Downregulation of miR-493 promoted melanoma proliferation by suppressing IRS4 expression

Abstract

Accumulating evidence indicated that aberrantly expressed microRNAs play critical roles in the initiation and progression of human cancers. However, the underlying functions of miR-493 in human melanoma remains unknown. Here, our study found that miR-493 expression was downregulated in human melanoma tissues and cells. Overexpression of miR-493 suppressed cell proliferation and cell cycle in human melanoma cell line A375. IRS4 was defined as a target for downregulation by miR-493 and was confirmed by luciferase assay. We also found that knockdown of IRS4 counteracted the proliferation promotion by miR-493 inhibitor. In summary, these results demonstrated that miR-493 acts as a tumor suppressor and inhibits cell proliferation and cell cycle in human melanoma by directly targeting IRS4.

Introduction

Human melanoma represents the most aggressive form and is the most common cause of death of skin cancer in humans. ¹ Although there is an advanced improvement in the treatment options, the prognosis of patients with malignant melanoma has not obviously improved.^2,3 Much interest has been taken in investigating new treatment options for human melanoma.^4,5

Growing evidence demonstrated that microRNAs (miRNAs) are a class of non-coding RNA molecules that contribute to various biological processes, including cell proliferation, cell cycle, cell migration, cell invasion, and cell apoptosis.^6–9 MiRNA functions by mediating the translation of messenger RNAs (mRNAs) by binding directly to the 3′-untranslated region (3′-UTR).^10–12 Recent studies have revealed that miR-493 was aberrantly expressed and plays critical roles in many types of cancers.^13–15 In gastric cancer, miR-493 was strongly downregulated and suppressed the proliferation and invasion by RhoC. ¹⁶ MiR-493 was found to suppress liver metastasis in colon cancer cells by regulating mitogen-activated protein kinase kinase 7 (MKK7). ¹⁷ Gu et al. ¹⁵ indicated that miR-493 suppressed tumor growth, invasion, and metastasis by regulating E2F1 in lung cancer. However, the underlying functions of miR-493 in human melanoma remain largely unexplored. In this study, miR-493 was shown to be frequently downregulated in human melanoma tissues and cells. Ectopic expression of miR-493 in malignant melanoma cells led to the suppression of cell proliferation and cell cycle. Mechanistically, we found that IRS4 was a putative target of miR-493 and was confirmed by luciferase reporter assays. Silencing of IRS4 in A375 cells after being transfected with miR-493 inhibitor (miR-493-in) reversed the promotion effects of miR-493-in. Taken together, miR-493 may be a promising therapeutic target for patients with malignant melanoma.

Materials and methods

Clinical specimens

Human Melanoma tissues and adjacent non-tumor tissues were obtained from patients and histopathologically diagnosed at the Department of Dermatology, Yanbian University Hospital (Yanji, People’s Republic of China). All tissues were confirmed by pathology. Ethical approval for the study was approved by the ethics committee of Yanbian University Hospital (Yanji, People’s Republic of China). All patients provided their consent and agreement.

Cell culture and cell transfection

Primary human epidermal melanocytes (PEM; PromoCell, Germany) were purchased from ScienCell (USA) and were grown in serum- and phorbol myristate acetate (PMA)-free melanocyte growth medium M2 (PromoCell, Germany). Human melanoma cells lines, including SK-MEL-28, WM-115, UACC257, A375, A7, MeWo, and NHEM were purchased from National Rodent Laboratory Animal Resource (Shanghai, People’s Republic of China) and were maintained in RPMI-1640 medium, containing 10% fetal bovine serum (HyClone, Pittsburgh, USA), 100 µg/mL penicillin, and 100 U/mL streptomycin (Sigma-Aldrich, St. Louis, MO, USA). All cells were cultured in a humidified incubator at 37°C with 5% CO₂.

The miR-493 mimic and miR-493-in were synthesized by GeneCopoeia (Guangzhou, People’s Republic of China), and the transfection of miR-493 or miR-493-in or negative control (NC) was performed using the Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) as per the manufacturer’s instructions.

Two small interfering RNAs (siRNAs) targeting IRS4 were purchased from purchased from GeneCopoeia (Guangzhou) and the cells were transfected with siRNAs using lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol.

RNA isolation and quantitative reverse transcription polymerase chain reaction

Total RNA was isolated from human melanoma cells and clinical tissues using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. MiR-493 expression was detected by TaqMan MicroRNA Assays (Applied Biosystems, Foster City, CA, USA) and U6 acted as an internal control. The mRNA expression of p21 and p27 was analyzed using the Applied Biosystems 7500 Sequence Detection system with the following primers which were synthesized by GeneCopoeia—p21 (HQP000331), p27 (HQP000342), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (HQP006940)—acted as an internal control. The quantitative reverse transcription polymerase chain reaction (qRT-PCR) results were defined based on the threshold cycle (Ct), and relative expression levels were calculated using the 2^−ΔΔCt method.

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays

The in vitro cell viability of A375 cells was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Sigma-Aldrich). After transfection, A375 (5 × 10³/well) cells were plated on 96-well plates and were incubated at 37°C. After 1, 2, 3, 4, and 5 days, 20 µL of MTT (5 mg/mL; Sigma, St. Louis, MO, USA) was added to each well for 4 h followed by removal of the medium and addition of 150 µL of dimethyl sulphoxide (DMSO; Sigma). Cellular viability was determined by measuring the absorbance of the converted dye at 495 nm.

Anchorage-independent growth assay

A375 cells (1.5 × 10³) were transfected with miR-493 mimic or miR-493-in or the relative controls, and were suspended in 1 mL of complete medium containing 0.66% agar (Sigma-Aldrich). Cells were incubated for 2 weeks at 37°C until colony formation, and colonies greater than 0.1 mm were counted using an inverted microscope after staining with 1% crystal violet (Sigma-Aldrich).

Luciferase assays

According to the manufacturer’s recommendation of the Lipofectamine 2000 Reagent (Invitrogen), the IRS4 3′-UTR wild type was amplified and cloned into the pGL3 luciferase assays vector (Promega, Madison, WI, USA) and then, the cells were co-transfected with miR-493 or miR-493-in or mutated miR-493 (miR-493-mut) or negative controls. Lysates were collected 48 h after transfection, and luciferase activity was detected using Dual-Luciferase Reporter Assay System (Promega).

Western blotting

Equal total protein extracts were loaded and separated by 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were immunoblotted with the following antibodies: anti-IRS4 (ab52622; Abcam, Cambridge, MA, USA), anti-p21 (ab109520; Abcam), anti-p27 (ab32034; Abcam), and anti-α-tubulin (ab7291; Abcam) acted as a loading controls followed by incubation with horseradish peroxidase–linked secondary antibodies (Abcam). The proteins were detected by ECL reagents (Pierce, Rockford, IL, USA).

Bromodeoxyuridine incorporation assays

The transfected cells (4 × 10³) were cultured on cover slips (Thermo Fisher Scientific, Pittsburgh, PA, USA) and incubated with bromodeoxyuridine (BrdU) for 60 min and then, they were incubated with an anti-BrdU antibody (1:200; Abcam) according to the manufacturer’s instructions. Gray level images were visualized using a fluorescence microscope (Olympus 600 auto-biochemical analyzer; Olympus, Tokyo, Japan).

Cell cycle analysis

Transfected cells were harvested after 48 h of transfection. The cells were fixed in 70% ethanol, washed once with phosphate-buffered saline (PBS), and then labeled with propidium iodide (Sigma-Aldrich) in the presence of RNase A (Sigma-Aldrich) for 30 min in the dark (50 g/mL). Samples were run on a FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA), and the percentages of cells within each phase of the cell cycle were analyzed using BD Cell Quest software, USA.

The cells were suspended in 0.5 mL solution containing 20 µg/mL propidium iodide and 50 µg/mL RNase and then analyzed using flow cytometry (BD Biosciences, San Jose, CA, USA). Histograms were used to represent the percentage of cells in each phase of the cell cycle (G₁/G₀, S, and G₂/M).

Statistical analysis

All statistical analyses were performed using the SPSS 17.0 (SPSS Inc., Chicago, IL, USA) and one-way analysis of variance (ANOVA). Two-tailed Student’s t test was used to calculate the statistical differences. p < 0.05 was considered to be statistically significant.

Results

MiR-493 expression was downregulated in human melanoma cell lines and tissues

ArrayExpress dataset GSE25653 indicated that miR-493 expression was significantly decreased in 52 human melanoma tissues compared with two adjacent non-cancerous tissues (Figure 1(a)). Real-time PCR analyses indicated that miR-493 was significantly downregulated in all the seven examined cell lines (SK-MEL-28, WM-115, UACC257, A375, A7, MeWo, and NHEM) compared with the PEM (Figure 1(b)). The expression of miR-493 in human melanoma tissues and adjacent non-tumor tissues which were obtained from our hospital was detected by real-time PCR. As shown in Figure 1(c), miR-493 was downregulated in human melanoma tissues compared with the paired adjacent non-tumor tissues. Taken together, these data indicated that miR-493 expression was frequently downregulated in human melanoma.

Figure 1.

Expression of miR-493 in human melanoma cell lines and tissues. (a) The expression levels of miR-493 in human melanoma tissues from GSE dataset (p < 0.001). (b) Real-time PCR analysis of miR-493 expression in primary human epidermal melanocytes (PEM) cell lines and human melanoma cell lines, including SK-MEL-28, WM-115, UACC257, A375, A7, MeWo, and NHEM. (c) Relative miR-493 expression levels in eight paired primary melanoma tissues (T) and the paired adjacent non-tumor tissues (ANT) from the same patient were detected by PCR analysis. Each bar represents the mean of three independent experiments (*p < 0.05). Each bar represents the mean of three independent experiments.

MiR-493 suppressed, while miR-493-in promoted, cellular proliferation and cellular cycle of human melanoma

To explore whether miR-493 contributed to tumor progression of human melanoma, human melanoma cell line A375 was stably transfected with miR-493 or miR-493-in and the relative controls, and the transfection efficiency was detected by qRT-PCR (Figures 2(a) and 3(a)). MTT assays revealed that miR-493-transfected cells had a significant decrease in the growth rate (Figure 2(b)). Anchorage-independent growth assay revealed that overexpression of miR-493 significantly enhanced more and larger sized colonies of cancer cells than control cells (Figure 2(c)). BrdU incorporation assays indicated that BrdU incorporation rate was decreased in A375 cells that are stably overexpressing miR-493 compared to control cells (Figure 2(d)). Analysis of cell cycle distribution showed that miR-493 significantly increased the proportions of cells in G₁/G₀ phase. Moreover, A375 cells stably transfected with miR-493-in showed increased cell growth compared to the control (Figure 3(b)). Anchorage-independent growth assay revealed that transfection with miR-493-in significantly promoted the anchorage-independent growth ability of A375 cells compared with those transfected with the control (Figure 3(c)). Similar results were also confirmed by BrdU incorporation assays (Figure 3(d)). Result of cell cycle indicated that miR-493-in significantly decreased the proportions of cells in G₁/G₀ phase in A375 cells (Figure 3(e)). Collectively, our data revealed that miR-493 functioned as a tumor suppresser in human melanoma.

Figure 2.

Upregulation of miR-493 suppressed human melanoma cell proliferation. (a) Validation of miR-493 expression levels after transfection by PCR analysis. (b) MTT assays revealed that inhibition of miR-493 suppressed growth of A375 cell line. (c) Upregulation of miR-493 inhibited the anchorage-independent growth of A375 cells. Representative micrographs (left) and quantification of colonies that were >0.1 mm (right). (d) Representative micrographs (left) and quantification (right) of the BrdU incorporation assay in A375 cells. (e) Flow cytometric analysis of the indicated A375 cells transfected with NC or miR-493. Each bar represents the mean of three independent experiments (*p < 0.05).

Figure 3.

Inhibition of miR-493 promoted human melanoma cell proliferation. (a) Validation of miR-493 expression levels after transfection by PCR analysis. (b) MTT assays revealed that miR-493-in promoted A375 cell growth. (c) Inhibition of miR-493 inhibited the anchorage-independent growth of A375 cells. Representative micrographs (left) and quantification of colonies that were >0.1 mm (right). (d) Representative micrographs (left) and quantification (right) of the BrdU incorporation assays in A375 cells. (e) Flow cytometric analysis of the indicated A375 cells transfected with NC or miR-493-in. Each bar represents the mean of three independent experiments (*p < 0.05).

MiR-493 targeted IRS4 directly in A375 cells

To further investigate the role of miR-493 in human melanoma, we searched for putative miR-493 targets using TargetScan (http://www.targetscan.org/vert_71/). IRS4 was identified as a potential target of miR-493 (Figure 4(a)). Then, we analyzed IRS4 expression in A375 cells after being transfected with miR-493, miR-493-in, or the relative controls using Western blot analysis. As shown in Figure 4(b), IRS4 expression was significantly reduced in A375 cells stably transfected with miR-493, while IRS4 expression clearly increased when miR-493 was knocked down by miR-493-in. In addition, we performed a luciferase reporter assay to confirm whether IRS4 is a direct target of miR-493, we found that transfection with miR-493 significantly increased the firefly luciferase activity in A375 cells, while miR-493-in resulted in a significant increase in the luciferase activity of the construct containing the wild type 3′-UTR of IRS4 (Figure 4(c)). Furthermore, miR-493-mut showed no effect on the firefly luciferase activity in A375 cells (Figure 4(c)), suggesting miR-493-mediated regulation of IRS4 expression depended on its binding to a specific seed region in its 3′-UTR.

Figure 4.

MiR-493 suppressed IRS4 expression by directly targeting its 3′-UTR and altered the levels of proteins related to cell proliferation and cell cycle in A375 cells. (a) Predicted miR-493 target sequence in the 3′-UTR of IRS4 (IRS4-3′-UTR) and positions of three mutated nucleotides (green) in the 3′-UTR of miR-493 (miR-493-mut). (b) Western blot analysis of IRS4 expression in A375 cells transfected with miR-493 or the miR-493-in; α-Tubulin served as the loading control. (c) Luciferase reporter assay of the indicated cells transfected with the pGL3-IRS4-3′-UTR reporter and miR-493 or miR-493-in or miR-493-mut (p < 0.05). (d) Real-time PCR analysis of expression of p21 and p27 in indicated A375 cells. (e) Western blot analysis of expression of p21 and p27 protein in A375 cells. α-Tubulin served as the loading control (*p < 0.05).

As miR-493 promoted cell proliferation, we further examined the cell proliferation and cell cycle relative proteins, p21 and p27. Results of RT-PCR and Western blot analysis indicated that p21 and p27 expression was downregulated after treatment with the miR-493, while the expression was upregulated in A375 cells stably transfected with miR-493-in (Figure 4(d) and (e)).

Silencing IRS4 expression could counteract miR-493-in-mediated cell proliferation promotion

To further determine whether IRS4 repression is involved in cell proliferation of human melanoma, specific siRNAs of IRS4 were used to inhibit expression of IRS4 in A375 cells stably transfected with miR-493-in. IRS4 expression was confirmed by Western blot analysis (Figure 5(a)). Results of anchorage-independent growth assay revealed that knockdown of IRS4 expression in miR-493-in transfected cells dramatically decreased cell proliferation (Figure 5(b)). BrdU incorporation assays indicated that the BrdU positive rate was decreased in A375 cells stably transfected with miR-493-in after being transfected with IRS4 siRNAs (Figure 5(c)).

Figure 5.

IRS4 downregulation counteracted the proliferation promotion by miR-493-in. (a) Western blot analysis verified that silencing of IRS4 effectively decreased the expression of IRS4 in miR-493-in-transfected A375 cells. (b) MiR-493-in-transfected A375 cells after transfection with IRS4 siRNAs promoted the anchorage-independent growth. Representative quantification of colonies that were >0.1 mm. (c) Representative micrographs (left) and quantification (right) of the BrdU incorporation assays. Each bar represents the mean of three independent experiments (*p < 0.05).

Discussion

Growing evidence demonstrated that miRNAs play essential roles in tumor development and progression. In this study, we observed that miR-493 was significantly downregulated in human melanoma and found that miR-493 suppressed cell proliferation of human melanoma. Furthermore, we confirmed that the miR-493 modulates cell proliferation and cell cycle by targeting enhancer of zeste homolog 2 (EZH2) expression.

Previous studies have illustrated that miRNAs are associated with various human cancer processes, including human melanoma. Qiu et al. ¹⁸ indicated that miR-769 expression was remarkably upregulated in human melanoma clinical tissues and cells and promoted human melanoma cell proliferation. MiR-138 was reported to suppress proliferation, invasion, and glycolysis of malignant melanoma cells. ¹⁹ Our data may add a new miRNA marker in diagnosis of human melanoma.

To date, the role of miR-493 in cancers has not been well understood. In gastric cancer, miR-493 promoted cell proliferation, invasion, and chemo-resistance. ¹³ MiR-493 has been reported to act as a tumor suppressor and decreases cell motility and migration ability. ²⁰ Okamoto et al. ²¹ indicated that miR-493 suppresses liver metastasis via inducing cell death of metastasized cells. The function of miR-493 in human melanoma remains unknown. In our study, result of ArrayExpress dataset GSE25653 revealed that level of miR-493 expression remarkably decreased in human melanoma clinical tissues. Further analysis indicated that miR-493 is significantly downregulated in human melanoma cell lines and melanoma tissues of our hospital. Based on in vitro experiments, we confirmed that miR-493 inhibits cell proliferation and cell cycle of human melanoma cells, suggesting that miR-493 acts as a tumor suppressor in human melanoma.

MiRNAs typically functions by directly binding to its targeted mRNAs. In lung cancer, miR-493 inhibits tumor growth, invasion, and metastasis by regulating E2F1. ¹⁵ Sakai et al. ¹⁷ indicated that miR-493 suppresses liver metastasis of colon cancer cells by modulating MKK7. Our findings confirmed that miR-493 acts as a tumor suppressor in human melanoma, but the underlying mechanism remains unknown. Therefore, IRS4 is being identified as a target gene of miR-493 in human melanoma using TargetScan database, which was further confirmed by luciferase assay. Further analysis indicated that IRS4 downregulation could counteract miR-493-in-mediated cell proliferation promotion.

In summary, this study proved that miR-493 significantly downregulated and suppressed cell proliferation and cell cycle in human melanoma by silencing IRS4. Our study provided new insights into the pathogenesis of human melanoma and represented a potential therapeutic target for the treatment of human melanoma.