miR-125b promotes growth of prostate cancer xenograft tumor through targeting pro-apoptotic genes

Abstract

Background: Increasing evidence demonstrates that aberrantly regulated microRNAs (miRNAs) contribute to the initiation and progression of human cancer. We previously have demonstrated that miR-125b stimulated the growth of prostate cancer (CaP) cells. In this study, we further determined the influence of miR-125b on the pathogenesis of CaP.

Methods: To evaluate the effect of miR-125b on xenograft tumor growth, male athymic mice were subcutaneously injected with PC-346C-miR-125b cells that stably overexpressed miR-125b. Potential direct target transcripts of miR-125b were identified using a bioinformatics approach and three miR-125b targeted molecules were confirmed by means of biochemical analyses.

Results: Enforced expression of miR-125b promoted tumor growth in both intact and castrated male nude mice. In an effort to define the molecular mechanism(s) mediating its tumor growth properties, we found that miR-125b directly targets eight transcripts, including three key pro-apoptotic genes: p53, Puma, and Bak1. Increasing the abundance of miR-125b resulted in a dramatic decrease in the levels of these three proteins in CaP cells. A direct repressive effect on each of these was supported by the ability of miR-125b to significantly reduce the activity of luciferase reporters containing their 3'-untranslated regions of each gene encompassing the miR-125b-binding sites. Additionally, we found that repression of miR-125b activity was able to sensitize CaP cells to different therapeutic interventions.

Conclusion: Data obtained in this study demonstrate that miR-125b promotes growth of prostatic xenograft tumors by down-regulating three key pro-apoptotic genes. This suggests that miR-125b is oncogenic and makes it an attractive therapeutic target in CaP.

Fig. 1

miR-125b promotes androgen-dependent and castration-resistant growth in vivo. A) Intact male nude mice (5 per group) each were injected subcutaneously with 2×10⁶ PC-346C-miR-125b cells or PC-346C-vector control cells. Tumors were dissected from miR-125b mice (top) and control mice (bottom) five weeks after inoculation. B) Sixteen intact nude mice each were injected subcutaneously with 2×10⁶ PC-346C-miR-125b cells which overexpress of miR-125b. When their tumor size reached approximately 0.125 cm³ (~0.6 cm in the diameter), eight of these 16 mice were castrated. The arrow indicates the castration time. Eight intact nude mice were injected subcutaneously with 2×10⁶ PC-346C-vector control cells (vector). Each time point represents mean ± SD of 8 independent values.

Fig. 2

Downregulation of miR-125b activity induces apoptosis. A) Annexin V assay of apoptosis. PC-346C cells grown in charcoal-deprived medium were treated with 50 nM anti-miR-125b (anti-125b) or anti-miRNA negative control (anti-NC) for four days and stained with Annexin V and propidium iodide. Both early apoptotic cells (%) and late apoptotic/necrotic cells (%) are combined. Data are means ± SD from 3 independent experiments. Charcoal-deprived medium (medium), transfection reagent lipofectamine 2000 (lipo-2000) and doxorubicin (Doxo) were the controls. B) Wastern blot analysis of SMAC, cytochrome c (CytC) and pro-caspases 3 (proCas3). PC-346C cells were treated with 50 nM anti-miR-125b (anti-125b) or anti-miRNA negative control (anti-NC) for four days. The expression levels of proteins were analyzed with specific antibodies. Significant reduction of the pro-caspase 3 band indicates the activation of caspase-3 (49).

Fig. 3

miR-125b targets pro-apoptotic genes. A) Western blot analyses of the expression levels of p53, Puma and Bak1 in lenti-miR-125b infected PC-346C cells (left panel), and in LNCaP cells grown in 10% FBS medium and in cds2 cells grown in androgen-deprived medium (right panel). Lent-V, lentiviral vector. B) Western blot analysis of the expression of p53, p21, Puma and Bak1 in miR-125bm-treated LNCaP cells. Cells were first transfected with 50 nM of chemically-modified miR-125b mimic (125bm) and 24 hours later irradiated with 10 Gys (IR). Eight hours after irradiation, cells were lysed and protein was extracted for Western blot analysis of p53, p21, Puma and Bak1. The controls include untreated cells (untreat.) and miRNA negative control (miR-NC)-treated cells. C) Detection of the expression levels of p53, Puma and Bak1 in LNCaP cells by immunostaining. LNCaP cells were grown for 24 hours on sterile slides in 100-mm Petri dishes in 10% FBS medium. Cells were first transfected with 50 nM of chemically-modified miR-125bm and 24 hours later irradiated with 10 Gys. Eight hours later, cells were fixed. p53, Puma or Bak1 were stained using specific 1st antibodies followed by a HRP-labeled 2nd antibody. Then, addition of the substrate (DAB) led to generate the brown color. The enzymatic approach shows that p53 locates mainly in nuclei, and Puma and Bak1 in cytoplasm. The white squares indicate the areas of magnified images. D) PC3 cells were transfected with 50 nM of miR-125bm (125bm) and 24 hours later treated with 100 µM resveratrol. Cells were lysed next day and protein was isolated for Western blot analysis of Puma and Bak1. Untreated cells (untreat.) and vehicle-treated cells are used as controls. β-actin is a loading control.

Fig. 4

Effect of androgen on the expression of p53, Puma and Bak1. A) LNCaP cells were treated with 5.0 nM of R1881 or 100 nM anti-miR-125b (anti-125b) for 48 hours. Subsequently cells were harvested for Western blot analysis of p53, Puma and Bak1. B) LNCaP cells were treated with 5.0 nM R1881 for 48 hours followed by 10 Gys of irradiation (IR). Eight hours after irradiation, the cellular levels of p53 and Puma were analyzed by Western blotting. Controls include untreated cells (untreat.) and ethanol vehicle. β-actin is a loading control.

Fig. 5

MiR-125b binds to the 3’UTRs of p53 and PUMA. A) Luciferase analysis of the 3’UTRs of p53 and PUMA in DU145 cells. Individual truncated 3’UTRs of p53 and PUMA were used as controls (no binding site) in these experiments. The assay was repeated three times with each assay being performed in three wells and similar results were obtained each time. The representative results are shown as M ± SD (n = 3). B) Western blot analysis of p53 protein in 5.0nM R1881-treated LNCaP cells and LNCaP-R273H cells.

Fig. 6

Effect of down-regulation of miR-125b on anti-cancer drugs. A) WST-1 assay of growth inhibition. Castration-resistant cds2 cells were grown in androgen-deprived medium and treated for 5 days using GCP (75µg/ml) and anti-miR-125b (anti-125b, 50 nM) in alone or in a combination. The controls include both vehicle and anti-miRNA-negative control (anti-NC). WST-1 analysis was used to measure the cell growth. The results are expressed as the growth inhibition relative to that treated with vehicle (100%), and shown as M ± SD (n=4). B) Annexin V assay of apoptosis. PC-346C cells cultured in medium containing 10% FBS were treated using Cisplatin (Cis, 5 µM) and anti-miR-125b (anti-125b, 50 nM) for two days in alone or in a combination. Cells were harvested and analyzed on the FACScan flow cytometer. Both early apoptotic cells (%) and late apoptotic/necrotic cells (%) are combined. Data are means ± SD from 3 independent experiments.