Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Cheeloo College of Medicine, Shandong University, Jinan 250012, China.
Department of Clinical Laboratory Medicine, The Second Hospital of Shandong University, Jinan 250033, China.
Asian J Androl. 2018 Sep-Oct;20(5):511-517. doi: 10.4103/aja.aja_36_18.
We sought to investigate the underlying mechanism of action of the long noncoding RNA (lncRNA) LOC283070 in the development of androgen independence in prostate cancer. The interactions between LOC283070 and target proteins were investigated by RNA pull-down and RNA-binding protein immunoprecipitation (RIP) assays. Subcellular fractionation and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) were used to detect the subcellular localization of LOC283070. Western blotting was performed to detect the expression of prohibitin 2 (PHB2). Luciferase activity assays were performed to evaluate the effects of LOC283070 and PHB2 on the androgen receptor (AR) signaling pathway. A methyl thiazolyl tetrazolium (MTT) assay and a growth curve assay were used to test cell viability. Flow cytometry was performed to analyze cell cycles. A transwell assay was employed to test cell migration. We identified PHB2 as an interaction partner of LOC283070 in the pull-down and RIP experiments. Furthermore, we confirmed that the enrichment of LOC283070 with PHB2 in androgen-independent LNCaP (LNCaP-AI) cells was much greater than that in LNCaP cells. Moreover, the expression of PHB2 was not significantly different between the two cell lines, and the expression of LOC283070 in the nuclei of the LNCaP-AI cells was significantly greater than that in the LNCaP cells. In vitro data revealed that PHB2 overexpression significantly inhibited AR activity and cell proliferation and migration and induced accumulation of prostate cancer cells in G0/G1 phase. Moreover, the overexpression of LOC283070 fully abrogated the effects of PHB2 overexpression. In conclusion, we found that LOC283070 can bind to PHB2 located in the nucleus and inhibit its effect, and this is one of the mechanisms by which LOC283070 is involved in the transition of LNCaP cells into androgen-independent cells.
我们试图研究长链非编码 RNA（lncRNA）LOC283070 在前列腺癌雄激素非依赖性发展中的作用机制。通过 RNA 下拉和 RNA 结合蛋白免疫沉淀（RIP）实验研究 LOC283070 与靶蛋白的相互作用。通过亚细胞分离和定量逆转录聚合酶链反应（qRT-PCR）检测 LOC283070 的亚细胞定位。通过 Western blot 检测抑制素 2（PHB2）的表达。通过荧光素酶活性测定评估 LOC283070 和 PHB2 对雄激素受体（AR）信号通路的影响。通过甲基噻唑基四唑（MTT）测定和生长曲线测定检测细胞活力。通过流式细胞术分析细胞周期。通过 Transwell 测定检测细胞迁移。我们在下拉和 RIP 实验中鉴定 PHB2 为 LOC283070 的相互作用伙伴。此外，我们证实 LOC283070 与 PHB2 在雄激素非依赖性 LNCaP（LNCaP-AI）细胞中的富集明显高于 LNCaP 细胞。此外，两种细胞系之间 PHB2 的表达没有显著差异，而 LNCaP-AI 细胞核中 LOC283070 的表达明显高于 LNCaP 细胞。体外数据显示，PHB2 过表达显著抑制 AR 活性和细胞增殖和迁移，并诱导前列腺癌细胞在 G0/G1 期积累。此外，LOC283070 的过表达完全消除了 PHB2 过表达的作用。总之，我们发现 LOC283070 可以与位于核内的 PHB2 结合并抑制其作用，这是 LOC283070 参与 LNCaP 细胞向雄激素非依赖性细胞转化的机制之一。
