An androgen-regulated miRNA suppresses Bak1 expression and induces androgen-independent growth of prostate cancer cells

Abstract

Although prostate cancer (CaP) is the most frequently diagnosed malignant tumor and the second leading cause of cancer deaths in American men, the mechanisms explaining the development and progression of CaP remain largely unknown. Recent studies have shown that some aberrantly expressed microRNAs (miRNAs) are involved in tumorigenesis. Although aberrant expression of certain miRNAs has been discovered in CaP, their function in this disease has not yet been defined. In this study, we found differential expression of miR-125b in androgen-dependent and independent CaP cells, as well as in benign and malignant prostate tissues. Furthermore, androgen signaling was able to up-regulate the expression of miR-125b. In addition, transfection of synthetic miR-125b stimulated androgen-independent growth of CaP cells and down-regulated the expression of Bak1. Our results suggest that miR-125b acts as an oncogene, contributing to the pathogenesis of CaP.

Fig. 1.

Northern blot analyses of miRNA expression in LNCaP line and androgen-independent cds lines. Two miRNAs were down-regulated (A) and five were upreregulated (B) in cds lines compared with their parental LNCaP line.

Fig. 2.

In situ hybridization detection of miR-125b abundance in CaP samples. Two human CaP samples (B and D) with Gleason score 7 and one benign prostate tissue (F) were stained by using the DIG-LNA-miR-125b probe. According to the intensity of staining, the miR-125b abundance was categorized as high (B), moderate (D), or low level (F). These tissues were also stained with hematoxylin and eosin (A, C, and E).

Fig. 3.

In situ hybridization analyses of miR-125b expression in prostatic cell lines. Cds1 (A), LNCaP (B), and pRNS-1-1-AR^WT cells (C) were hybridized by using a Fitc-LNA-miR-125b probe in the absence (Upper) or the presence (Lower) of the synthetic androgen R1881. The fluorescent signals are cytoplasmic in location (green). The nuclei were stained by using DAPI (blue). The experiment was performed twice with similar results.

Fig. 4.

ChIP analysis of the AR loading to the 5′ DNA region of miR-125b. (A) Schematic representation of miR-125b-2 and its 5′ DNA region. A TATA box (TATAAA) is located at −172 upstream of miR-125b-2. ●, Potential ARE clusters (labeled A–D). The arrows indicate the locations of primers used to amplify the −558/−368 fragment in cluster A and the −2666/−2388 fragment in cluster C. (B) PCR amplification of the 5′ DNA of miR-125b-2. IgG- or anti-AR-precipitated chromatin DNA from PC3, DU145, and R1881- or estradiol (E2)-treated LNCaP cells was amplified by using the primers indicated in Fig. 4A. Input is the control reaction of genomic DNA. The numbers under the gels are the fold changes of the −558/−368 fragment (Top) and the −2666/−2388 fragment (Middle Upper) relative to untreated LNCaP cells (Untreat.). Fold changes were calculated by scanning the bands of PCR products and normalizing for input.

Fig. 5.

Effects of miR-125b on the growth of LNCaP and cds1 cells. (A) WST-1 analysis of the growth of LNCaP cells that were transfected with synthetic premiR-125b (miR-125bm) or miR-NC. (B) WST-1 analysis of the growth of cds1 cells that were transfected with synthetic anti-miR-125b or anti-miR-NC. In both A and B, the experiments were repeated at least three times with similar results obtained each time. The representative results are shown as M ± SD (n = 3). No transfected cells (medium) and transfection reagent-treated cell (lip. 2000) are additional controls. The bars represent SDs. (C) Morphology of LNCaP cells after transfection with miR-125bm or miR-NC. (D) Morphology of cds1 cells after transfection with anti-miR-125b or anti-miR-NC. (E) Flow cytometry assay of S-phase fraction in miR-125bm-treated LNCaP cells (filled) and anti-miR-125b-treated cds1 cells (open). The assay was repeated three times, and similar results were obtained. The representative results are shown as M ± SD (n = 3).

Fig. 6.

MiR-125b regulates Bak1. (A) RT-PCR analysis of BAK1 mRNA in miR-125bm-treated LNCaP cells. RAS was used as a loading control. (B) Western blot analysis of Bak1 expression in miR-125bm-treated LNCaP cells (Upper) and cds1 cells (Lower). (C) Western blot analysis of Bak1 expression in LNCaP cells that had been transfected with anti-miR-125b (anti-125b). (D) Western blot analysis of Bak1 in LNCaP cells that were treated with 1.0 nM R1881 for 48 h. In B, C, and D, β-actin is a loading control, and the numbers are percentages relative to untransfected cells (B and C, no treat.), calculated by scanning the Bak1 bands followed by normalization for β-actin. (E) Luciferase analysis in DU145 cells. 1, DU145 cell; 2, luciferase vector (LUC-V); 3, LUC-V plus 50 nM miR-125bm; 4, BAK1 3′ UTR antisense LUC-V (LUC-V-AS); 5, LUC-V-AS plus 50 nM miR-125bm; 6, BAK1 3′ UTR sense LUC-V (LUC-V-S); 7, LUC-V-S plus 50 nM miR-NC; 8, LUC-V-S plus 25 nM miR-125bm; 9, LUC-V-S plus 50 nM miR-125bm; 10, LUC-V-S plus 100 nM miR-125bm. The percentages represent enzyme activity in miR-125bm-treated LUC-V-S transfectants relative to that in untreated LUC-V-S transfectants. The assay was repeated three times with each assay being performed in four wells, and similar results were obtained each time. The representative results are shown as M ± SD (n = 4). RLU, relative luciferase unit.