Antiproliferative and Pro-Apoptotic Effects of MiR-4286 Inhibition in Melanoma Cells

Introduction MicroRNAs are essential regulators of gene expression at the post-transcriptional level. Their expression is altered in cancer tissues, and evaluation of these alterations is considered a promising tool used to diagnose and identify prognostic markers. Materials and methods The microRNA expression profiles of formalin-fixed, paraffin-embedded melanoma and melanocytic nevi samples were estimated with a microarray and subsequently validated by real-time PCR. Melanoma cells were transfected with miR-4286 inhibitor to evaluate the influence of this microRNA on the viability, proliferation, apoptosis, migration, and invasion of melanoma cells. Results The microarray revealed that the expression of 1,171 microRNAs was altered in melanoma samples compared to melanocytic nevi. Real-time PCR validation experiments found the microRNA expression levels to correspond to the melanoma/melanocytic nevi microarray results. The pathway analysis identified 52 modulated pathways in melanoma. Moreover, the application of miR-4286 inhibitor to BRO melanoma cells resulted in a 2.6-fold increase in the apoptosis rate and a 1.7-fold decrease in the cell proliferation/viability but did not affect the invasiveness and migration of these cells. Furthermore, the use of miR-4286 inhibitor altered the mRNA expression of several miR-4286 gene targets: folylpolyglutamate synthase, RNA polymerase I-specific transcription initiation factor, apelin, G-protein-coupled receptor 55, and high-mobility group A1 protein, which have been implicated in cell proliferation/apoptosis regulation. Lastly, the transiently transfected SK-MEL-1 cells with miR-4286 inhibitor decreased proliferation rate and modulated folylpolyglutamate synthase rates of these cells. Conclusion Our results demonstrate that miR-4286 mediates proliferation and apoptosis in melanoma cells, these findings may represent a novel mechanism underlying these processes.

Introduction MicroRNAs are small (19-24 nucleotides), non-coding RNA molecules that regulate gene expression at the post-transcriptional level and hundreds of microRNAs have been discovered and investigated to date [1]. A single microRNA can regulate the expression of more than 100 genes. In turn, a gene may be targeted by several microRNAs. Given the large number of miR-NAs annotated in the human genome, 30%-80% of human genes are predicted to be regulated by miRNAs [2; 3; 4; 5; 6; 7]. Specifically, aberrant microRNA profiles have been observed in various cancer types, which may be a result of epigenetic alterations of miRNA promoters, the altered activity of transcriptional factors or dysregulated enzymes involved in miRNA biogenesis [8]. Furthermore, altered microRNA expression contributes to cancer development by down-regulating tumor suppressor genes or up-regulating tumor-promoting oncogenes [9].
Melanoma is an aggressive skin cancer whose incidence rate has been increasing within white-skinned populations [10]. The survival of patients with melanoma depends on tumor thickness, which is one of the most important prognostic factors. Therefore, the early detection of the tumor is crucial for a successful treatment, and the detection of changes in the micro-RNA profile in tumor tissue may serve as a tool for identification of novel molecular markers. Recent studies showed that melanoma progression is accompanied by significant dysregulation of the microRNA expression pattern [11,12], but a specific microRNA has not yet been shown as an adequate marker to diagnose and predict the prognosis of patients with this disease. This challenge may be due to tumor heterogeneity, which complicates the differentiation of microRNA expression pattern between tumor and tumor microenvironment cells. Conversely, the tissue microRNA expression pattern is relatively stable and specific under different pathological states. Specifically, a recent study [13] identified the following five microRNAs to be strongly associated with the outcome of patients with melanoma: miR-142-5p, miR-150-5p, miR-342-3p, miR-155-5p, and miR-146b-5p. Unfortunately, the serum-derived microRNAs of these patients correlate only with the microRNA tissue pattern at the late stage but not with primary tumors which currently complicates the use of microRNA identification as prognostic or diagnostic approach for melanoma [14]. Thus, additional studies are necessary to clarify the exact role of altered microRNAs in tumor development and progression as well as their clinical importance. MiR-4286 was shown to be up-regulated in different types of malignancies such as pancreatic cancer [15], glioma [16], and esophageal adenocarcinoma [17]. Furthermore, Sand M. et al. included miR-4286 in a list of significantly up-regulated microRNAs in melanoma whose role in melanomagenesis has not been studied [18].
The aim of this study was to compare the microRNA expression profiles in melanoma and melanocytic nevi in order to identify a specific pattern and study the function of aberrantly expressed microRNAs in melanoma.

Tissue samples
The study was approved by the Krasnoyarsk State Medical University Local Ethics Committee (protocol №59/2014 issued on December 2, 2014). Written consent was approved by the Local Ethics Committee and obtained from all participants in the study before obtaining samples. Treatment-naive primary melanoma and benign melanocytic tumor samples were obtained from patients at the Krasnoyarsk Regional Oncology Center, which is named after A.I. Kryzhanovsky. Sixteen melanoma and 3 melanocytic nevi samples were fixed in formalin and embedded in paraffin. The ages of patients with melanoma ranged from 35 to 81 years. Men comprised 56% and females-44% of the cohort ( Table 1). The ages of patients with melanocytic nevi ranged from 29 to 55 years. Two patients had nevi on the face, whereas one was with nevus on the breast ( Table 2). Samples of melanocytic lesions containing less than 70% of studied cells were macrodissected to separate the tissues from melanoma cells and tumor microenvironment. The percentage of target tissue in each sample was evaluated by hematoxylin/eosin staining and subsequent light microscopy.
Samples were processed according to a standard protocol that included incubation in 10% neutral buffered formalin for 24 h and embedding in paraffin blocks. The diagnosis was made by a certified pathologist at the Krasnoyarsk Pathologic Anatomy Bureau. After fixation, the samples were stored in the Archive of Pathologic Anatomy Bureau at room temperature for up to 5 years.

RNA isolation
Total RNA was isolated using the RecoverAll™ Total Nucleic Acid Isolation kit (Ambion, Life Technologies, Vilnius, Lithuania). Formalin-fixed and paraffin-embedded (FFPE) samples were sectioned at a thickness of 15 μm, deparaffinized with xylene, and washed twice with ethanol according to the standard procedure. The tissues were then suspended in 200 μl of Digestion buffer. RNA was isolated according to manufacturer's instructions for RNA isolation and eluted with 50 μl of nuclease-free water. The microRNA concentration was measured on Qubit1 2.0 fluorimeter (Invitrogen by Life Technologies, Singapore, Singapore) using the Qubit1 microRNA Assay kit (Ref. Q32880, Eugene, Oregon, USA). The concentration of microRNA in the samples submitted to the microarray analysis exceeded 16.3 ng/μl.

TaqMan miRNA real-time PCR
In this work, the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines was applied for data reporting [20]. The reverse transcription reaction was performed in a reaction mixture containing 0.4 μl of 5x primers miRNA TaqMan assays specific to the investigated microRNA (Cat. № 4427975, Applied Biosystems, Foster City, CA, USA) or Random-primers for mRNA expression analysis, 0.4 μl of OT-buffer, 0.05 μl revertase enzyme, and 0.15μl of RNA-eluent solutions from the "Reverta" kit (Ampli-Sens, Moscow, Russia) per 1 μl of isolated RNA. The reactions were carried out at 37˚C for 30 min. cDNA was immediately used for the RT-PCR reaction or stored at -20˚C. Briefly, the obtained cDNA was added to a PCR cocktail containing 8 μl of the 2.5-fold RT-PCR reaction mixture and ROX reference dye (Syntol, Moscow, Russia), 1 μl of 20x primers, miRNA Taq-Man assay probe mix, and deionized water to a total volume of 20μl. U6snRNA and RNU6B (Cat. № 001973, Assay ID 001973 and 4427975 respectively, Applied Biosystems, Foster City, CA, USA) were used as endogenous controls to estimate the relative microRNA expression levels; beta-actin (Cat. № 4331182, Assay ID Hs01060665_g1) and HPRT-1 (hypoxanthinephosphoribosyltransferase 1) (Cat. № 4331182, Assay ID Hs01003267_m1) were used as endogenous controls to evaluate mRNA levels. The reaction was performed on a StepOne™ Real-Time PCR System (Applied Biosystems, Singapore, Singapore) with the following temperature cycling protocol: 50˚C for 2 min and 95˚C for 10 min, followed by 40 cycles of 95˚C for 15 sec and 60˚C for 1 min with FAM/VIC detection. Three replicates were performed for each experiment. The microRNA or mRNA expression was normalized to the geometric mean of U6 snRNA and RNU6B or beta-actin and HPRT-1, respectively, as described previously [21]. The data were analyzed using the ΔΔC t method.

Cell culture and transfection
The human melanoma cell line BRO [22; 23]  The 50 mM anti-miR water solution was diluted in 500 μl of cell-containing culture medium to a final concentration of 25 nM. The medium was replaced every 24 h. To evaluate the transfection efficiency, anti-miR TM hsa-let-7c miRNA inhibitor (Cat. № 4392431, Ambion, Carlsbad, CA, USA) and anti-miR TM negative control #1 (Cat. № AM17010, Ambion, Carlsbad, CA, USA) were transfected into Bro and SK-MEL-1 melanoma cells in a 24-well plate in concentration of 1×10 5 cells/ml using 1.5 μl Lipofectamine 2000 reagent (Invitrogen, Life Technologies, Carlsbad, CA, USA) per well. The transfection efficiency was estimated by detecting changes in the expression of high-mobility group AT-hook 2 (HMGA2, Assay ID Hs00171569_m1, Cat № 4331182, Applied Biosystems, Foster City, CA, USA) in the transfected cells using real-time PCR detection. Because hsa-let-7c specifically down-regulates the expression of HMGA2, the effective inhibition of let-7c increases the HMGA2 mRNA level in cells. Moreover, the miR-4286 expression levels were evaluated in both melanoma cell lines after transfection with hsa-miR-4286 anti-miR TM miRNA inhibitor (Cat. № AM17000, Assay ID AM 17048, Ambion, Life Technologies, Carlsbad, CA, USA) using real-time PCR and 5X specific from primers of Taq-Man MicroRNA Assay (Cat. № 4427975, Assay ID 241500_mat, Applied Biosystems, Foster City, CA, USA), as described above. The experiments were performed in triplicate.
Fluorescent microscopy. Cells transfected with hsa-miR-4286 anti-miR TM miRNA inhibitor (Cat. № AM17000, Ambion, Life Technologies, Carlsbad, CA, USA) or anti-miR TM negative control #1 (Cat. № AM17010, Ambion, Life Technologies, Carlsbad, CA, USA) were cultured in 100 μl of RPMI-1640 medium at a final concentration of 10 5 cells/ml in 96-well plates. The cells were incubated at 37˚C in 5% CO 2 . After 48-h incubation, fresh RPMI medium containing L-glutamine and 10% FBS was mixed with NucBlue1 Live fluorescent reagents from the Ready Probes1 Cell Viability Imaging Kit (Blue/Red) (Molecular probes, Life Technologies, Eugene, Oregon, USA) and CellEvent1 Caspase-3/7 Green (Molecular probes, Life Technologies, Eugene, Oregon, USA) according to the manufacturer's protocol. The culture medium in the wells was then replaced with a medium containing both fluorescent reagents mentioned above according to the manufacturer's protocol. After 30 min of incubation, cells positive for Caspase-3/7 were visualized and photographed using Floid1 Cell Imaging Station (Life Technologies, Bothell, WA, USA). The nuclei of living cells were stained in blue by NucBlue1 Live reagent (Molecular probes, Life Technologies, Eugene, Oregon, USA). The cytoplasm of Caspases-3/7 positive cells appeared green.

Cell viability/proliferation MTT-test
Cells transfected with hsa-miR-4286 anti-miR TM miRNA inhibitor (Cat. № AM17000, Ambion, Life Technologies, Carlsbad, USA) or anti-miR TM negative control #1 (Cat. № AM17010, Ambion, Life Technologies, Carlsbad, CA, USA) were cultured for 24 h, detached with 0.25% trypsin solution in HBSS (Gibco1, Paisley, UK), and replaced in 96-well plates in a final medium volume of 100 μl per well and a concentration of 3Á10 4 cells per ml. MTT assays were performed 24, 48, 72, and 96 h after transfection. To this end, the culture medium was replaced and 10 μl of 2-(4,5-dimethyltriazol2yl)-2,5-diphenyltetrazolium bromide (MTT) solution was added to each well. The cells were incubated with MTT for 4 h at 37˚C in 5% CO 2 . The solution was then removed, followed by dissolving the pellet with 200 μl of DMSO per well before being incubated at room temperature for 30 min. The absorbance of stained supernatants was measured with an EFOS-9305 spectrophotometer (Shvabe-photosystems, Moscow, Russia) at 560 nm. Cell viability/proliferation was directly proportional to the absorbance. The relative cellular growth was assessed based on the ratio of average absorbance of hsa-miR-4286 anti-miR TM miRNA inhibitor cells to the average absorbance of anti-miR TM negative control #1 cells. The experiment was performed in triplicate. KEGG pathway analysis of miRNA target genes DIANA-mirPath v.3.0 was used for the KEEG pathway analysis of the miRNA signature. MiRNA targets were predicted based on DIANA-microT-CDS and heatmaps were generated using the log-transformed enrichment p-values as features for each miRNA. The threshold (Pvalue 0.05 and false discovery rate (FDR) 0.05) was calculated using Fisher's exact test.

Migration and invasion assay
To predict the gene targets of hsa-miR-4286, three different algorithms, TargetScan 7.0, miRWalk 2.0, and miRTarBase v.4.5 were applied to identify the validated targets of hsa-miR-4286. To reduce the false-positive results, only genes identified by all three methods were selected as miRNA targets to be subsequently analyzed. Cumulative context score was applied to choose most targeted genes for miR-4286. The PANTHER™ v.10.0 classification system was used to interpret the biological function of the validated targets of hsa-miR-4286.

Statistical analysis
The Expression Console and Transcriptome Analysis Console 2.0 (Affymetrix, USA) software were used for quality control, statistical analysis and miRNA annotation. The data were automatically statistically analyzed using ANOVA test and FDR-corrected values. MicroRNAs expression differences were assessed using a Mann-Whitney U-test and considered significant at p 0.05. The hierarchical clustering of differently expressed microRNAs was generated by the Transcriptome Analysis Console 2.0 software (Affymetrix, Santa Clara, CA, USA). Realtime PCR data were analyzed by using the ΔΔC t method and a nonparametric Mann-Whitney U-test with the Statistica 6.1 software (StatSoft, Moscow, Russia). The data on cell proliferation, viability, apoptosis, migration, and invasion were analyzed using a nonparametric Mann-Whitney U-test. The differences were considered significant at p < 0.05.
The efficiency of cell transfection with anti-miR-4286 was estimated based on a real-time PCR analysis of HMGA2 expression in the transfected cells. Specifically, transfected BRO and SK-MEL-1 cells with let-7c miRNA inhibitor resulted in a 30% increase in HMGA2 expression compared to the negative control (p = 0.0495), (Table 3). Furthermore, real-time PCR determined that the miR-4286 levels were down-regulated in both anti-miR-4286 transfected cell types compared to the negative control (p = 0.0495), (data are presented in Fig 3, S3 Table).

Signal pathway analysis
The study of FFPE-embedded tissues identified 52 signaling pathways as significantly modulated. Specifically, fatty acid biosynthesis, elongation, metabolism, AMPK signaling, actin cytoskeleton regulation, and melanoma pathways were altered in melanoma compared to benign melanocytic lesions.
Furthermore, melanoma samples exhibited alterations in the Hippo, viral carcinogenesis, TGF-beta signaling, cell cycle and adherens junction pathways. The full list of signaling pathways associated with differently expressed microRNAs is presented in S4 Table. MiR-4286 Furthermore, miR-4286 was more than 17.8-fold up-regulated in melanoma compared to benign melanocytic tumors in our study. Thus, we carried out a biometrical analysis of target genes of miR-4286 and a functional study of its activity using a miR-4286 specific inhibitor that was transfected in melanoma cells.
Apoptosis detection. To further investigate the effect of miR-4286 on apoptosis in cancer cells we transfected cells with the miR-4286 inhibitor and examined its effect on melanoma cells. Flow cytometry showed that miR-4286 inhibition in BRO melanoma cells increased the apoptosis rate 2.6-fold and the number of melanoma cells in the pre-apoptotic phase also increased 2-fold ( Fig 5, S7 Table). A fluorescence microscopy assay revealed increase in the caspase 3/7 activity in miR-4286-inhibitor transfected cells.
Migration, invasion analysis. We performed an in vitro cell invasion and migration assay to further determine if miR-4286 affects cell invasion and migration. The inhibition of miR-   Table. These results suggest that the down-regulation of miR-4286 does not inhibit the migratory and invasive capacities of melanoma cells in vitro.
MiR-4286 gene targets validation by real-time PCR after miR-4286 inhibitor application. To understand the mechanism by which miR-4286 affects cell proliferation and apoptosis, we studied the expression levels of miR-4286 target genes that regulate these processes. Indeed, miR-4286 inhibition resulted in degradation of folylpolyglutamate synthase, RNA polymerase I-specific transcription initiation factor, apelin, G-protein-coupled receptor 55, and high-mobility group A1 protein mRNA in BRO melanoma cells, as measured by real-time PCR (Fig 7, S9 Table). In SK-MEL-1 cells, only the expression of folylpolyglutamate synthase was down-regulated (Fig 8, S9 Table).
The present study identified several novel microRNAs aberrantly expressed in melanoma compared to melanocytic nevi. Specifically, the investigation of FFPE melanocytic tumors showed that miR-4454 and miR-7975, whose functions have not yet been studied, were overexpressed.
A pathway analysis revealed that fatty acid biosynthesis, elongation, degradation, and metabolism were altered in melanoma samples compared to nevi samples. The previous studies showed that the fatty acids play a role in melanomagenesis. In contrast to most normal cells, fatty acid synthase is overexpressed in a variety of human cancers, including cutaneous melanoma, in which its elevated expression levels are associated with tumor invasion and poor prognosis. Furthermore, fatty acid synthase inhibition induced apoptosis in melanoma cells [34]. Other aberrantly activated pathways in melanoma compared to melanocytic nevi include the AMP-activated protein kinase (AMPK), hippo and transforming growth factor-beta (TGFbeta) signaling pathways. AMPK is known as an important regulator of cellular energy homeostasis and has been shown to control tumor progression by regulating the cell cycle, protein synthesis, cell growth and survival [35].
Hippo signaling was first described to suppress proliferation and induce apoptosis. Moreover, the Hippo signaling pathway is also responsible for cell-cell adhesion interactions during metastasis development, and it has been also described to play a role in cancer stem cell regulation [36]. TGF-beta is a pleiotropic regulator of cancer cell activity that affects cell proliferation, apoptosis, differentiation, and immune function [37]. Specifically, TGF-beta induces epithelial-to-mesenchymal-like transitions in melanoma cells and peritumoral angiogenesis [38; 39]; recent studies by Fedorenko et al. showed that treating melanoma cells with vemurafenib induce fibroblasts activity by releasing TGF-beta, which leads to therapeutic resistance [40].

Impact of miR-4286 on melanoma cells behavior
The inhibition of miR-4286 led to diminishing cell proliferation rate in both melanoma cell lines and triggered an increase in BRO apoptotic melanoma cells as indicated by elevated numbers of Annexin V+/7AAD-detected by flow cytometry. Unfortunately, Annexin V/7AAD staining does not allow to clearly differentiate late apoptosis and necrosis. Our data show that inhibition of miR-4286 increases the apoptosis and necrosis of BRO melanoma cells that is partially referable to the apoptosis induction. Moreover, the inhibition of miR-4286 down-regulated the expression of its targets. Taken together, these findings suggest that miR-4286 is involved in cell proliferation and apoptosis regulation. Apelin modulates these processes via PI3/Akt signaling pathway [41,42], and G protein-coupled receptor 55 triggers MAPK cascade [43]. Furthermore, high-mobility group A1 protein is involved in Wnt/β-catenin pathway [44]. SK-MEL-1 melanoma cells proliferation was altered after miR-4286 inhibitor application, which matched the changes in the levels of folylpolyglutamate synthase (FPGS), an enzyme crucial for survival of dividing cells. FPGS expression levels showed different tendencies in BRO and SK-MEL-1 cells. This phenomenon can be explained by different action of FPGS in melanoma cells due to its oxidative status. Melanoma metastases of visceral organs (BRO melanoma cells) are more subjected to oxidative stress that leads to FPGS activation as folate metabolism is a major source of NADPH for oxidative stress reactions. On the contrary, primary tumors and regional lymph nodes metastasis (SK-MEL-1) showed a reduced expression of folate metabolism enzymes because oxidative stress reactions are not present [45]. MiR-4286 does not target p53 since its levels were not altered after miR-4286 inhibitor application that may be due to the activation of p53-independent apoptosis in cells.

Conclusion
The obtained data demonstrate that miR-4286 contributes to the regulation of melanoma cell proliferation and apoptosis by triggering several pathways, suggesting that it exerts a pleotropic effect.
Supporting Information S1