微小 RNA-99b-5p 靶向 mTOR/AR 轴，诱导自噬并抑制前列腺癌细胞增殖。
MicroRNA-99b-5p targets mTOR/AR axis, induces autophagy and inhibits prostate cancer cell proliferation.
机构信息
Julius L. Chambers Biomedical Biotechnology Research Institute, North Carolina Central University Durham, NC, USA.
Children's Hospital of Philadelphia Research Institute, Pennsylvania, PA, USA.
出版信息
Tumour Biol. 2022;44(1):107-127. doi: 10.3233/TUB-211568.
OBJECTIVES
MicroRNAs (miRNAs) are the small non-coding regulatory RNA molecules involved in gene regulation via base-pairing with complementary sequences in mRNAs. The dysregulation of specific miRNAs, such as miR-99b-5p (miR-99b), is associated with prostate cancer (PCa) progression. However, the mechanistic role of miR-99b in PCa remains to be determined. In this study, we aimed to investigate the functional and clinical significance of miR-99b in PCa.
STUDY DESIGN
The expression of miR-99b and its downstream targets mTOR/AR in the PCa samples were analyzed by RT/qPCR. The effects of miR-99b overexpression/inhibition on PCa cell survival/proliferation, spheroid formation, and cell migration were examined by specific assays. Luciferase reporter assays were performed to determine the binding of miR-99b to 3' untranslated region (UTR) of the mTOR gene. The effects of miR-99b on the expression of mTOR, AR, and PSA proteins, as well as on AKT/mTOR signaling, autophagy, and neuroendocrine differentiation markers were analyzed by western blotting. The expression of miR-99b, mTOR, AR, PSA in AR-negative PC3 and AR-positive LNCaP cells was analyzed by RT/qPCR. The effect of miR-99b on global gene expression in PC3 cells was analyzed by RNA-seq.
RESULTS
The expression of miR-99b was downregulated in tumor samples from PCa patients, whereas the expression of mTOR and AR was upregulated. In PCa cell lines, overexpression of miR-99b inhibited cell proliferation and cell colony/spheroid formation; induced apoptosis, and increased sensitivity towards docetaxel (DTX). In contrast, inhibition of miR-99b by miR-99b inhibitor resulted in increased cell growth in PCa cells. Mechanistically, miR-99b inhibited the expression of the mammalian target of the rapamycin (mTOR) gene by binding to its 3' UTR and induced autophagy. Furthermore, miR-99b inhibited androgen receptor (AR) activity in LNCaP cells and induced apoptosis. Activation of AR signaling by dihydrotestosterone (DHT) downregulated miR-99b expression and promoted cell PCa cell growth/survival, whereas inactivation of mTOR by rapamycin or AR by enzalutamide decreased miR-99b mediated PCa cell growth.
CONCLUSION
Our data suggest that miR-99b functions as a tumor suppressor by targeting the mTOR/AR axis in PCa cells, implicating miR-99b as a novel biomarker and therapeutic target for PCa management.
目的
MicroRNAs（miRNAs）是通过与 mRNA 中的互补序列碱基配对来参与基因调控的小非编码调节 RNA 分子。特定 miRNAs（如 miR-99b-5p（miR-99b））的失调与前列腺癌（PCa）的进展有关。然而，miR-99b 在 PCa 中的作用机制仍有待确定。在这项研究中，我们旨在研究 miR-99b 在 PCa 中的功能和临床意义。
研究设计
通过 RT/qPCR 分析 PCa 样本中 miR-99b 及其下游靶标 mTOR/AR 的表达。通过特定的测定法检查 miR-99b 过表达/抑制对 PCa 细胞存活/增殖、球体形成和细胞迁移的影响。通过荧光素酶报告基因测定法确定 miR-99b 与 mTOR 基因 3'非翻译区（UTR）的结合。通过 Western blot 分析 miR-99b 对 mTOR、AR 和 PSA 蛋白表达以及 AKT/mTOR 信号转导、自噬和神经内分泌分化标志物的影响。通过 RT/qPCR 分析 AR 阴性 PC3 和 AR 阳性 LNCaP 细胞中 miR-99b、mTOR、AR 和 PSA 的表达。通过 RNA-seq 分析 miR-99b 对 PC3 细胞中全局基因表达的影响。
结果
miR-99b 在来自 PCa 患者的肿瘤样本中表达下调，而 mTOR 和 AR 的表达上调。在 PCa 细胞系中，miR-99b 的过表达抑制了细胞增殖和细胞集落/球体形成；诱导细胞凋亡，并增加了对多西他赛（DTX）的敏感性。相比之下，miR-99b 抑制剂抑制 miR-99b 的表达会导致 PCa 细胞中的细胞生长增加。在机制上，miR-99b 通过结合其 3'UTR 抑制哺乳动物雷帕霉素靶蛋白（mTOR）基因的表达并诱导自噬。此外，miR-99b 在 LNCaP 细胞中抑制雄激素受体（AR）活性并诱导细胞凋亡。二氢睾酮（DHT）激活 AR 信号转导下调 miR-99b 的表达并促进 PCa 细胞生长/存活，而雷帕霉素抑制 mTOR 或恩扎卢胺抑制 AR 可降低 miR-99b 介导的 PCa 细胞生长。
结论
我们的数据表明，miR-99b 通过靶向 PCa 细胞中的 mTOR/AR 轴发挥肿瘤抑制作用，提示 miR-99b 作为一种新的生物标志物和治疗靶点用于 PCa 的管理。
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