m6A RNA Methylation Promotes the Melanoma Metastasis Mediated by Extracellular Vesicle miR-23a-5p

Simple Summary

Melanoma is an aggressive skin cancer with a high tendency to metastasize. This study reveals that highly metastatic melanoma cells can enhance the metastatic ability of less aggressive tumor cells by sending extracellular vesicles (EVs) containing a molecule called miR-23a-5p. Once inside the recipient cells, miR-23a-5p reduces levels of the protein METTL14, which alters m6A modifications on RNA and silences two tumor suppressor genes, Mtus1 and Prrg4. This process ultimately increases the invasive behavior of melanoma cells. These findings provide new insights into how tumor cells communicate to drive metastasis and suggest potential targets for future diagnosis and therapy.

Abstract

Background/Objectives: Melanoma, characterized by high rates of metastasis and recurrence, is a particularly aggressive malignant tumor. The underlying mechanisms driving its progression remain enigmatic. The close interplay between tumor and non-tumor cells is pivotal, significantly shaping the tumor microenvironment. Extracellular vesicles emerge as a crucial vector influencing this environment, as they can modulate cellular mechanisms and biological functions—marking a key frontier in tumor mechanism research. However, the potential impact of intercellular communication on tumor cell biology remains largely unexplored. Methods: In the study, we employed a pair of cell lines derived from melanoma M14 cells, designated as highly metastatic cells (POL cells) and the low metastatic cells (OL cells), and elucidate their characteristics. Results: Our findings revealed that POL cells can potentiate the metastatic potential of OL cells through the transfer of extracellular vesicles, which harbor functional microRNAs, specifically miR-23a-5p in this context. Upon entering OL cells, the EV-miR-23a-5p orchestrates changes in the m6A modification levels of the mRNA of tumor suppressor genes Mtus1 and Prrg4. Conclusions: This modulation subsequently influences the expression of these genes and, in turn, the invasive behavior of OL cells.

1. Introduction

Melanoma has high incidence and mortality rates due to its metastatic propensity and frequent recurrence [1], yet its etiology remains elusive. The field of melanoma research is extensive, covering areas such as immunotherapy [2], BRAF/NRAS mutations [3] and checkpoint blockade [4]. Currently, researchers are increasingly focusing on histone lactylation, a modification that regulates methylation processes and influences tumor initiation and progression [5]. The regulation of autophagy also plays a role in the advancement of melanoma [6]. Additionally, alterations in the tumor microenvironment are a pivotal area of investigation [7], particularly concerning the mechanisms behind these changes [8,9,10,11,12,13]. Research into melanoma’s tumor microenvironment currently encompasses the impact of tumor cells on growth and development through interactions with normal cells via extracellular vesicles [14]; adoptive cell therapy, utilizing in vitro-amplified tumor-infiltrating lymphocytes, can help eliminate or reduce metastatic melanoma [15]; and miRNAs derived from highly invasive melanoma cells can enhance the invasive capacity of less invasive melanoma cells, at least in part via EV-mediated miR-411-5p transfer [16].

Investigations into the complex interactions between neoplastic cells and their healthy somatic microenvironment are central to delineating the intricate mechanisms that drive the initiation and progression of tumors. While the study of inter-tumor dynamics remains in its nascent stages, compelling research has identified a significant role for extracellular vesicles. These EVs, originating from melanoma primary lesions, have been shown to stimulate lymphangiogenesis, thus establishing a favorable landscape for the metastasis and adhesion of tumor cells [17]. Our laboratory has successfully established melanoma cell lines exhibiting high and low invasive and metastatic tendency to investigate the mechanisms of inter-tumor communication (POL cells and OL cells). Our studies reveal that POL cells can transduce high invasive and metastatic characteristics to OL cells via microRNA-enriched EVs. This transmission modulates tumor-related signaling pathways, furthering cancer progression. This exemplifies a common pattern of extracellular vesicle-mediated intercellular communication observed across different cancer types. For instance, we noted a correlation with findings from other research where highly invasive and metastatic liver cancer cells were reported to have a pronounced effect on transforming normal fibroblasts into cancer-associated fibroblasts (CAFs). These transformative effects are mediated through microRNAs in secreted EVs, which specifically target the β1-integrin-NF-κB signaling pathway [18].

Despite the underexplored domain of m6A in investigating the crosstalk between POL cells and OL cells, it is a critical post-transcriptional modification of mRNA in eukaryotic organisms. m6A regulates the expression of oncogenes and tumor suppressor genes. This regulation can influence the proliferation and metastatic abilities of tumor cells, thereby significantly impacting the progression of tumorigenesis [19]. Malignant tumors are characterized by metabolic reprogramming, with m6A modification influencing nearly all stages of RNA metabolism. Consequently, m6A plays a pivotal role in tumor metabolism [20,21]. The impact of m6A on RNA metabolism is inevitably linked to the alterations in key factors that govern tumor initiation and progression, underscoring the significant influence of m6A on both tumor onset and development [22,23].

To address this gap, we established melanoma cell lines with differing invasive capacities (POL and OL). We then investigated whether POL-derived, microRNA-enriched EVs regulate m6A modification in recipient tumor cells and thereby modulate the stability and turnover of tumor suppressor mRNAs, ultimately promoting melanoma migration and invasion. Therefore, this study aimed to determine whether EV miR-23a-5p promotes melanoma metastasis via m6A-dependent regulation of tumor suppressor Mtus1 and Prrg4 mRNAs.

2. Materials and Methods

2.1. Cell Culture

Green Fluorescent Protein (GFP)-tagged M14 cells were human melanoma sublines provided by Dr. Robert Hoffman (University of California San Diego) through a generous contribution to our research [24,25]. Derived from the M14 cell line, OL, POL, and their various engineered variants (POL-shRab27a, OL-mimics-miR-23a-5p, POL-inhibitor-miR-23a-5p, OL-agomir-miR-23a-5p, OL-OEMettl14 and OL-shMettl14) exhibit distinct molecular characteristics vital for our investigation into melanoma cell behavior. These cell lines were cultured in Dulbecco’s Modified Eagle Medium (DMEM) high-glucose medium enriched with 10% Fetal Bovine Serum (FBS) to maintain optimal growth conditions. The cell lines were incubated at 37 °C in a humidified atmosphere containing 5% CO₂.

2.2. Animal Experiments

All animal experiments were approved by the Ethics Committee of Chongqing Medical University and conducted in accordance with international and national guidelines for laboratory animal welfare. The study was conducted in an SPF facility using IVC cages, with controlled environmental parameters (temperature, humidity, light cycle). Eight-week-old female NOD/SCID mice were randomly assigned to comparative groups, each comprising 5 animals, to elucidate the effects of miR-23a-5p on melanoma cells. The groups were as follows: 1. OL-agomir-NC vs. OL-agomir-miR-23a-5p; 2. OL-agomir-miR-23a-5p-OENC vs. OL-agomir-miR-23a-5p-OEMettl14. In compliance with the approved experimental protocol, each of the eight-week-old female NOD/SCID mice was administered a controlled volume of 5 × 10⁵ tumor cells via intravenous injection through the tail vein, with precision dosing to allow for systematic observation and monitoring of the disease progression. On the 25th day post-inoculation, the mice were euthanized by cervical dislocation following appropriate anesthesia, in accordance with institutional animal care guidelines. Lung metastases in the mice were observed using Sellstrom Z87 fluorescent goggles, equipped with an LDP 470 nm light source for exciting fluorescent proteins.

2.3. Cell Transfection

The miR-23a-5p mimics, agomir of miR-23a-5p, Cy5-labeled miR-23a-5p mimics, and inhibitor of miR-23a-5p, essential for the experimental components of this study, were procured from GenePharma (Shanghai, China). Melanoma cells were seeded in 6-well plates and cultured until they reached 70–80% confluency. For each well, 50 pmol of miRNA mimics, agomir, or Cy5-labeled mimics, and 100 pmol of miRNA inhibitor, were used. These RNA oligonucleotides were diluted in 250 µL of Opti-MEM^® reduced serum medium (Gibco, Waltham, MA, USA). Separately, 5 µL of Lipofectamine™ 2000 (Invitrogen, Carlsbad, CA, USA) was diluted in 250 µL of Opti-MEM^®. After 5 min incubation at room temperature, the diluted RNA and diluted Lipofectamine™ 2000 were combined, mixed gently, and incubated for another 20 min at room temperature to allow complex formation. The entire mixture was then added dropwise to each well containing complete culture medium without antibiotics. The final concentrations were 25 nM miRNA mimics, 25 nM agomir and 25 nM Cy5-labeled mimics, and 50 nM miRNA inhibitor. Following the 48 h incubation period post-transfection, the efficiency of the transfection process was quantitatively assessed.

After confirming transfection efficiency, subsequent experiments were performed. The lentiviral particles for both Mettl14 overexpression and knockdown were obtained from GeneChem (Shanghai, China). Cells were infected with a multiplicity of infection (MOI) of 10 according to the manufacturer’s protocol. Cells in logarithmic growth phase were seeded at a density of 5 × 10⁴ cells/well in 24-well plates. Subsequently, each well received an inoculation of 5 µL of the corresponding lentiviral particle reagent. After a 3-day incubation period, cells were prepared for quantitative real-time polymerase chain reaction (qRT-PCR) to evaluate the efficiency of Mettl14 modulation.

2.4. EV Isolation

Cells (OL and POL) were maintained in DMEM supplemented with 10% FBS for 48 h. The conditioned medium was centrifuged at 800× g for 5 min and then at 2000× g to clear cellular fragments. The ultracentrifuge we used was the Beckman Coulter Optima L-100XP, equipped with a fixed-angle rotor. The k factor for this rotor is 951. The supernatant was further processed with Beckman Coulter’s Optima L-100XP at 10,000× g for 30 min followed by ultracentrifugation at 100,000× g for 70 min. Precipitates were washed in PBS, centrifuged at 100,000× g for 70 min at 4 °C, resuspended in PBS, filtered (0.22 micron), and stored at −80 °C.

2.5. TEM

EVs were prepared for transmission electron microscopy (TEM) by fixation with 2.5% glutaraldehyde at 4 °C overnight. Post-washing in deionized water, they were deposited on formvar/carbon-coated grids and stained with phosphotungstic acid for 1 min. For TEM analysis, approximately 6–8 μg of total protein (based on BCA assay) in a volume of 20 μL was loaded onto each grid. The TEM analysis was conducted using a JEM-1400PLUS microscope from JEOL.

2.6. Nanoparticle Tracking Analysis

EV concentrations and sizes were assessed via Nanoparticle Tracking Analysis (NTA) by following the ZetaVIEW S/N 17-310’s user manual. Samples were suspended in 1 mL PBS and analyzed with the ZetaVIEW instrument (Particle Metrix GmbH, Munich, Germany). Their sizes and Brownian motion were determined, with ZetaView 8.04.02 software processing the results.

2.7. miRNA Expression Profiling

RNA was extracted, examined for quality, and processed for library construction and sequencing by BGI in Wuhan, China. ReadCount values were obtained, and sample uniformity between OL-EVs and POL-EVs was assessed using the Hmisc package in R. A correlation heatmap was generated with the pheatmap package. Differential expression analysis was performed using the limma package to calculate log fold changes (logFC) and p-values, enabling the identification of differentially expressed miRNAs. To investigate how POL cells may promote OL cell metastasis through cancer-promoting miRNAs carried by EVs, we selected miRNAs with Log₂(POL-EVs/OL-EVs) > 1 as candidates highly enriched in POL-EVs.

2.8. Western Blot

Cell lysates were prepared by incubating in 1% PMSF-treated RIPA buffer on ice. A total of 5–20 μg of protein was added to each well on 12% polyacrylamide gels (Beyotime, Shanghai, China). The gels were first electrophoresed at 80 V for 30 min, followed by 120 V for 1 h, and then transferred to PVDF membranes by wet transfer at a constant current of 300 mA for 1 h. The membrane was blocked for 1 h at room temperature with 5% milk in Tris-buffered saline, followed by overnight incubation with primary antibodies (anti-ALIX, CD81, CD63, 1:1000, ALBUMIN 1:5000, from Abcam; goat anti-rabbit IgG, goat anti-mouse IgG, both at 1:5000, from Proteintech Group, Inc., Rosemont, IL, USA) at 4 °C and a subsequent 1 h incubation with secondary antibodies at room temperature. Band analysis was conducted using ImageJ 1.50e software (National Institutes of Health, Bethesda, MD, USA), and the process was repeated for three independent trials to ensure statistical reliability.

2.9. qRT-PCR Analysis

RNA was isolated using TRIzol Reagent from Takara Biotechnology. mRNA levels were quantified after reverse transcription of 1 μg total RNA with PrimeScript RT Master Mix. Quantitative RT-PCR (qRT-PCR) was conducted using the SYBR Green Real-Time PCR Master Mix with a program consisting of pre-incubation at 95 °C for 30 s, followed by 39 cycles at 95 °C for 5 s and 60 °C for 30 s. We used the Agilent AriaMx Real-Time PCR System. The mRNA expression levels were determined via the 2^−ΔΔCT method. For miRNA analysis, the Mir-X miRNA First-Strand Synthesis Kit facilitated cDNA synthesis, and subsequent qRT-PCR was similarly performed with SYBR Green. The expression of U6 served as an endogenous control for normalization. The oligonucleotide sequences used are detailed in Supplementary Materials: Primers used in qRT-PCR.

2.10. Transwell Migration and Invasion Assays

Cell invasion was assessed using Transwell inserts equipped with 8-micrometer polycarbonate filters. For invasion assays, Matrigel diluted to 1:8 in serum-free medium (30 μL, supplied by BD Biosciences) was applied to the filters. A control migration assay was set up without Matrigel. A volume of 500 μL serum-free medium supplemented with 3 × 10⁴ cells was placed in the upper chamber, while 800 μL culture medium enriched with 20% FBS was added to the lower chamber. The apparatus was incubated for 20 h. Post-incubation, cells adhering to the membrane’s lower surface were fixed with 70% ethanol for 20 min at 4 °C and then stained with crystal violet for 10 min. Any cells remaining on the upper surface were gently wiped off with a cotton swab. Images of the cells that had migrated and invaded were captured using light microscopy. For subsequent statistical analysis, we randomly selected nine fields of view in the Transwell chambers and counted the number of migrated and invaded cells. Three independent replicates were performed under consistent conditions.

2.11. EV Labelling and Visualization of Cellular Uptake

We transfected POL cells with Cy5-tagged miR-23a-5p mimics and cultured them for three days, allowing them to secrete fluorescent EVs. These EVs, marked with Sigma’s PKH26 dye, were collected and added to OL cells. The EVs’ interaction with OL cells was explored through steps of 6 h co-culture periods. The cells were fixed with 4% paraformaldehyde, stained with DAPI to visualize the nuclei, and then observed under a TCS SP2 LEICA confocal microscope. Distinct interactions between cells and fluorescently labeled EVs were documented to assess the miR-23a-5p mimics’ mobility and influence on OL cells.

2.12. Bioinformatics Analysis

miRDB (miRDB—MicroRNA Target Prediction Database) and miRWalk (Home—miRWalk (uni-heidelberg.de)) were deployed to forecast the potential targets of miR-23a-5p. We applied the DAVID (https://david.ncifcrf.gov/ (accessed on 15 October 2025)) platform to conduct Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. RMVar (database of functional variants involved in RNA modification (renlab.org)) and GEO (GSE154952 and GSE134388, Home—GEO—NCBI (nih.gov)) datasets were used to find the genes related to m6A and Mettl14 (logFC > 1, p < 0.05).

2.13. m6A Methylation-Level Detection

The experiment utilized the EpiQuikTM m6A RNA Methylation Quantification Kit, where fresh cells were sourced for RNA extraction. For each reaction, 200 ng of total RNA was added per well. RNA was immobilized and purified within wells before staining. Relative quantification was achieved through spectral analysis at 400 nm, comparing RNA samples to both negative and positive controls.

2.14. RNA-Binding Protein Immunoprecipitation (RIP)

Harvest cells into centrifuge tubes for lysing, then prepare the resulting protein solution for future use. Magnetic beads should be pre-cleaned with three washes in RIPA buffer. Following this, co-incubate the protein solution with magnetic beads at 4 °C with gentle stirring at 10 r/min for 2 h. Subsequently, clear unbound proteins from the beads, and proceed to an overnight m6A antibody (Proteintech, Cat No. 68055-1-Ig) incubation under the same temperature and rotation settings. Once the proteins have been specifically tagged with antibodies, clean the beads again and resuspend the content in RIPA buffer. Split the mixture into two: one aliquot goes towards the isolation of RNA for subsequent qRT-PCR analysis, and the other towards the acetone-induced precipitation aimed for protein fractionation.

2.15. Co-Culture Experiments

Seed OL cells at 60% density in a 6-well plate and allow 2 h for adhesion. Then, position a 0.4 um pore-sized (Corning) cross well co-culture chamber on the plate, populating it with 5 × 10⁴ POL cells. Cultivate the POL cells in DMEM supplemented with 10% FBS. After a 24 h co-culture period, detach the chamber and apply trypsin to OL cells to facilitate Transwell migration/invasion and qRT-PCR studies.

2.16. Ethical Approval

The in vivo research was conducted with a high standard of animal care and ethics, following the stringent protocols reviewed and approved by Chongqing Medical University (Ethics Approval ID: 2024055; Date Approved: 20 March 2024). Their guidelines ensured that all animal work was ethically sound and conformed to the best practices in animal welfare.

2.17. Statistical Analysis

All experiments were independently repeated at least three times, and the data were subjected to statistical analysis. Quantitative results are expressed as mean ± standard error of the mean (SEM). Data analysis was performed using the two-tailed Student’s t-test in GraphPad Prism version 8.0 (GraphPad Software Inc., San Diego, CA, USA). Significance levels are denoted as * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.

2.18. MISEV2023 Compliance Statement

All EV-related experiments in this study were conducted in accordance with the Minimal Information for Studies of Extracellular Vesicles 2023 (MISEV2023) guidelines [26]. This includes adherence to recommended nomenclature, isolation protocols, orthogonal characterization methods (TEM, NTA, immunoblotting) and functional study controls to ensure rigor and reproducibility. Detailed experimental parameters are provided in the relevant sections to align with MISEV2023 principles.

3. Results

3.1. EVs from POL Cells Can Promote the Migration and Invasion of OL Cells

OL and POL cells derived from the M14 melanoma cell line represented low metastasis ability and high metastasis ability, respectively [24,25]. At first, we carried out characterization of the isolated EVs, including transmission electron microscopy (TEM) analysis, which revealed their typical double-layered membrane vesicles; nanoparticle tracking analysis, which confirmed their nanometer scale (100–200 nm); and immunoblotting to identify associated proteins such as ALIX, CD81, CD63, and ALBUMIN (Figure 1A–C, Document S1). To assess the influence of EVs from POL cells on OL cells, we adopted two experimental approaches. Directly adding POL-derived EVs to OL cells resulted in a noticeable elevation in both in vitro migration and invasion capabilities of the latter, an effect that was statistically significant (Figure 1D, p < 0.01). Co-culturing these cells yielded similar results, with OL cells’ migration and invasion abilities being noticeably improved (Figure 1E, p < 0.05). To ascertain whether the observed effects were mediated by POL EVs, we knocked down the Rab27A, which could promote EV secretion in POL cells. Consequently, the reduction in secretion was mirrored by a decrease in the aggressive phenotype of OL cells, succinctly proving the catalytic role of POL EVs in cell migration (Figure 1F, p < 0.001) and invasion (Figure 1F, p < 0.01).

All of these results indicated that POL cell-derived EVs potentiate the metastatic potential of OL cells in vitro.

3.2. EV miR-23a-5p Is a Key Regulatory Factor That Promotes the Migration and Invasion Ability of OL Cells

Our hypothesis posits that microRNAs (miRNAs), specifically those residing within POL cell EVs, hold a pivotal role in modulating the metastatic potential of OL cells, as evidenced by previous studies [16,17]. To validate this, we conducted exhaustive sequencing of EVs, revealing distinct miRNA expression profiles between high-metastatic POL and low-metastatic OL cells (Figure 2A). Notably, miR-23a-5p stood out for its marked upregulation in POL EVs, a finding that aligns with its documented association with cancer progression. To confirm this observation, we employed quantitative reverse transcription PCR (qRT-PCR), which corroborated the elevated expression of miR-23a-5p in POL cells (Figure 2B, p < 0.001) and further demonstrated its enhanced expression in OL cells upon exposure to POL-derived EVs (Figure 2C, p < 0.001), underscoring its pivotal function. To delve deeper into miR-23a-5p’s modus operandi within OL cells, we utilized confocal microscopy. By tagging miR-23a-5p with a CY5 green fluorescence marker and staining EVs with PKH26 red fluorescence, we observed its packaging into EVs in POL cells and subsequent transfer into OL cells during a 6 h co-culture period (Figure 2D). This finding positions miR-23a-5p as a prime candidate for functional exploration. To assess the functional implications of miR-23a-5p in cancer cell migration and invasion, we conducted a battery of functional assays. Overexpression of miR-23a-5p in OL cells significantly augmented their migratory (Figure 3A, p < 0.01) and invasive (Figure 3A, p < 0.001) capabilities in vitro, hinting at a pro-metastatic role. Conversely, when miR-23a-5p was knocked down in POL cells and their EVs were subsequently administered to OL cells, we observed a notable decrease in OL cell migration (Figure 3B, p < 0.05) and invasion (Figure 3B, p < 0.01), emphasizing the influence of miR-23a-5p-depleted EVs. To extend our findings to an in vivo setting, we injected miR-23a-5p-overexpressing OL cells into immunodeficient NOD/SCID mice via tail vein injection. Given that OL cells typically induce pulmonary oligometastasis, whereas POL cells elicit multi-organ metastasis [18], we aimed to discern the effect of miR-23a-5p upregulation on OL cell metastasis. After 25 days of monitoring, we performed cervical dislocation and lung dissection, followed by histological analysis using Hematoxylin and Eosin (H&E) staining. Our findings revealed a striking enhancement in the metastatic potential and colonization of OL cells (Figure 3C,D), conclusively demonstrating that miR-23a-5p overexpression promotes OL cell metastasis in vivo.

In conclusion, our study underscores the ability of highly metastatic POL cells to bolster the metastatic prowess of low-metastatic OL cells through the extracellular vesicle transfer of the oncogenic miR-23a-5p both in vitro and in vivo.

3.3. miR-23a-5p Promotes Transfer Properties Through Mettl14

Through miRDB and miRWalk, we pinpointed potential targets of miR-23a-5p, as depicted in Figure 4A. Subsequently, we harnessed the power of KEGG pathway analysis through DAVID, zeroing in on methylation-related pathways that exhibited a notable connection to miR-23a-5p’s actions. Recognizing the pivotal role of m6A methylation amidst transcriptome dynamics, we decided to delve deeper into its mechanisms. To unravel the pivotal regulatory player in the interplay between m6A genes and miR-23a-5p, we embarked on a study using a univariate approach, analyzing sequencing data from OL and POL cells. Mettl14 emerged as a central figure in this narrative. Subsequent qRT-PCR assays uncovered a marked downregulation of Mettl14 in POL cells (Figure 4C), with a similar trend observed in OL cells upon miR-23a-5p overexpression (Figure 4D). By cross-referencing with the NCBI database, we precisely located the miR-23a-5p binding site on Mettl14 and introduced a mutation for a dual-luciferase reporter assay. Our findings unequivocally showed that miR-23a-5p significantly repressed the expression of Mettl14 with the wild-type sequence, as opposed to the mutated version (Figure 4E–G).

To evaluate Mettl14’s contribution to tumor progression, we knocked down its expression in OL cells and then conducted both in vitro Transwell assays and in vivo metastasis studies. Notably, Mettl14 knockdown significantly enhanced the metastatic potential of OL cells under ex vivo conditions (Figure 5A). Furthermore, tail vein injection of OL sh-Mettl14 cells in mice facilitated lung tumor colonization, reinforcing its crucial in vivo role (Figure 5B,C). To further explore the relationship between miR-23a-5p and Mettl14, we concurrently manipulated the expression levels of in OL cells. Our results indicated that elevating Mettl14 levels could effectively modulate the oncogenic effects elicited by miR-23a-5p both in vitro and in vivo, as evidenced in Figure 5D–F.

3.4. Mtus1 and Prrg4 Are the Main Pathways for Inhibiting Tumor Metastasis

The N6-methylation of adenosine (m6A) stands as the predominant internal modification within eukaryotic mRNA, playing a pivotal role in regulating cellular processes such as differentiation, invasion, and apoptosis by modulating mRNA expression profiles [19,20,21]. Our investigation uncovered that POL cancer cells harbored notably lower m6A levels compared to OL cells, with miR-23a-5p overexpression augmenting these levels, potentially fostering cancer progression (Figure 6A). Then, we leveraged the RMVar and GEO resource database datasets, specifically GSE154952 and GSE134388, focusing on melanoma and Mettl14. This comprehensive analysis enabled us to identify and correlate differentially expressed genes regulated by m6A with Mettl14, ultimately narrowing down our focus to 19 positively correlated genes (Figure 6B–D). Notably, Mtus1 and Prrg4 emerged as key genes from our correlation studies, exhibiting a favorable correlation with patient prognosis. Intriguingly, our experiments utilizing the transcription inhibitor actinomycin D revealed that m6A modification stabilized the mRNA of Mtus1 and Prrg4. This finding was further underscored by a subsequent decrease in their expression levels upon miR-23a-5p overexpression, highlighting the intricate interplay between m6A and these genes (Figure 6E,F). To confirm the regulation of these genes by m6A modification, we conducted RIP assays employing an m6A-specific antibody. Our results unequivocally demonstrated that both Mettl14 suppression and miR-23a-5p overexpression led to reduced expression of Mtus1 and Prrg4, reinforcing the pivotal role of m6A in their regulation (Figure 6G).

4. Discussion

Metastatic melanoma’s rising incidence and variable prognosis are largely influenced by EVs, which disseminate factors like miRNAs that foster tumor progression by altering the recipient cell’s microenvironment. While earlier studies targeted tumor impacts on angiogenesis and cell inhibition, the role of EV-mediated intercellular tumor communication remains underexplored [22,23,24]. This study illuminates EVs’ influence on the metastatic potential of tumor cells, particularly how miR-23a-5p released from POL cells affects m6A levels and inhibits tumor suppressors Mtus1 and Prrg4 in OL cells, boosting their metastatic and invasive properties. The findings suggest therapeutic potentials in melanoma via miR-23a-5p or methylation interventions.

Research indicates that tumor EVs facilitate signal transduction between tumor and non-tumor cells, thereby driving the reconfiguration of the tumor microenvironment [27]. Stem cells can modulate the tumor microenvironment through EVs, contributing to drug resistance in cancer therapy [28]. Furthermore, EVs from tumor cells can induce M2 polarization in macrophages, thereby enhancing tumor invasion by altering the tumor microenvironment [29,30]. However, this article delves into the relationship between highly invasive and low invasive cells within tumor cells. It underscores that while the tumor microenvironment’s significance lies in interactions between tumor and normal cells, it also holds a crucial role within the tumor itself.

m6A modulation exhibits a bi-directional effect in tumors [31], acting to either promote cancer progression by influencing the translation of specific proteins or inhibit tumor growth through the regulation of distinct mechanisms [32,33,34,35]. This dual functionality of m6A is evident in its varied impact on different types of cancer, as it governs the expression of downstream genes. For instance, research has demonstrated that m6A can suppress melanoma development by modulating the expression levels of certain key proteins [36]. In our study, we investigate how m6A regulates the expression of downstream genes to inhibit the progression of melanoma.

Our studies on miR-23a-5p in EVs found that it significantly advances melanoma metastasis. Due to this role, miR-23a-5p holds promise as a biomarker for diagnosis, with miRNA mimics offering potential for extracellular vesicle diagnostics. Most notably, targeting miR-23a-5p could offer a therapeutic intervention for treating melanoma. Restoring Mettl14-dependent m6A on Mtus1 and Prrg4 may impede melanoma progression. If miR-23a-5p and reduced methylation promote tumor growth, further methylation modulation of Mtus1 and Prrg4 could amplify therapeutic efficacy.

While our study elucidates a novel EV–miRNA–m6A axis in melanoma metastasis, several limitations should be acknowledged. First, the experiments were conducted primarily in a single genetic background using M14-derived cell lines, which may not fully capture the heterogeneity of melanoma. Second, the reliance on immunodeficient tail vein metastasis models focuses on late metastatic stages and does not recapitulate the early steps of the metastatic cascade within an intact immune microenvironment. Third, the absence of patient-derived samples or clinical validation limits the direct translation of our findings to human disease. Finally, our mechanistic insights are limited by the reliance on total RNA m6A measurements, as we did not directly assess the m6A status of the two downstream tumor suppressor gene transcripts. Consequently, the observed correlations between global m6A modification levels and target gene stability remain indirect and require further locus-specific validation to establish causality. Addressing these limitations in future studies will strengthen the biological and clinical relevance of the proposed pathway.

5. Conclusions

In summary, this study demonstrates that highly metastatic melanoma cells enhance the aggressiveness of low metastatic cells by delivering miR-23a-5p via extracellular vesicles. Mechanistically, miR-23a-5p downregulates the m6A methyltransferase METTL14, leading to reduced m6A modification of the tumor suppressors Mtus1 and Prrg4. This results in destabilization and increased degradation of Mtus1 and Prrg4 mRNA, thereby downregulating their expression and ultimately contributing to the cancer-promoting effects of miR-23a-5p. These findings reveal a new EV–miRNA–m6A axis that promotes melanoma metastasis through intercellular communication, highlighting miR-23a-5p and m6A modulation as potential targets for diagnosis and therapy.