Regulation and Methylation of Tumor Suppressor MiR-124 by Androgen Receptor in Prostate Cancer Cells

<div><p>Prostate cancer (PCa) is the most frequently diagnosed cancer for men in the developed world. Androgen receptor signaling pathway plays an important role in prostate cancer progression. Recent studies show that microRNA miR-124 exerts a tumor suppressive function in prostate cancer. However, the relationship between AR and miR-124 is unclear. In the present study, we found a negative feedback loop between AR and miR-124 expression. On one hand, miR-124 was a positively regulated target gene of the AR, on the other hand, overexpression of miR-124 inhibited the expression of AR. In addition, we found that miR-124-2 and miR-124-3 promoters were hypermethylated in AR-negative PCa cells. Furthermore, overexpression of miR-124 inhibited proliferation rates and invasiveness capacity of PCa cells <i>in vitro</i>, and suppressed xenograft tumor growth <i>in vivo</i>. Taken together, our results support a negative feedback loop between AR and miR-124 expression. Methylation of miR-124-2 and miR-124-3 may serve as a biomarker for AR-negative PCa cells, and overexpression of miR-124 might be of potential therapeutic value for the treatment of PCa.</p></div

Methylation Status of MiR-124-1, MiR-124-2 and MiR-124-3 CpG Islands.

<p>Schematic summary of CpG sites in the miR-124-1, miR-124-2 and miR-124-3 promoter regions. (A) Methylation analysis was done in 10 clones from each cell line. Each row of circles represents a single clone, and each circle represents a single DNA methylated or demethylated site. (B) The methylation percentages of 10 clones from each of the cell lines are summarized in the bar graph. Data are shown as the means ± SEM. Asterisks indicate P<0.05, double asterisks indicate P<0.001.</p

A Negative Feedback Loop Between MiR124 and AR Expression.

<p>(A) PC3/AR cells are a stable cell line overexpressing human AR cDNA; PC3/neo cells are used as a control. (B) LNCaP-sh-AR cells are AR-knockdown cells, in which LNCaP cells were infected with lentivious AR shRNA; LNCaP-sh-control cells are used as a control. (C) 0 nM, 1 nM and 10 nM DHT were added to LNCaP cells and cultured for 12 hour. (D) LNCaP cells were infected with a control lentivirus (LNCaP-control cells) or plenti-CMV-mir-124 lentivirus (LNCaP-miR-124 cells). Relative expression of miR-124 in LNCaP cells was determined by qRT-PCR and corrected to RUN44 levels. Values mean fold-changes normalized to (A) PC3/neo cells; (B) LNCaP-sh-AR cell; (C) LNCaP cells (0 nM DHT) and (D) LNCaP-control cells. (E) Relative expression of AR was determined by qRT-PCR and normalized to β-actin levels. Values mean fold-changes normalized to LNCaP-control cells. (F) A schematic diagram of the pathway described in the study. Data are shown as the means ± SEM from 3 independent experiments, each of which were performed in triplicates</p

Overexpression of MiR-124 Suppresses LNCaP Cells Proliferation, Migration and Xenograft Tumors Growth.

<p>(A) LNCaP-control cells and LNCaP-miR-124 cells were plated in 96 wells and examined at 48, 72, 96 hours using CCK8 assay. Cell proliferation was assayed by the value of OD. Data are shown as the means ± SEM (n = 12). Group data (B) and representative images (C,D) are shown. (B) Number of migratory cells per field of LNCaP-control cells and LNCaP-miR-124 cells. Representative photomicrographs illustrating migration of LNCaP-control cells (C) and LNCaP-miR-124 cells (D). Five nude mice per group were injected subcutaneously with 4x10⁶ LNCaP-control cells or LNCaP-miR-124 cells. (E) The volume of xenograft tumors were measured every 10 days from 2 weeks after inoculation. (F) Tumor weights were measured at the time of sacrifice on 34 day after tumor cell injection. (G) Tumors were excised at the time of sacrifice on 34 day post tumor cell injection. Data are presented as means ± SEM. Asterisks indicate P<0.05, double asterisks indicate P<0.001.</p