发现一种新型长非编码 RNA 与 LCK 基因重叠，可调节前列腺癌细胞生长。
Discovery of a novel long noncoding RNA overlapping the LCK gene that regulates prostate cancer cell growth.
机构信息
Departments of Microbiology Immunology, and Cancer Biology, University of Virginia, Charlottesville, Virginia, 22908, USA.
College of Pharmacy Pharmaceutics and Pharmaceutical Chemistry, The Ohio State University, Columbus, OH, 43210, USA.
出版信息
Mol Cancer. 2019 Jun 28;18(1):113. doi: 10.1186/s12943-019-1039-6.
BACKGROUND
Virtually all patients with metastatic prostate cancer (PCa) will relapse and develop lethal castration-resistant prostate cancer (CRPC). Long noncoding RNAs (lncRNAs) are emerging as critical regulatory elements of many cellular biological processes, and may serve as therapeutic targets for combating PCa progression. Here, we have discovered in a high-throughput RNAi screen a novel lncRNA in PCa, and assessed the oncogenic effects of this lncRNA.
METHODS
Rapid amplification of cDNA ends and sequencing was utilized to identify a previously unannotated lncRNA lying within exon six and the 3'UTR of the lymphocyte-specific protein tyrosine kinase (LCK) gene. The levels of HULLK in the presence or absence of hormone and/or enzalutamide or coregulator inhibitors were measured by quantitative PCR (qPCR). The determination of HULLK transcription and localization were characterized by strand-specific qPCR and cellular fractionation followed by qPCR, respectively. The correlation between HULLK expression and prostate cancer Gleason score was analyzed by droplet digital PCR. CyQuant assays were conducted to evaluate the effects of knocking down HULLK with shRNAs or overexpressing HULLK on cell growth.
RESULTS
In this study, a previously unannotated lncRNA lying within exon six and 3'UTR of the LCK gene was dramatically upregulated by androgen in a dose-dependent manner, and the anti-androgen enzalutamide completely blocked this hormone-induced increase. Therefore, we labeled this lncRNA "HULLK" for Hormone-Upregulated lncRNA within LCK. Binding sites for two AR coregulators p300 and Brd4 reside near the HULLK transcriptional start site (TSS), and inhibitors of these coregulators downregulated HULLK. HULLK is transcribed from the sense strand of DNA, and predominantly localizes to the cytoplasm. HULLK transcripts are not only expressed in prostate cancer cell lines, but also prostate cancer patient tissue. Remarkably, there was a significant positive correlation between HULLK expression and high-grade PCa in multiple cohorts. shRNAs targeting HULLK significantly decreased PCa cell growth. Moreover, cells overexpressing HULLK were hypersensitive to androgen stimulation.
CONCLUSIONS
HULLK is a novel lncRNA situated within the LCK gene that may serve as an oncogene in PCa. Our data enhances our understanding of lncRNA biology and may assist in the development of additional biomarkers or more effective therapeutic targets for advanced PCa.
背景
几乎所有转移性前列腺癌（PCa）患者都会复发并发展为致命的去势抵抗性前列腺癌（CRPC）。长链非编码 RNA（lncRNA）作为许多细胞生物学过程的关键调节因子而出现，可能成为对抗 PCa 进展的治疗靶点。在这里，我们在高通量 RNAi 筛选中发现了 PCa 中的一种新型 lncRNA，并评估了该 lncRNA 的致癌作用。
方法
利用快速扩增 cDNA 末端和测序技术鉴定了一个以前未注释的 lncRNA，该 lncRNA位于淋巴细胞特异性蛋白酪氨酸激酶（LCK）基因的外显子六和 3'UTR 内。通过定量 PCR（qPCR）测量激素和/或恩扎鲁胺或共调节剂抑制剂存在或不存在时 HULLK 的水平。通过特异性 qPCR 和细胞分离后 qPCR 分别对 HULLK 的转录和定位进行特征描述。通过液滴数字 PCR 分析 HULLK 表达与前列腺癌 Gleason 评分之间的相关性。通过 shRNA 敲低或过表达 HULLK 进行 CyQuant 测定，评估对细胞生长的影响。
结果
在这项研究中，位于 LCK 基因外显子六和 3'UTR 内的一个以前未注释的 lncRNA 被雄激素以剂量依赖性方式显著上调，并且抗雄激素恩扎鲁胺完全阻断了这种激素诱导的增加。因此，我们将这个 lncRNA 标记为“ HULLK”，表示 LCK 内的激素上调 lncRNA。两个 AR 共调节剂 p300 和 Brd4 的结合位点位于 HULLK 转录起始位点（TSS）附近，这些共调节剂的抑制剂下调了 HULLK。HULLK 从 DNA 的正义链转录，主要定位于细胞质。HULLK 转录本不仅在前列腺癌细胞系中表达，而且在前列腺癌患者组织中也表达。值得注意的是，在多个队列中，HULLK 表达与高级别 PCa 之间存在显著的正相关关系。靶向 HULLK 的 shRNA 显著降低了 PCa 细胞的生长。此外，过表达 HULLK 的细胞对雄激素刺激更加敏感。
结论
HULLK 是位于 LCK 基因内的一种新型 lncRNA，它可能是 PCa 中的癌基因。我们的数据增强了对 lncRNA 生物学的理解，并可能有助于开发用于治疗晚期 PCa 的其他生物标志物或更有效的治疗靶点。
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