The antiapoptotic function of miR-96 in prostate cancer by inhibition of FOXO1

Abstract

microRNAs (miRNAs) are small molecules that regulate gene expression posttranscriptionally. In a previous study, we identified miR-96 to be upregulated in prostate cancer specimens in comparison to normal adjacent tissue and to be an independent marker of biochemical relapse in a multivariate prediction model. Therefore, we investigated the functional role of miR-96 in prostate carcinogenesis. LNCaP and DU145 prostate cancer cells were transiently transfected with miR-96 precursors and phenotypic changes were analyzed. The miR-96 increased proliferation and impaired apoptosis induced by camptothecine in these cells. In silico target prediction analysis identified FOXO1 as potential pro-apoptotic miR-96 target. miR-96 was able to bind to both bindings sites in the FOXO1 3' UTR in a luciferase reporter gene assay. Overexpression of miR-96 in LNCaP cells resulted in a reduced FOXO1 expression. Overexpression of FOXO1 induced a strong apoptotic phenotype that was partially rescued by coexpression of miR-96. RT-qPCR and immunohistochemistry of 69 prostate cancer specimens revealed a downregulation of FOXO1 and an inverse correlation of miR-96 and FOXO1 protein expression. In conclusion, we show that miR-96 can regulate apoptosis in prostate cancer, by inhibiting the FOXO1 transcription factor.

Figure 1. Functional role of miR-96 in vitro.

LNCaP and DU145 cells were transfected with 10 nM pre-miR-96, anti-miR-96, pre-miR-NC #1 or combination of pre-miR-96 and anti-miR-96. The effect in (A) LNCaP and (B) DU145 on cell proliferation under serum starvation was measured by MTT assay over three days. All data were normalized on proliferation of control cultures on day 1 and are shown as mean (±SD) of three independent assays. *P <0.05, Two-way ANOVA. Cell cycle transition was measured by PI staining in transfected (C) LNCaP and (D) DU145 cells. Cells were serum starved one day after transfection for another 24 hrs and subsequently fixed and stained. Black, G1-phase; light grey, S-phase; dark grey, G2/M-phase. Data are shown as mean of three independent assays. Apoptosis in CPT-treated (E) LNCaP and (F) DU145 cells. 24 h after transfection cells were treated with 10 µM CPT for 24 h. Cells were stained with Annexin V-FITC and PI. Fraction of early (black bars) and late (white bars) apoptotic cells was measured by flow cytometry. Data are shown as mean (+SD) of three independent assays. *P <0.05; Bonferroni post-test (P < 0.001, Two-way ANOVA).

Figure 2. Binding of miR-96 in the 3’ UTR of FOXO1.

LNCaP cells were transfected with 10 nM pre-miR-96, anti-miR-96, pre-miR-NC #1 or combination of pre-miR-96 and anti-miR-96 as well as 500 ng pMiR report β-Galactosidase control plasmid and 500 ng pMiR report plasmid for FOXO1 3’ UTR A) binding site 1 at position 264-270 and B) binding site 2 at position 2138-2145. * P <0.05, **P <0.01, One-way ANOVA and Tukey’s multiple comparison post test.

Figure 3. Effect of miR-96 overexpression on FOXO1 expression.

Prostate cancer cells were transfected with 10 nM pre-miR-96, anti-miR-96, pre-miR-NC #1 or combination of pre-miR-96 and anti-miR-96. miR-96 expression in (A) LNCaP and (B) DU145 cells and FOXO1 expression in (C) LNCaP and (D) DU145 cells. Data are shown as mean (+SD) of three independent assays. ** P <0.01, *** P <0.001, One-way ANOVA. FOXO1, AKT, and pAKT protein expression in (E) LNCaP and (F) DU145 cells was visualized by western blotting. β-Actin was used as a loading control.

Figure 4. FOXO1 overexpression in prostate cancer cell lines.

LNCaP and DU145 cells were transfected with 500 ng FOXO1 full length clone or empty vector, 10 nM pre-miR-96 or pre-miR-NC #1. FOXO1 expression in (A) LNCaP and (B) DU145 cells. Data are shown as mean (+SD) of three independent assays. *** P <0.001, One-way ANOVA. FOXO1 protein expression in (C) LNCaP and (D) DU145 cells was visualized by western blotting. β-Actin was used as a loading control. Apoptosis in CPT-treated (E) LNCaP and (H) DU145 cells. 24 h after transfection cells were treated with 10 µM CPT for 24 h. Cells were stained with Annexin V-FITC and PI. Fraction of early (black bars) and late (white bars) apoptotic cells was measured by flow cytometry. Data are shown as mean (+SD) of three independent assays. ns, P>0.05,*P <0.05,**P<0.01,***P<0.001, Two-way ANOVA, Bonferroni’s post test). Cell cycle transition was measured by PI staining in transfected (F) LNCaP and (I) DU145 cells. Cells were serum starved one day after transfection for another 24 hrs and subsequently fixed and stained. Black, G1-phase; light grey, S-phase; dark grey, G2/M-phase. Data are shown as mean of three independent assays. ***P<0.001, One-way ANOVA. Analysis of subG1 peak in (G) LNCaP and (J) DU145 cells. Data are shown as mean of three independent assays. ***P<0.001, One-way ANOVA.

Figure 5. miR-96 expression and FOXO1 expression in PCa specimens.

(A) miR-96 and FOXO1 mRNA expression was measured by RT-qPCR in 69 matched PCa (PC) and normal adjacent tissue (PN). All data were normalized to efficiency and interplate control. FOXO1 expression was normalized to the reference gene TUBA1B [19] and miR-96 expression was normalized to miR-130b [6]. Data are shown as median expression (+ interquartile range). FOXO1 expression is displayed on the left y-axis, miR-96 expression on the right y-axis. ***P <0.001; Wilcoxon signed rank test. (B) Haematoxilin-eosin (H/E) staining was performed to identify tumor ducts. (C+D) FOXO1 expression was detected by immunhistochemistry on a tissue microarray containing tissue cores corresponding to 69 PCa specimen. Each core had a diameter of 1.5 mm.