School of Biomedical Sciences, Faculty of Health, Institute of Health and Biomedical Innovation, Queensland University of Technology, Australian Prostate Cancer Research Centre-Queensland (APCRC-Q), Translational Research Institute, Brisbane, Australia.
University of Queensland Diamantina Institute (UQDI), Faculty of Medicine, Translational Research Institute, University of Queensland, Brisbane, Australia.
Clin Chem. 2019 Jun;65(6):771-780. doi: 10.1373/clinchem.2018.295824. Epub 2019 Apr 24.
MicroRNAs mediate biological processes through preferential binding to the 3' untranslated region (3' UTR) of target genes. Studies have shown their association with prostate cancer (PCa) risk through single-nucleotide polymorphisms (SNPs), known as miRSNPs. In a European cohort, 22 PCa risk-associated miRSNPs have been identified. The most significant miRSNP in the 3' UTR of () created a binding site for miR-3162-5p. Here we investigated the miR-3162-5p-KLK interaction and the clinical implication of miR-3162-5p in PCa.
We tested the role of miR-3162-5p in PCa etiology using IncuCyte live-cell imaging and anchorage-independent growth assays. The effect of miR-3162-5p on KLK and androgen receptor (AR) expression was measured by RT-quantitative (q)PCR and target pulldown assays. KLK3 proteolytic activity was determined by DELFIA immunoassay. Mass spectrometry identified pathways affected by miR-3162-5p. miR-3162-5p expression was measured in clinical samples using RT-qPCR.
miR-3162-5p affected proliferation, migration, and colony formation of LNCaP cells by regulating the expression of KLK2-4 and AR by direct targeting. KLK3 protein expression was regulated by miR-3162-5p consistent with lower KLK3 proteolytic activity observed in LNCaP-conditioned media. KLK/AR pulldown and mass spectrometry analysis showed a potential role of miR-3162-5p in metabolic pathways via KLK/AR and additional targets. Increased miR-3162-5p expression was observed in prostate tumor tissues with higher Gleason grade.
Our study provides an insight into possible involvement of miR-3162-5p in PCa etiology by targeting KLKs and AR. It highlights clinical utility of miR-3162-5p and its interactive axis as a new class of biomarkers and therapeutic targets for PCa.
微小 RNA 通过优先结合靶基因的 3'非翻译区（3'UTR）来调节生物过程。通过单核苷酸多态性（SNP），即 miRSNPs，研究表明它们与前列腺癌（PCa）风险相关。在一个欧洲队列中，已经确定了 22 个与 PCa 风险相关的 miRSNPs。在 （）3'UTR 中最显著的 miRSNP 为 miR-3162-5p 创造了一个结合位点。在这里，我们研究了 miR-3162-5p-KLK 的相互作用以及 miR-3162-5p 在 PCa 中的临床意义。
我们使用 IncuCyte 活细胞成像和非锚定依赖性生长测定来测试 miR-3162-5p 在 PCa 病因学中的作用。通过 RT-定量（q）PCR 和靶标下拉测定来测量 miR-3162-5p 对 KLK 和雄激素受体（AR）表达的影响。通过 DELFIA 免疫测定法测定 KLK3 蛋白水解活性。质谱鉴定受 miR-3162-5p 影响的途径。使用 RT-qPCR 测量临床样本中的 miR-3162-5p 表达。
miR-3162-5p 通过直接靶向调节 KLK2-4 和 AR 的表达，影响 LNCaP 细胞的增殖、迁移和集落形成。miR-3162-5p 调节 KLK3 蛋白表达，与观察到的 LNCaP 条件培养基中较低的 KLK3 蛋白水解活性一致。KLK/AR 下拉和质谱分析表明，miR-3162-5p 通过 KLK/AR 和其他靶标在代谢途径中可能发挥作用。在具有较高 Gleason 分级的前列腺肿瘤组织中观察到 miR-3162-5p 表达增加。
我们的研究通过靶向 KLKs 和 AR，提供了 miR-3162-5p 可能参与 PCa 病因的深入了解。它突出了 miR-3162-5p 及其交互轴作为 PCa 新一类生物标志物和治疗靶点的临床实用性。
