The POU2F1/miR-4490/USP22 axis regulates cell proliferation and metastasis in gastric cancer

Abstract

Purpose: Growing evidence indicates that aberrant expression of microRNAs contributes to tumor development. However, the biological role of microRNA-4490 (miR-4490) in gastric cancer (GC) remains to be clarified.

Methods: To explore the function of miR-4490 in GC, we performed colony formation, EdU incorporation, qRT-PCR, Western blotting, in situ hybridization (ISH), immunohistochemistry (IHC), flow cytometry, ChIP and dual-luciferase reporter assays. In addition, the growth, migration and invasion capacities of GC cells were evaluated.

Results: We found that miR-4490 was significantly downregulated in primary GC samples and in GC-derived cell lines compared with normal controls, and that this expression level was negatively correlated with GC malignancy. Exogenous miR-4490 expression not only reduced cell cycle progression and proliferation, but also significantly inhibited GC cell migration, invasion and epithelial-mesenchymal transition (EMT) in vitro. Mechanistically, we found that miR-4490 directly targets USP22, which mediates inhibition of GC cell proliferation and EMT-induced metastasis in vitro and in vivo. Moreover, we found through luciferase and ChIP assays that transcription factor POU2F1 can directly bind to POU2F1 binding sites within the miR-4490 and USP22 promoters and, by doing so, modulate their transcription. Spearman's correlation analysis revealed a positive correlation between USP22 and POU2F1 expression and negative correlations between miR-4490 and USP22 as well as miR-4490 and POU2F1 expression in primary GC tissues.

Conclusion: Based on our results we conclude that miR-4490 acts as a tumor suppressor, and that the POU2F1/miR-4490/USP22 axis plays an important role in the regulation of growth, invasion and EMT of GC cells.

Keywords: Epithelial-mesenchymal transition; Gastric cancer; Metastasis; MiR-4490; POU2F1.

Fig. 1

MiR-4490 is downregulated in GC cells and tissues. (a) Expression of miR-4490 in human GC-derived cells compared with human normal gastric epithelial GES-1 cells. **, p < 0.05. (b) Relative expression of miR-4490 measured using qRT-PCR in 70 pairs of GC samples and corresponding non-cancerous samples; the results were normalized to endogenous U6 RNA. **, p < 0.05. (c) Expression of miR-4490 in paired GC tissues and matched normal tissues. (d) Expression of miR-4490 in different clinical GC stages. The patients were staged in accordance with the 7th Edition of the AJCC Cancer Tumor Node Metastasis Classification. **, p < 0.05. stages I-II vs. stages III-IV. (e) The miR-4490 expression level is lower in T3–4 than in T1–2 GC samples. **, p < 0.05. (f) ISH analysis of miR-4490 expression in normal gastric mucosa and GC tissues. Data are shown as the means ± SD from at least three independent experiments. Scale bars, 50 μm in F

Fig. 2

MiR-4490 suppresses GC cell growth in vitro. (a) Representation (left) and quantification (right) of colony formation assay showing the growth of GC cells transfected with NC or miR-4490 mimics or inhibitors. ****, p < 0.001. (b) The proliferation of miR-4490 mimic-transfected GC cells is lower compared with the corresponding m-NC-transfected cells, and downregulation of miR-4490 reverses this effect. ***, p < 0.01; ****, p < 0.001. (c) The EdU-positive cell rate is decreased after miR-4490 mimic transfection, whereas miR-4490 inhibitor-treated GC cells show a higher proliferation rate. ****, p < 0.001. (d) Flow cytometric analysis of the effect of miR-4490 on GC cell cycle progression; the percentages in G0-G1, S and G2-M phase GC cells were quantified. The figures are representative of three independent experiments. (e) Protein expression detected by Western blotting. Scale bars, 100 μm in C

Fig. 3

MiR-4490 suppresses GC cell migration/invasion via EMT in vitro. (a) Scratch wound healing assays were used to detect GC cell motility following transfection with NC and miR-4490 mimics or a miR-4490 inhibitor. Wound closure percentages are shown in the top panel. ****, p < 0.001. (b) Representation and quantification of matrigel assay indicating the invasive capacity of GC cells transfected with NC, miR-4490 mimics, or a miR-4490 inhibitor. ****, p < 0.001. Error bars represent the means ± SD from three independent experiments. (c) GC cell morphology observed under an inverted phase-contrast microscope. (d) Expression levels of E-cadherin, γ-cadherin, vimentin, ZEB1, N-cadherin and MMP2 in GC cells analyzed by Western blotting. GAPDH was used as a loading control. (e) Immunofluorescence analysis of E-cadherin and vimentin expression in human GC cells. Scale bars, 200 μm in C and 20 μm in E

Fig. 4

USP22 is a direct downstream target of miR-4490 in GC cells. (a) miRmap, miRWalk, miRanda and TargetScan databases were used to predict miR-4490 targets; USP22 was identified as a potential target. (b) Putative miR-4490-binding sequence within the 3′UTR of USP22. Mutations in the complementary site for the seed region of miR-4490 in the 3′UTR of USP22 are indicated. (c) Reporter plasmids containing either the wild-type 3′UTR or a mutated 3′UTR of the USP22 gene were co-transfected into GC cells with a miR-4490-encoding plasmid, after which luciferase activity was measured. WT, wild type; MUT, mutated. *, p > 0.05; ****, p < 0.001. The data are presented as the means ± SD of three experiments. (d) USP22 expression in m-NC and miR-4490 mimic-transfected or i-NC and miR-4490 inhibitor-treated GC cells analyzed by Western blotting. GAPDH was used as a loading control. (e) Representative micrographs of crystal violet-stained cell colonies formed by the indicated GC cells at 12 days after seeding. (f) Assessment of the invasive activity of GC cells transfected with a USP22 expression plasmid and/or miR-4490 mimics. (g) USP22, ERK1/2, p-ERK1/2, Akt, and p-Akt protein expression in GC cells transfected with miR-4490 mimic, inhibitor or NC plasmid. (h) Expression levels of USP22, E-cadherin and vimentin detected by Western blotting in GC cells transfected with USP22 expression plasmid, miR-4490 mimic and/or its control plasmid. The figures are representative of three independent experiments

Fig. 5

MiR-4490 suppresses USP22-mediated GC cell growth and invasion in vivo. (a) Representative images of tumors in nude mice after inoculation of BGC-823 cells stably expressing LV-m-NC, LV-miR-4490, LV-vector, LV-USP22 and LV-4490/USP22 (n = 3). (b) Tumor volumes measured after GC cell inoculation in each group. ****, p < 0.001, m-NC vs. miR-4490; ****, p < 0.001, miR-4490 vs. miR-4490/USP22. (c) and (d) Immunohistochemical (IHC) detection and quantification of Ki-67 expression in subcutaneous tumors from mice injected with GC cells. Student’s t test. ****, p < 0.001, m-NC vs. miR-4490; ***, p < 0.01, miR-4490 vs. miR-4490/USP22 and Ki-67. (e) Lungs of mice orthotopically transplanted with GC cells (n = 3 in each group). (f) Quantification of the number of metastatic loci in the lungs. ***, p < 0.01, m-NC vs. miR-4490; **, p < 0.05, miR-4490 vs. miR-4490/USP22. (g) E-cadherin expression in tumors derived from GC cells measured by qRT-PCR; **, p < 0.05, m-NC vs. miR-4490 and miR-4490 vs. miR-4490/USP22. (h) Immunohistochemical (IHC) detection of E-cadherin and vimentin expression in lung metastases. Scale bars, 200 μm in C and H

Fig. 6

MiR-4490 is directly regulated by POU2F1. (a) and (b) Expression of miR-4490 in GC cells overexpressing of YY1, c-JUN or POU2F1 determined by qRT-PCR. *, p > 0.05; **, p < 0.05. (c) Luciferase (Luc) reporter constructs containing a miR-4490 promoter fragment with a potential POU2F1 binding site upstream of a luciferase gene. (d) Luciferase activity of the miR-4490 promoter construct after transfection of POU2F1 or vector plasmid in GC cells. ****, p < 0.001. (e) ChIP assay showing direct binding of POU2F1 to the miR-4490 promoter in GC cells. (f) and (h) Effects of POU2F1 and POU2F1/miR-4490, or POU2F1 siRNA and POU2F1 siRNA/miR-4490 on GC cell proliferation using a colony formation assay. (g) and (i) Effects of POU2F1 and POU2F1/miR-4490, or POU2F1 siRNA and POU2F1 siRNA/miR-4490 on GC cell invasion using a transwell assay. (j) POU2F1, E-cadherin, γ-catenin, Vimentin, ZEB1, p-Akt, Akt, p-ERK1/2 and ERK1/2 protein expression levels in GC cells transfected with POU2F1, POU2F1 plus miR-4490 or m-NC plasmids. The figures are representative of three independent experiments. (k) and (m) Mice were injected subcutaneously or systemically via the tail vein with BGC-823/Vector/m-NC, BGC-823/POU2F1/m-NC and BGC-823/POU2F1/miR-4490 cells, respectively. (l) Representative IHC images of Ki-67 expression in mouse tumor tissues. Scale bars, 100 μm. (n) Quantification of the numbers of metastatic loci in the lungs. ****, p < 0.001, vector vs. POU2F1; **, p < 0.01, POU2F1 vs. POU2F1/miR-4490 respectively

Fig. 7

Identification of a POU2F1/miR-4490/USP22 axis in primary human GC samples. (a) USP22 and POU2F1 protein levels detected by Western blotting in 12 paired GC tissues; GAPDH was used for normalization. (b) Average tumor/adjacent normal gastric epithelium (N/T) ratios of USP22 (a) POU2F1 and (b), miR-4490 expression quantified by qRT-PCR and normalized against GAPDH or U6 (n = 12). **, p < 0.05; ***, p < 0.01. (c) Correlation between miR-4490 and USP22 expression, between miR-4490 and POU2F1 expression, or between USP22 and POU2F1 expression in 12 GC tissues. (d) MiR-4490, USP22, POU2F1, E-cadherin, and Vimentin expression patterns in human GC tissues and normal adjacent epithelial tissues determined by in situ hybridization (ISH) or IHC staining. Scale bars, 50 μm