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ORIGINAL ARTICLE
Year : 2018  |  Volume : 14  |  Issue : 8  |  Page : 60-64

MiR-186 inhibits cell proliferation and invasion in human cutaneous malignant melanoma


1 Department of Dermatology, Xinxiang Central Hospital, Xinxiang, Henan, China
2 Department of Head and Neck, and Breast Surgery, Xinxiang Central Hospital, Xinxiang, Henan, China

Date of Web Publication26-Mar-2018

Correspondence Address:
Bei-bei Su
Department of Dermatology, Xinxiang Central Hospital, 56 Jinsui Avenue, Xinxiang Henan - 453 000
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.157340

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 > Abstract 


Aims: MicroRNA-186 (miR-186) has been shown to be involved in various types of cancer. The purpose of this study was to investigate the expression level and functional role of miR-186 in human cutaneous malignant melanoma cells.
Subjects and Methods: Expression of miR-186 was analyzed in human cutaneous malignant melanoma (CMM) cell lines SK-MEL-1, G-361, A375 and A875, and human normal epidermal melanocytes cell line HEMn-LP by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Additionally, the functional role of miR-186 on melanoma cells was investigated by transfection of miR-186 mimic followed by analyses of cell proliferation, apoptosis, and metastasis.
Results: We found that the expression levels of miR-186 were decreased in CMM cell lines compared with normal epidermal melanocytes cell line. Moreover, overexpression of miR-186 inhibited cells proliferation through abrogating the G1–S transition, and reduced cells migration and invasion.
Conclusions: Our findings suggested that miR-186 exhibit an inhibitory effect on CMM cell proliferation, migration, and invasion; thus, may serve as a potential therapeutic target for human CMM intervention.

Keywords: Cutaneous malignant melanoma, invasion, miR-186, proliferation


How to cite this article:
Su Bb, Zhou Sw, Gan Cb, Zhang Xn. MiR-186 inhibits cell proliferation and invasion in human cutaneous malignant melanoma. J Can Res Ther 2018;14, Suppl S1:60-4

How to cite this URL:
Su Bb, Zhou Sw, Gan Cb, Zhang Xn. MiR-186 inhibits cell proliferation and invasion in human cutaneous malignant melanoma. J Can Res Ther [serial online] 2018 [cited 2019 Jun 16];14:60-4. Available from: http://www.cancerjournal.net/text.asp?2018/14/8/60/157340




 > Introduction Top


Cutaneous malignant melanoma (CMM) is a highly aggressive cancer arising from uncontrolled proliferation of melanocytes, accounts for approximately 10% of skin malignancies.[1] Although new drugs such as ipilimumab, vemurafenib, dabrafenib, and trametinib, have been approved for the treatment of patients with metastatic melanoma, these targeted agents has revolutionized the treatment of this disease, it is generally still incurable.[2] A better understanding of the underlying mechanism of melanoma development and the expression profile and function of various genes has become basis for development of new treatments.

It has been shown that microRNA (miRNAs) play an important role in the development of malignancies including melanoma. By directly regulating oncogenes or tumor-suppressor genes, miRNAs exhibit oncogenic or tumor suppressive role in melanoma.[3],[4] Previous work on miRNA microarray analysis of human melanoma blood samples revealed that miRNA-186 (miR-186) was upregulated in blood cells of melanoma patients (7.9-fold of the nontumor miR-186),[5] and it has been shown to play a suppressive role in multiple types of human malignancies.[6],[7],[8],[9] However, the biological functions of miR-186 in melanoma cells remain largely unclear.

In this present study, we observed a significant reduced expression of miR-186 in human CMM cell lines compared with human normal epidermal melanocytes cell line. In addition, we showed that upregulation of miR-186 expression by miRNA mimics in melanoma cell lines decreased cell proliferation by inducing G1–S checkpoint arrest, and inhibited cell migration and invasion in vitro. Our data demonstrated that miR-186 may play an important role as tumor suppressor in human CMM.


 > Subjects and Methods Top


Cell lines and culture conditions

Human CMM cell lines SK-MEL-1, G-361, A375, and A875; and human normal epidermal melanocytes cell line HEMn-LP were purchased from the Cell Bank of Central South University. The base medium for SK-MEL-1 and G-361 were Eagle's Minimum Essential Medium (EMEM; Invitrogen, Carlsbad, CA, USA) and McCoy's 5A Medium Modified, respectively. A375 and A875 cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Carlsbad, CA, USA). The HEMn-LP cells were cultured in M-254 basal medium (Invitrogen, Carlsbad, CA, USA) supplemented with human melanocyte growth supplement-2 (HMGS-2) and amphotericin B (0.25 μg/ml). All culture media were supplemented with 10% FBS (fetal bovine serum; Gibco, Carlsbad, CA, USA), 100 U/mL penicillin, and 100 mg/mL streptomycin (Invitrogen, Carlsbad, CA, USA). Cells were incubated at 37°C and supplemented with 5% CO2 in the humidified chamber.

Extraction of total RNA and real-time quantitative polymerase chain reaction

Total RNA was isolated using TRIzol ® Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. The reverse transcription (RT) reaction was carried out using the TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). RT reaction was processed at 16°C for 30 min, 42°C for 30 min, and 85°C for 5 min. Gene expression levels were quantified using the ABI 7900 Real-Time PCR system (Applied Biosystems, Foster City, CA, USA) at 95°C for 10 min, followed by 40 cycles of 95°C for 15s, and 60°C for 60s. RNU6B served as the internal control. PCRs of each sample were conducted in triplicate. The relative levels of gene expression were presented as Δ Ct = Ctgene − Ctreference, and the fold change of gene expression was calculated by the 2−ΔΔCt method.

Transfection

Transfection was performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's instructions. For miR-186 functional analysis, melanoma A375 and A875 cells were transfected with scrambled miRNA as a negative control, or miR-186 mimics (Invitrogen, Carlsbad, CA, USA).

Cell proliferation assay and cell cycle analysis

The cell proliferation assay was evaluated using a Cell Counting Kit-8 assay (Dojindo, Gaithersburg, MD) according to the manufacturer's protocol. Briefly, 3 × 103 cells/well incubated in 100 μL culture medium in 96-well plates and treated with 10 μL/well of Cell Counting Kit-8 solution during the last 4 h of culture. The absorbance at 450 nm was measured in a ThermoMax Microplate Reader (Molecular Devices). For cell cycle analysis, after transfection for 48 h, cells were harvested and fixed with ethanol overnight, then stained with propidium iodide (PI, Calbiochem), and examined with a fluorescence-activated cell sorting (FACS) flow cytometer (Beckman Coulter, Pasadena, CA, USA).

Annexin-V-fluorescein isothiocyanate (FITC) apoptosis assay

After transfection for 48 h, cells were collected and the translocation of phosphatidylserine was detected by using the Annexin-V-FLUOS Staining Kit (Roche Applied Science, Mannheim, Germany). Briefly, the harvested cells were suspended in 500 mL of binding buffer and incubated at room temperature in the dark for 15 min after being labeled with 5 mL of Annexin-V-FITC and 5 mL of Propidium Iodide (PI). The labeled cells were then analyzed by flow cytometry.

Wound healing assay

Cells transfected with miR-186 mimics or negative control were seeded at a density of 4 × 105 cells/mL in six-well plates. When cells grew to confluence, a scratch was traced with a pipette tip. Cells were then washed with serum-free medium and incubated with DMEM. The wound was photographed at 0 and 24 h.

Cell invasion assay

Cell invasion analysis was done using Matrigel invasion assay (BD Biosciences). In brief, A375 and A875 cells transfected with miR-186-mimics or control for 48 h were harvested, suspended (4 × 104/well) in 250 μL serum-free medium and then loaded onto the upper compartment of chamber, and 500 μL of complete medium was added to the lower compartment. After 24 h incubation, cells were fixed with 10% trichloroacetic acid at 4°C for 1 h. Noninvaded cells were removed from the upper surface of the filter carefully with a cotton swab. Invaded cells on the lower side of the filter were stained with 0.5% crystal violet for 2 h at room temperature and counted from 10 random fields under a microscope at × 200 magnification.

Statistical analysis

Each experiment was performed in triplicate, and the data represent the mean ± standard deviation (SD) of all measurements. The program Statistical Package for Social Sciences (SPSS) 20.0 software package (IBM, Armonk, NY, USA) and GraphPad Prism 5.0 statistical software (GraphPad Software Inc., La Jolla, CA, USA) were utilized for all statistical analyses. RNA expression levels were calculated based on 2ΔΔCt. The difference between groups was estimated by Student's t-test. P < 0.05 was considered statistically significant.


 > Results Top


MiR-186 is downregulated in CMM cells

As shown in [Figure 1], expression levels of miR-186 were investigated in human CMM cell lines SK-MEL-1, G-361, A375, and A875 and normal epidermal melanocytes HEMn-LP, and miR-186 was found to be significantly downregulated in CMM cells as compared with the normal epidermal melanocytes (All p<0.05).
Figure 1: Micro-ribonucleic acid-186 (MiR-186) expression was downregulated in CMM cell lines. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay was performed to detect the average level of miR-186 expression, using RNU6B as a normalization control. MiR-186 expression in human melanoma cell lines A375, A875, G361, and SK-MEL-1; and human immortalized skin keratinocyte cell lines HEMn-LP. *P < 0.05

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MiR-186 suppresses melanoma cancer cell growth and caused cell-cycle arrest

To explore the functions of miR-186 in CMM cell lines, we overexpressed miR-186 by transfecting miR-186 mimics in human CMM cell lines A375 and A875 [Figure 2]a. Initially, we explored miR-186 role in cell growth, and found that compared with control, miR-186 overexpression significantly inhibit the growth of A375 and A875 cells [Figure 2]b. Secondly, we examined the effect of miR-186 on cell cycle distribution. Accompanying with this inhibition of cell proliferation, our result showed a significant increase in the percentages of cells in G1-phase, but decreased proportions of S-phase cells in both miR-186-mimics transfected A375 and A875 cells [Figure 2]c. Finally, we found that overexpression of miR-186 had no significant effect on the early and late apoptosis, or total apoptotic cell death in A375 and A875 cells [Figure 3]. These results indicated that miR-186 abrogating the G1/S transition of cell-cycle progression and consequently suppressed CMM cell proliferation.
Figure 2: Overexpression of miR-186 suppressed melanoma cell proliferation and caused cell-cycle arrest. (a) Ectopic expression of miR-186 in melanoma cell lines A375 and A875 were evidence by qRT-PCR after transfection of miRNA mimics. (b) Cell Counting Kit (CCK)-8 assays revealed cell growth curves of indicated cells. (c) Flow cytometric determination of proportion of indicated cells in distinct cell-cycle phases. *P < 0.05, **P < 0.01

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Figure 3: MiR-186 had no significant effect on the melanoma cell apoptosis. After transfection for 48 h, Annexin-V-FITC and PI co-staining were used for flow cytometric analysis. FITC = Fluorescein isothiocyanate, PI = Propidium iodide

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MiR-186 suppresses CMM cell migration and invasion

To investigate whether the declined expression of miR-186 can affect the migration and invasion of CMM cells, we examined the rate of miR-186-mimics transfected A375 and A875 cells migration by wound healing assay. Our results revealed that both cells have a marked decrease in cell migration compared with their control counterparts [Figure 4]a. Accordingly, miR-186 resulted in about 26.8% decrease of migrated A375 [Figure 4]b, left] and 30.6% of A875 cells [Figure 4]b, right] into wound area compared with the negative control cells. We also examined the ability of miR-186-mimics transfected cells to invade through the Matrigel matrix using the Boyden chamber assay. We found that overexpression of miR-186 significantly reduced both A375 and A875 cells invasion comparing with the control group [Figure 4]c. Accordingly, miR-186 resulted in a 60.9% decrease of invaded A375 [Figure 4]d, left] and 50.9% of A875 cells [Figure 4]d, right] into wound area compared with the negative control cells. These data suggest that miR-186 downregulation might be an important event in the metastasis of CMM cells.
Figure 4: MiR-186 inhibited melanoma cell migration and invasion. (a) After transfection with miR-186 mimics for 48 h, cells were subjected to wound healing assays and images were taken at 0 and 24 h (×100). (b) Quantitation of (a), *P < 0.05. (c) For Boyden chamber assay, cells were counted from 10 random fields (×200), and the data are presented as the average number of cells per field (d) (mean ± standard deviation (SD)). *P < 0.05

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 > Discussion Top


Multiple miRNAs are involved in various human malignancies. MiR-186 has also been shown to be aberrantly expressed in different cancers.[8],[10],[11],[12],[13],[14],[15],[16],[17] However, to our knowledge, no study on the expression of miR-186 in human CMM cell lines, or its possible functions in melanoma formation and progression has been conducted so far. In the current study, our data revealed a significant reduction in miR-186 expression in CMM cell lines compared with normal epidermal melanocytes cell line. Furthermore, miR-186 inhibited melanoma cell proliferation and caused cell-cycle arrest, suppresses melanoma cell migration and invasion, suggest that miR-186 might act as a tumor suppressor during CMM development.

Our study results were contrary to Leidinger and colleagues,[5] they showed that miR-186 was overexpressed in melanoma patients in compared to healthy controls. However, different with our selected cutaneous cells, Leidinger et al., enrolled peripheral blood cells as subjects. In addition to active release of tumor cells, circulating miRNAs can also derived from the apoptotic and necrotic cells, and circulating cell lysis, differences of sources may lead to the opposite miR-186 expression pattern.[18] Similarly, the tissue specificity of miRNA expression pattern and tumor heterogeneity well interpret the discrepancy that miR-186 was overexpressed in pancreatic [10] and endometrial [11] cancer tissues. Certain miRNAs (for example, miR-9 and the miR-17-92 clusters) can act as oncogenic or tumor-suppressor miRNAs, depending on the tissue and context.[19] Similar contradictory expression pattern and functions have been reported for miR-186. miR-186 is reported to repress expression of the tumor suppressor gene FOXO1[11] and AKAP12,[13] downregulate expression of the proapoptotic purinergic P2x7 receptor;[20] suggesting that miR-186 harbors protumorigenic properties and can behave as an oncogene. However, other studies have reported the exact opposite role for miR-186: Induce cellular senescence by targeting α subunit of protein kinase CKII in human colorectal cancer cells,[12] inhibited the proliferation of non-small cell lung cancer (NSCLC) cells by inhibiting cyclin D1 (CCND1) and restoring cyclin-dependent kinase (CDK) 2,[6] modulate migration and invasion of NSCLC cells by targeting PTTG1[8] and ROCK1.[7] Cancer is a multistep, multifactorial, complicated disease; numerous genes regulate each other and form complex interactions that result in cancer development and progression. Therefore, the opposing expression profile and role of miR-186 in various kinds of cancers are not surprising.

Although miR-186 can regulate the progression of CMM, little is known about the underlying molecular mechanisms. Previous studies have reported that miR-186 impact cellular functions through various mechanisms, such as targets CCND1 and CDK2, to suppress the growth of NSCLC cells.[6] Moreover, miR-186 could involve in tumor metastasis by targeting ROCK1 and PTTG1.[7],[8] We speculate that miR-186 may influence many cellular processes, including proliferation, cell cycle, migration, and invasion, by targeting different protein-coding genes in different stage of CMM. In future work, high-throughput techniques should be applied to obtain a global view of the changes in protein-coding RNAs and to elucidate the specific mechanisms of regulation of miR-186. Furthermore, whether miR-186 represent a potential target for gene therapy, needs to be validated in some more clinically meaningful experiments such as three-dimensional models in vitro and/or animal experiments.

In conclusion, we determined that miR-186 expression was significantly downregulated in CMM cell lines. Furthermore, we demonstrated the inhibitory effect of miR-186 on melanoma cell growth, migration, and invasion in vitro. MiR-186 may play a key role in CMM tumorigenesis and progression. Our data suggest that miR-186 might exert tumor suppressor function in human CMM.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
 > References Top

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4]



 

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