MicroRNA-99b-5p targets mTOR/AR axis, induces autophagy and inhibits prostate cancer cell proliferation

Abstract

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.

Downregulation of miR-99b-5p and Upregulation of Nuclear mTOR Cooperatively Promotes the Tumor Aggressiveness and Drug Resistance in African American Prostate Cancer

“…Previous studies have shown that the tumor suppressive miR-99b-5p is downregulated in various cancers including PCa [ 15 , 29 , 30 , 31 , 32 ]. In contrast, mTOR is upregulated in a variety of cancer types [ 15 , 16 , 33 ].…”

“…Overexpression of miR-99b-5p has been reported to negatively regulate the protein expression of mTOR, AR and prostate specific antigen (PSA), consequently inhibiting the cell proliferation, migration, inducing autophagy, promoting apoptosis, and sensitizing the docetaxel-induced cytotoxicity in PCa [ 32 ]. AR has also been implicated in the coordination of protein complex diversity and subcellular localization of mTOR, including regulating the translocation of mTOR to the nucleus and the mTOR-mediated gene networks [ 34 , 38 ].…”

High miR-99b expression is associated with cell proliferation and worse patient outcomes in breast cancer

“…miR-99b inhibits mechanistic target of rapamycin (mTOR) signaling, which regulates gene transcription and protein synthesis involved in cell proliferation, immune cell differentiation, and tumor metabolism [8]. It inhibits prostate cancer cell growth via autophagy induction [9,10]. miR-99b also negatively regulates insulin-like growth factor (IGF)-1 receptor expression, and in turn suppresses gastric cancer growth [11].…”

Role of Nivolumab in the Modulation of PD-1 and PD-L1 Expression in Papillary and Clear Cell Renal Carcinoma (RCC)

“…However, still no evidence indicates a relationship between AR and spheroid formation. Nevertheless, Niture et al [ 46 ] described an increased ability of prostate cancer cells to create spheroids after microRNA-99b-5p silencing, which was accompanied by the upregulation of the AR axis. However, the relationship between AR expression and spheroid formation remains open.…”