM二氢睾酮（DHT）处理0、24、96小时。整体实验设计：共包含18个样本，分为6组实验条件（每组设置3次生物学重复）：shCTL-0hr、shCTL_24hr、shCTL_96hr、shRARG-0hr、shRARG-24hr、shRARG-96hr