MicroRNA-214 targets PTK6 to inhibit tumorigenic potential and increase drug sensitivity of prostate cancer cells

Abstract

Prostate cancer is the most commonly diagnosed cancer in men with African American men disproportionally suffering from the burden of this disease. Biomarkers that could discriminate indolent from aggressive and drug resistance disease are lacking. MicroRNAs are small non-coding RNAs that affect numerous physiological and pathological processes, including cancer development and have been suggested as biomarkers and therapeutic targets. In the present study, we investigated the role of miR-214 on prostate cancer cell survival/migration/invasion, cell cycle regulation, and apoptosis. miR-214 was differentially expressed between Caucasian and African American prostate cancer cells. Importantly, miR-214 overexpression in prostate cancer cells induced apoptosis, inhibiting cell proliferation and colony forming ability. miR-214 expression in prostate cancer cells also inhibited cell migration and 3D spheroid invasion. Mechanistically, miR-214 inhibited prostate cancer cell proliferation by targeting protein tyrosine kinase 6 (PTK6). Restoration of PTK6 expression attenuated the inhibitory effect of miR-214 on cell proliferation. Moreover, simultaneous inhibition of PTK6 by ibrutinib and miR-214 significantly reduced cell proliferation/survival. Our data indicates that miR-214 could act as a tumor suppressor in prostate cancer and could potentially be utilized as a biomarker and therapeutic target.

Figure 1

Effects of miR-214 on cell proliferation and colony formation in prostate cancer cells. (A) RWPE-2, PC3, DU145, MDA-PCa-2b, and LNCaP cells were cultured for 30 h, and the relative endogenous expression of miR-214 was analyzed by RT/qPCR. (B) RWPE-2, PC3, DU145, MDA-PCa-2b cells, and LNCaP were transfected with negative control (NC) mimic miRNA or miR-214 mimic for 48 h. miR-214 expression levels were then determined by RT/qPCR. (C) RWPE-2, PC3, DU145, MDA-PCa-2b, and LNCaP cells were transfected with NC or miR-214 mimic. After 48 h, the effect of the miR-214 expression on cell viability was determined by MTT assay. (D) The time-dependent effects of miR-214 expression on cell viability were determined by MTT assay. (E) The effect of NC or miR-214 mimic on cell colony formation was determined by colony formation assay (left). The number of colonies were measured by ImageJ software. The data represent the colony count ± SEM (right). One-way and two-way ANOVA followed by Tukey’s multiple comparisons test were used to determine statistical significance for RT/qPCR and MTT assay, respectively. Results are representative of three independent experiments. *P < 0.05, **P < 0.005, ***P < 0.0005 compared with NC-transfected cells.

Figure 2

Effects of miR-214 overexpression on cell cycle progression and apoptosis. (A) RWPE-2, PC3, DU145, and MDA-PCa-2b cells were transfected with NC or miR-214 mimic. The percentages of cells in the G₀/G₁, S, and G₂/M cell cycle phases for each cell line are shown. The red numbers indicate a significant increase in cell cycle phase in the miR-214-transfected cells compared to NC-transfected cells. B) RWPE-2, PC3, DU145, and MDA-PCa-2b cells were transfected with NC or miR-214 mimic with and without 1 nM docetaxel (DTX). Flow cytometry was used to determine the percentages of Annexin V-FITC+ cells (right lower quadrant, early apoptosis) and Annexin V-FITC+/PI+ cells (right upper quadrant, late apoptosis). Representative plots (left) and the corresponding quantification (right) are shown. C) RWPE-2, PC3, DU145, and MDA-PCa-2b cells were transfected with NC or miR-214 mimic. Cells were also treated with DTX or doxorubicin (DOX). The effects of miR-214 mimic alone or in combination with the drugs above on C-PARP and GAPDH expression were analyzed by western blotting. One-way ANOVA with Tukey’s multiple comparisons test was used to determine statistical significance. The data represent mean ± SEM and results are representative of three independent experiments *P < 0.05, ***P < 0.0005.

Figure 3

Effects of miR-214 overexpression on prostate cancer cell migration and invasion. (A) Wound healing assay: RWPE-2, PC3, DU145, and MDA-PCa-2b cells were transfected with NC or miR-214 mimic for 48 h. The effect of the miR-214 expression on cell migration was analyzed using the wound healing assay. Representative images of the wound healing assay (top) and the calculated scratch area (bottom) are shown. (B) 3D spheroid invasion assay: RWPE-2, PC3, DU145, and MDA-PCa-2b cells were transfected with NC or miR-214 mimic and then analyzed using the 3D spheroid assay. 3D spheroid images (top) and the quantified 3D invasion area (bottom) were determined by subtracting pre-invasion (day 3) from post-invasion (day 10) area. (C) Trans-well invasion assay: PC3 cells were transfected with NC or miR-214 mimic. After 48 h, the effect of miR-214 mimic on cell invasion was analyzed by trans-well invasion assay in miR-214-transfected PC3 cells stained with Calcein-AM (Green fluorescence, top) and quantified (bottom). The invasion assay was performed in duplicate, and results are representative of three independent experiments. (D,E) The effects of NC or miR-214 mimic transfection on the expression of E-Cadherin (top, green) and N-Cadherin (bottom, green) in PC3 cells were analyzed by (D) immunofluorescence and (E) western blotting. *P < 0.05, **P < 0.005, ***P < 0.0005 compared with NC-transfected cells.

Figure 4

miR-214 targets PTK6 and inhibits its expression. (A) The binding sites of miR-214 in the 3′UTR region of PTK6 were analyzed by TargetScan (http://www.targetscan.org/vert_72/) and presented (top). We mutated the binding nucleotides of miR-214 to the 3′UTR region of PTK6 to generate a mutant miR-214 mimic. Constructs of the wild-type and mutant miR-214 mimics are shown (bottom). (B) Luciferase reporter assay: PC3 cells were first transfected with one µg of PTK6-3′UTR-Luciferase reporter construct for 18 h and then transfected with NC, wild-type miR-214, or mutant miR-214 mimic for an additional 24 h. Transfected cells were lysed, and luciferase activity was then measured. **P < 0.005 compared with NC or wild-type miR-214 mimic. (C) RWPE-2, PC3, DU145, and MDA-PCa-2b cells were transfected with NC or miR-214 mimic. After 48 h, relative PTK6 expression was analyzed by RT/qPCR. ***P < 0.0005 compared with NC-transfected cells. (D) RWPE-2, PC3, DU145, and MDA-PCa-2b cells were transfected with NC or miR-214 mimic. After 48 h, PTK6 protein levels were analyzed by western blotting (top) and quantified using ImageJ (bottom). ***P < 0.0005 compared with NC-transfected cells. (E) PC3 cells were transfected with NC or miR-214 mimic for 48 h, and then PTK6 expression (green) was visualized by immunofluorescence as described in the materials and methods section. (F) RWPE-2, PC3, DU145, and MDA-PCa-2b cells were transfected with PTK6 plasmid or empty vector plasmid, and expression of PTK6 protein levels were analyzed by western blotting (upper panel). Effect of PTK6 overexpression on cell viability of prostate cancer cells was measured by MTT assay and plotted (lower panel). The data represent mean ± SEM. One-way ANOVA with Tukey’s multiple comparisons test was used to determine statistical significance for RT/qPCR and western blot analysis.

Figure 5

Effects of PTK6 overexpression on prostate cancer cell proliferation and colony formation. (A) Western blot analysis: PC3, MDA-PCa-2b, and LNCaP cells were co-transfected with empty vector (EV) or PTK6 plasmids as indicated for the first 24 h and then transfected with NC or miR-214 mimic for an additional 24 h. Cell lysates (40 µg) were immunoblotted with anti-PTK6 and anti-GAPDH antibodies. (B) MTT assay: PC3, MDA-PCa-2b, and LNCaP cells were transfected as indicated in (A) and after 48 h, cell proliferation was analyzed by MTT assay. (C) Cell colony formation assay: PC3, MDA-PCa-2b, and LNCaP cells were transfected with EV or PTK6 plasmids for 24 h and then transfected NC or miR-214 mimic for an additional 24 h. Cells were then trypsinized, counted, and re-plated in 6-well plates for analysis using colony formation assays. The data represent mean ± SEM. One-way ANOVA followed by Tukey’s multiple comparisons test was used to determine statistical significance for the cell viability and colony formation assays. Results are representative of three independent experiments. *P < 0.05, **P < 0.005, ***P < 0.0005 compared with NC mimic or PTK6 transfected cells.

Figure 6

Effects of ibrutinib on prostate cancer cell proliferation. (A) Ibrutinib inactivates PTK6 activity in vitro (as indicated in red). (B) RWPE-2, PC3, DU145, MDA-PCa-2b, and LNCaP cells were treated with increasing concentrations of ibrutinib for 48 h, and then cell proliferation was measured by MTT assay. *P < 0.05, **P < 0.005, ***P < 0.0005 compared with vehicle-treated cells. (C) RWPE-2, PC3, DU145, MDA-PCa-2b, and LNCaP cells were transfected with NC or miR-214 mimic for 24 h and then treated with increasing concentrations of ibrutinib for an additional 24 h. Cell survival was measured by MTT assay. *P < 0.05, **P < 0.005, ***P < 0.0005 compared with NC/miR-214-transfected and ibrutinib-treated cells. One-way ANOVA and two-way ANOVA, followed by Tukey’s multiple comparisons test were used to determine statistical significance. Results are representative of three independent experiments.