Inhibition Of Prostate Cancer Proliferation By Androgen Receptor-miR-124 Auto-regulatory Loop Feedback

Objective: Prostate cancer(PCa) is one of the most frequently diagnosed cancers in male urogenital system. Androgen receptor(AR) signaling pathway plays an important role in prostate cancer progression. AR impacts prostate cancer progression through regulating the expression of multiple downstream genes including coding and non coding genes such as microRNA. Recent studies have shown that microRNA-124(miR-124) exerted a tumor suppressive function in PCa. However, the upstream regulator of mi R-124 and the relevant molecular mechanisms are unclear. In this study, based on the former researches, we attempt to explore the inhibitory mechanisms of PCa proliferation through AR-mi R-124 interaction.Methods: In AR- PCa cell line PC3, AR restoration subclone cell line PC3/AR and its control PC3/neo were constructed. In AR+ PCa cell line LNCaP, AR knock down cell line LNCaP-sh-AR and its control LNCaP-sh-control were obtained by lentivirus infection, which includes AR targeted shRNA or control respectively. Also in LNCaP, stable overexpression of mi R-124 cell line LNCaP-mi R-124 and its control LNCaP-control were obtained by lentivirus infection, which includes mature miR-124 or control respectively. The expression levels of miR-124 and AR were detected by real-time quantitative reverse transcriptional polymerase chain reaction(qRT-PCR) in relevant cell lines respectively to evaluate the auto-regulatory effect of these two genes. Androgen-responsive assay was carried out to analyse whether miR-124 expression was responsed to androgen. The tumor suppressive function of miR-124 was evaluated by CCK-8 cell proliferation assay, transwell assay in vitro and subcutaneous xenograft model in nude mice in vivo. The methylation status of pri-miR-124-3 promoter was determined by Bisulfite-Sequencing PCR(BSP). Statistical analysis and graphes were done using Prism GraphPad5 software. Differences between groups were analyzed using the student’s t test. P < 0.05 is considered to be statistically significant.Results: In this study, we found an auto-regulatory loop feedback between AR and miR-124 expression. AR activation promoted miR-124 expression. Meanwhile, miR-124 inhibited the expression of AR at post-transcriptional level. Therefore, AR and miR-124 expression are homeostatic under physiological conditions. But in PCa, miR-124 expression is repressed and the homeostasis is interfered due to the hypermethylation in pri-miR-124-3 promoter and repression of AR-miR-124 interaction. Ectopic miR-124 overexpression inhibits proliferation and tumorigenesis of PCa both in vitro and in vivo.Conclusion: The AR-miR-124 auto-regulatory loop feedback is abrogated by hypermethylation in pri-miR-124-3 promoter. Restoration of mi R-124 expression inhibits PCa proliferation.