The putative tumour suppressor miR-1-3p modulates prostate cancer cell aggressiveness by repressing E2F5 and PFTK1

Abstract

Background: Previous studies report that miR-1-3p, a member of the microRNA-1 family (miR-1), and functions as a tumor suppressor in several different cancers. However, little is known regarding the biological role and intrinsic regulatory mechanisms of miR-1-3p in prostate cancer (PCa).

Methods: In this study, the expression levels of miR-1-3p were first examined in PCa cell lines and tumor tissues by RT-qPCR and bioinformatics. The in vitro and in vivo functional effect of miR-1-3p was examined further. A luciferase reporter assay was conducted to confirm target associations.

Results: We found that miR-1-3p was significantly downregulated in advanced PCa tissues and cell lines. Low miR-1-3p levels were strongly associated with aggressive clinicopathological features and poor prognosis in PCa patients. Ectopic expression of miR-1-3p in 22RV1 and LncaP cells was sufficient to prevent tumor cell growth and cell cycle progression in vitro and in vivo. Further mechanistic studies revealed that miR-1-3p could directly target the mRNA 3'- untranslated region (3'- UTR) of two central cell cycle genes, E2F5 and PFTK1, and could suppress their mRNA and protein expression. In addition, knockdown of E2F5 and PFTK1 mimicked the tumor-suppressive effects of miR-1-3p overexpression on PCa progression. Conversely, concomitant knockdown of miR-1-3p and E2F5 and PFTK1 substantially reversed the inhibitory effects of either E2F5 or PFTK1 silencing alone.

Conclusion: These data highlight an important role for miR-1-3p in the regulation of proliferation and cell cycle in the molecular etiology of PCa and indicate the potential for miR-1-3p in applications furthering PCa prognostics and therapeutics.

Keywords: Proliferation; Prostate cancer; Target gene; microRNA.

Fig. 1

Mature miR-1-3p expression levels in prostate cancer cell lines and tissues, and the prognostic value of miR-1-3p levels in patients with PCa. a Expression levels of miR-1-3p, (c) E2F5 and (e) PFTK-1 were examined by RT-qPCR in RWPE-1 cells and two PCa cell lines. GAPDH and U6 served as corresponding loading controls. Error bars represent the mean ± S.D. of three independent experiments (*P < 0.05, **P < 0.01 and ***P < 0.001 compared to RWPE-1 group). b Expression levels of mature miR-1-3p in 25 paired PCa and adjacent non-tumour tissues. Alteration of expression is shown as box plot presentations, with the y axis indicating miR-1-3p expression. The mean level of miR-1-3p expression in PCa tissues were significantly lower than that in non tumor tissues. (***P < 0.001, independent t test). d E2F5 and (f) PFTK-1 protein expression in primary prostate tissues was detected by Immunohistochemically staining assay. Scale bar: 50 μm. g 124 PCa patients from the Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, and (h) 402 PCa patients from the TCGA database. Left panel: X-tile plots automatically selected the cut-off point of miR-1-3p. Right panel: Kaplan–Meier analysis of survival and the COX proportional hazards model for the hazard ratio

Fig. 2

MiR-1-3p attenuates prostate cancer cell lines proliferation through inducing cell cycle arrest at G0/G1 phase. LnCap and 22RV1 cells were transfected with 100 nM indicated RNAs molecules respectively for 72 h. a and b MTS assays revealed cell growth curves of both PCa cells in every 24 h. c Representative micrographs and (d) relative quantification of crystal violet-stained cell colonies analyzed by clonogenic formation. e and f flow cytometric determination of proportion of LnCap and 22RV1 cells in distinct cell cycle phases. g and h Expression of CDK2 and CDK4 mRNA in LnCap and 22RV1 cells were assessed by RT-qPCR following transfection with 100 nm miR-1-3p mimics, miR-1-3p inhibitor, or their negative controls (NC). GAPDH served as a loading control. i Western blot analysis of the relative expression of CDK2 and CDK4 in response to indicated RNAs molecules. GAPDH served as a loading control. The results were plotted as the mean ± S.D. of three independent experiments. (*P < 0.05, **P < 0.01, ***P < 0.001, ^# P < 0.05 and ^## P < 0.01)

Fig. 3

E2F5 and PFTK-1 were identified as direct target genes of miR-1-3p and down-regulated by miR-1-3p in the prostate cancer cell. a and b Schematic diagram of the predicted target binding sites of miR-1-3p in the 3′-UTR of E2F5 and PFTK-1. The seed recognition site is denoted. All nucleotides of the 3′-UTR region of E2F5 and PFTK-1 that binds with miR-1-3p are highly conserved across species as predicated by TargetScan (http://www.targetscan.org/vert_72/). c and d The luciferase activity of the wild type E2F5/ PFTK-1 3’-UTR (Wt) and mutant E2F5/ PFTK-1 3’-UTR (Mut) co-transfected with miR-1-3p mimics or a miRNA negative control (miR-LacZ) was measured in LnCap cells. Relative luciferase activity was plotted as the mean ± S.D. of three independent experiments. e-g Expression of PFTK-1 and E2F5 mRNA in LnCap, 22RV1 and RWPE-1 cells were assessed by RT-qPCR following transfection with miR-1-3p mimics, miR-1-3p inhibitor, or their negative controls (NC). GAPDH served as a loading control. h and i Western blot analysis of the protein levels and relative expression of E2F5 and PFTK-1 in response to 100 nM of indicated RNAs molecules. GAPDH served as a loading control in LnCap and 22RV1 cells. (*P < 0.05, **P < 0.01)

Fig. 4

E2F5 and PFTK1 are involved to promote PCa cell proliferation and cell cycle progression in vitro. LnCap and 22RV1 cells were transfected with siRNAs of E2F5 and PFTK-1 respectively for 72 h; siControl served as negative control. a Representative photographs of colony formation assay and (b) quantification of the cell colonies formation were used to determine the proliferative ability of PCa Cells. c and d MTS assays revealed cell growth curves of indicated cells in every 24 h. e and f Flow cytometric determination of proportion of indicated cells in distinct cell cycle phases. g-j RT-qPCR and Western blot analysis the gene and protein expression of CDK2 and CDK4. LnCap cells were infected by lenti-E2F5 and PFTK1 to overexpress E2F5/PFTK1. Lenti-vector served as negative control. k and l The cell colony formation was used to determine the proliferative ability. m MTS assays revealed cell growth curves in every 24 h. n Western blot analysis the expression of CDK2 and CDK4 indicated cells. The results were plotted as the mean ± SEM of three independent experiments, with at least three replicates in each independent experiment. (*P < 0.05, **P < 0.01; #P < 0.05, ##P < 0.01)

Fig. 5

E2F5 and PFTK1 are a functional targets of miR-1-3p suppression of PCa cells proliferation and cell cycle progression. LnCap cells were co-transfected with miR-1-3p inhibitor and siRNA of E2F5 and PFTK-1 respectively for 72 h, inhibitor-NC and siControl served as respective negative control. a The effect of concomitant knockdown of miR-1-3p and E2F5 or PFTK1 on LnCap cells growth rates as measured by MTS assays. b and c Western blot analysis of E2F5 and PFTK1 protein levels in indicated cells. GAPDH was used as the loading control. d The effect of concomitant knockdown of miR-1-3p and E2F5 or PFTK1 on LnCap cells proliferative ability as determined by colony formation assays. e-f The proportion of indicated cells in distinct cell cycle phases as identified by FACS. Results were plotted as the mean ± SEM of three independent experiments, with at least three replicates in each independent experiment. (*P < 0.05, **P < 0.01)

Fig. 6

MiR-1-3p inhibits PCa tumor xenograft growth in vivo. a Representative photograph of tumor formation and (b) tumors excised 40 days after inoculation of stably transfected cells into nude mice. c Tumor volume was measured using a Vernier caliper on the indicated days. d Tumor weight in the miR-NC and miR-1-3p treated groups. e The relative expression of miR-1-3p in xenograft tumor tissue were identifed by RT-qPCR. f Immunohistochemical analysis of Ki-67,E2F5 and PFTK1 in xenografts tumors of miR-NC and miR-1-3p treated groups. Scale bar: 100 μm. (*P < 0.05, **P < 0.01)