Laboratório de Expressão Gênica Em Eucariotos, Instituto Butantan, Av. Vital Brasil 1500, São Paulo, SP, 05503-900, Brazil.
Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, SP, 05508-900, Brazil.
Cell Commun Signal. 2021 Jan 11;19(1):5. doi: 10.1186/s12964-020-00691-x.
Androgen receptor (AR) and polycomb repressive complex 2 (PRC2) are known to co-occupy the loci of genes that are downregulated by androgen-stimulus. Long intergenic non-coding RNA (lincRNA) PVT1 is an overexpressed oncogene that is associated with AR in LNCaP prostate cancer cells, and with PRC2 in HeLa and many other types of cancer cells. The possible involvement of PVT1 in mediating androgen-induced gene expression downregulation in prostate cancer has not been explored.
LNCaP cell line was used. Native RNA-binding-protein immunoprecipitation with anti-AR or anti-EZH2 was followed by RT-qPCR with primers for PVT1. Knockdown of PVT1 with specific GapmeRs (or a control with scrambled GapmeR) was followed by differentially expressed genes (DEGs) determination with Agilent microarrays and with Significance Analysis of Microarrays statistical test. DEGs were tested as a tumor risk classifier with a machine learning Random Forest algorithm run with gene expression data from all TCGA-PRAD (prostate adenocarcinoma) tumors as input. ChIP-qPCR was performed for histone marks at the promoter of one DEG.
We show that PVT1 knockdown in androgen-stimulated LNCaP cells caused statistically significant expression upregulation/downregulation of hundreds of genes. Interestingly, PVT1 knockdown caused upregulation of 160 genes that were repressed by androgen, including a significantly enriched set of tumor suppressor genes, and among them FAS, NOV/CCN3, BMF, HRK, IFIT2, AJUBA, DRAIC and TNFRSF21. A 121-gene-set (out of the 160) was able to correctly predict the classification of all 293 intermediate- and high-risk TCGA-PRAD tumors, with a mean ROC area under the curve AUC = 0.89 ± 0.04, pointing to the relevance of these genes in cancer aggressiveness. Native RIP-qPCR in LNCaP showed that PVT1 was associated with EZH2, a component of PRC2. PVT1 knockdown followed by ChIP-qPCR showed significant epigenetic remodeling at the enhancer and promoter regions of tumor suppressor gene NOV, one of the androgen-repressed genes that were upregulated upon PVT1 silencing.
Overall, we provide first evidence that PVT1 was involved in signaling a genome-wide androgen-dependent transcriptional repressive program of tumor suppressor protein-coding genes in prostate cancer cells. Identification of transcriptional inhibition of tumor suppressor genes by PVT1 highlights the pathway to the investigation of mechanisms that lie behind the oncogenic role of PVT1 in cancer. Video Abstract.
雄激素受体（AR）和多梳抑制复合物 2（PRC2）已知共同占据受雄激素刺激下调的基因的基因座。长链非编码 RNA（lncRNA）PVT1 是一种过表达的癌基因，在 LNCaP 前列腺癌细胞中与 AR 相关，在 HeLa 和许多其他类型的癌细胞中与 PRC2 相关。PVT1 是否参与介导前列腺癌细胞中雄激素诱导的基因表达下调尚未得到探索。
使用 LNCaP 细胞系。用抗 AR 或抗 EZH2 的天然 RNA 结合蛋白免疫沉淀，然后用 PVT1 的引物进行 RT-qPCR。用特异性 GapmeR（或用乱序 GapmeR 作为对照）敲低 PVT1，然后用 Agilent 微阵列和 Significance Analysis of Microarrays 统计检验确定差异表达基因（DEGs）。使用随机森林算法的机器学习对所有 TCGA-PRAD（前列腺腺癌）肿瘤的基因表达数据作为输入，对 DEGs 进行肿瘤风险分类器测试。用组蛋白标记 ChIP-qPCR 在一个 DEG 的启动子上进行。
我们表明，在雄激素刺激的 LNCaP 细胞中敲低 PVT1 会导致数百个基因的表达显著上调/下调。有趣的是，PVT1 敲低导致 160 个受雄激素抑制的基因上调，其中包括一组显著富集的肿瘤抑制基因，其中包括 FAS、NOV/CCN3、BMF、HRK、IFIT2、AJUBA、DRAIC 和 TNFRSF21。160 个基因中有 121 个基因集（来自 160 个）能够正确预测所有 293 个中高危 TCGA-PRAD 肿瘤的分类，平均 ROC 曲线下面积 AUC=0.89±0.04，表明这些基因与癌症侵袭性相关。在 LNCaP 中的天然 RIP-qPCR 表明 PVT1 与 EZH2 相关，EZH2 是 PRC2 的一个组成部分。PVT1 敲低后进行 ChIP-qPCR 显示，在 NOV 基因的增强子和启动子区域发生了显著的表观遗传重塑，NOV 是雄激素抑制的基因之一，在 PVT1 沉默后被上调。
总的来说，我们首次提供了证据表明，PVT1 参与了信号转导，导致前列腺癌细胞中广泛存在的雄激素依赖性肿瘤抑制蛋白编码基因转录抑制程序。PVT1 对肿瘤抑制基因转录抑制的鉴定突出了对 PVT1 在癌症中致癌作用背后机制的研究途径。
