Clinicopathological role of microRNA‑205, microRNA‑223 and microRNA‑590 in vulvar cancer

Introduction

Vulvar cancer is a relatively rare form of malignant tumor that develops in the external female genital organs. According to studies conducted by the International Agency for Research on Cancer, vulvar cancer ranks 29th among the most common malignant neoplasms in the general global population and 4th among the most common gynecological cancers. Its incidence varies by region, ranging from 0.8 per 100,000 women in Asian countries to 3.2 per 100,000 women in developing countries (1,2). Epidemiological data indicate that in developed countries, such as the USA and Western European nations, vulvar cancer occurs more frequently compared with developing countries; it most commonly affects older women, primarily after menopause, with the median age at diagnosis being 69 years. However, in recent years, there has been an increase in the number of cases among younger women, which may be associated with human papillomavirus (HPV) infections. Although vulvar cancer accounts for only ~4% of all female genital tract cancers, its significance in public health is considerable, mainly due to diagnostic challenges and late detection, both of which negatively affect patient prognosis (3).

The etiopathogenesis of vulvar cancer is complex and involves environmental, genetic and immunological factors. The main causes include HPV infection (particularly high-risk types, such as HPV-16 and HPV-18), chronic inflammation, immunosuppression and genetic predisposition. Persistent infection with oncogenic HPV types can lead to the integration of viral DNA into the host genome, resulting in the inactivation of tumor suppressor proteins tumor protein 53 (TP53) and retinoblastoma protein (RB1) and uncontrolled cell proliferation. Immunosuppressed patients, particularly those on chronic steroid therapy (such as in lupus), have a 3-fold increased risk of developing the disease (4,5).

In recent years, particularly over the past decade, research efforts have increasingly concentrated on identifying novel pathological mechanisms that contribute to tumor development. MicroRNAs (miRNAs/miRs), which are small non-coding RNA molecules consisting of 21–23 nucleotides, have emerged as a key focus of these investigations. These molecules have been identified not only in humans but also across a broad spectrum of species, including plants, animals and certain viral entities. The initial discovery of miRNAs dates back to 1990, when they were first identified in Caenorhabditis elegans (6,7). A growing body of evidence has established the involvement of miRNAs in cancer pathogenesis, as well as in various other medical conditions, such as asthma, rheumatic diseases, multiple sclerosis and depression. These diminutive RNA fragments are integral to the mechanisms of RNA degradation and impede the translation of messenger RNA (mRNA). They exert their influence on gene expression by binding to the 3′untranslated region (3′UTR) of target mRNAs. Notably, the dysregulation of miRNAs is a hallmark of numerous types of tumor; moreover, the altered expression levels of specific miRNAs are often associated with particular tumors and disease stages. In addition to their roles in tumor evolution, recent studies have highlighted that miRNAs also contribute to the modulation of the tumor microenvironment (TME). This adjustment facilitates processes, such as angiogenesis, immune system infiltration and metastasis, underscoring the multifaceted functions of miRNAs in oncological contexts (8,9).

miR-205 has gained increasing attention in cancer research due to its dual role as both a tumor suppressor and an oncogene, depending on the context and type of tumor. Located at chromosome 1q32.2, miR-205 expression levels may vary across various cancer types and stages, reflecting its complex involvement in tumorigenesis. In addition to its regulatory roles, studies have indicated that miR-205 also influences the TME, promoting processes, such as immune evasion and angiogenesis (10,11). The dysregulation of miR-205 often occurs in conjunction with the expression of various other miRNAs, suggesting a complex regulatory network that can either suppress or promote tumor characteristics based on the biological context (12). Research continues to explore the potential therapeutic implications of modulating miR-205 expression as a strategy for cancer treatment, emphasizing the need for a more comprehensive understanding of its regulatory mechanisms in tumor biology (13).

miR-223 has emerged as a pivotal molecule in various biological processes, including inflammation, cancer progression and tissue repair. It is primarily expressed in granulocytes and its dysregulation has been linked to several diseases, particularly liver-related disorders and various types of cancer (14,15).

miR-590 has been identified as a potential oncogene in vulvar squamous cell carcinoma (VSCC), promoting tumor growth and metastasis through the regulation of key signaling pathways. Its elevated expression is associated with more aggressive disease, suggesting its value as both a biomarker and a therapeutic target. Although its exact mechanisms of action remain unclear, further research into miR-590 may support the development of targeted treatments for vulvar cancer (16).

The aim of the present study was the evaluation of the expression of miR-205, miR-223 and miR-590 using reverse transcription-quantitative (RT-q) PCR in histopathological material from paraffin blocks obtained from patients who underwent surgery due to vulvar cancer at the Department of Operative, Endoscopic and Oncological Gynecology Polish Mother Memorial Hospital in Poland and its effect on surveillance, the advance of the cancer process at the time of diagnosis and tumor grading. In selecting specific miRNAs for expression analysis, both their potential oncogenic and tumor-suppressive roles were taken into account; nevertheless, existing studies present inconsistent findings concerning their predominant biological function. To date, only isolated studies have examined individual miRNAs, and no comprehensive profiling has been performed, at least to the to the best of the authors' knowledge, emphasizing the importance of continued research in this field.

Patients and methods

Patients

In the present retrospective study, 38 patients with vulvar carcinoma were included. Of these, 25 had successful RNA extraction from both tumor tissue and matched non-malignant vulvar tissue collected intraoperatively from a site distant from the tumor margin, with the non-malignant tissue histopathologically confirmed to originate from a margin >1 mm, whereas 13 patients contributed tumor tissue only due to insufficient RNA yield from the matched non-malignant specimen. The median follow-up time was 49 months (range, 2–128 months). Patients alive at the last recorded contact were censored. Of note, three patients with missing follow-up data were excluded from the survival analyses [n=35 for overall survival (OS) analyses]. Fold-change-based Kaplan-Meier analysis was restricted to the 20 patients with matched tumor and control specimens and available OS data. At the time of surgical treatment, all patients provided written informed consent for the surgical procedure, the storage of biological material (biobanking), and the use of their tissue samples and clinical data for scientific research purposes in accordance with institutional regulations. The protocol was approved by the Bioethical Committee of the Polish Mother's Memorial Hospital (approval no. 49/2021). Tissue material for analysis was collected from archival paraffin-embedded blocks derived from intraoperative specimens. The study cohort consisted of patients treated surgically at the Department of Operative Gynecology, Endoscopy and Gynecologic Oncology (Mother's Memorial Hospital-Research Institute, Lodz, Poland) between 2012 and 2020. Disease stage was classified according to the International Federation of Gynecology and Obstetrics (FIGO) system. Histopathological assessment was performed at the Department of Clinical Pathology of the Polish Mother's Memorial Hospital-Research Institute (Lodz, Poland), while the molecular analyses of miRNAs were carried out at the CoreLab (Medical University of Lodz, 92–215 Lodz, Poland). Participants were women >40-90 years, representing all stages of vulvar cancer progression (FIGO IA-IVB). Body mass index values were between 20 and 37; however, the majority of the patients had a normal body weight. The characteristics of the patients are presented Table I.

Table I. Characteristics of study group patients with vulvar cancer.

Table I.

Characteristics of study group patients with vulvar cancer.

CharacteristicCases
Mean age (range)	71.5 (48–90)
Body mass index (kg/m2)	24.42 (20–37.2)
<24.9 (normal)	19 (61%)
25-29.9 (overweight)	9 (29%)
>30 (obese)	3 (10%)
Staging (FIGO)
I	19 (59%)
II	4 (12.5%)
III	2 (6%)
IV	7 (22%)
Grading
G1	3 (9.4%)
G2	27 (84.4%)
G3	2 (6.3%)

[i] FIGO, International Federation of Gynecology and Obstetrics.

miRNA expression analysis

Total RNA, including small RNA, was isolated from FFPE tissues using the High Pure miRNA Isolation Kit (Roche Diagnostics GmbH; cat. no. 05080576001), according to the manufacturer's instructions. Briefly, 5–10 µm FFPE tissue sections were first deparaffinized with 800 µl 100% xylene (Roche Diagnostics GmbH) for 5 min, followed by the addition of 400 µl 100% ethanol. The mixture was centrifuged for 2 min at 13,000 × g at room temperature and the supernatant was carefully discarded. The samples were then washed with 1 ml 100% ethanol and dried at 55°C for 10 min to ensure the complete evaporation of ethanol. The dried tissue was lysed with 100 µl Paraffin Tissue Lysis Buffer, 16 µl 10% SDS and 40 µl Proteinase K working solution (Roche Diagnostics GmbH), followed by incubation at 55°C overnight to ensure complete digestion. Following lysis, RNA was purified on columns according to the Roche's protocol. To each lysate, 325 µl Binding Buffer and 205 µl Binding Enhancer (Roche Diagnostic GmbH) were added, and the mixture was loaded onto High Pure Filter Tubes and centrifuged for 30 sec at 13,000 × g at room temperature. Columns were washed sequentially with 500 and 300 µl Wash Buffer (Roche Diagnostic GmbH), followed by an additional centrifugation at 13,000 × g for 1 min at room temperature to remove residual liquid. RNA was eluted with 50 µl Elution Buffer and incubated for 1 min at room temperature prior to centrifugation at 13,000 × g for 1 min at room temperature. RNA yield and purity (260/280 optical density ratios) were determined using a Picodrop spectrophotometer (Picodrop Limited). Isolated RNA was stored at −80°C.

Reverse transcription of miRNA

Complementary DNA (cDNA) synthesis was performed in a 7 µl reaction using the miRCURY LNA RT kit (Qiagen, Inc.; cat. no. 339340) according to the manufacturer's instructions, with 7 ng total RNA per reaction. The reaction mix was incubated for 60 min at 42°C followed by 5 min at 95°C for reverses transcription inactivation. Reactions were carried out using the GeneAmp PCR System 9700 Thermal Cycler (Applied Biosystems; Thermo Fisher Scientific, Inc.). The synthesized cDNA was stored at −20°C until qPCR analysis.

qPCR

qPCR was performed using the miRCURY Probe PCR Kit (Qiagen, Inc.; cat. no. 339372) in combination with miRCURY LNA miRNA Probe Assays (Qiagen, Inc.; cat. no. 339350) specific for hsa-miR-223-5p (GeneGlobe ID ZP00000465), hsa-miR-590-5p (GeneGlobe ID ZP00001844) and hsa-miR-205-5p (GeneGlobe ID ZP00000415). The small nuclear RNA U6 snRNA (GeneGlobe ID ZP00030496) was used as an internal reference for normalization. qPCR reactions were prepared in 10 µl volumes containing 5 µl of 2X miRCURY Probe PCR Master Mix, 1 µl of 10X target-specific assay, 2 µl cDNA (prediluted 1:40 in nuclease-free water according to the manufacturer's instructions), and nuclease-free water added to reach the final volume. Amplification was carried out on a 7900HT Fast Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.) under the following thermocycling conditions: Initial activation at 95°C for 2 min, followed by 40 cycles of denaturation at 95°C for 10 sec and annealing/extension at 60°C for 60 sec. All reactions were performed in duplicate. Relative expression level was determined using to the 2−ΔΔCq method (17). Genomic DNA was extracted from FFPE specimens with the High Pure PCR Template Preparation kit (Roche Diagnostics GmbH), according to the manufacturer's instructions. The DNA concentration was measured using a PicoDrop spectrophotometer (Picodrop Limited) and subsequently either applied directly for PCR amplification or preserved at −20°C until use. Total RNA was isolated from FFPE samples using the High Pure miRNA Isolation kit (Roche Diagnostics GmbH) according to the manufacturer's protocol. Following deparaffinization, lysis and column purification, RNA was eluted in 50 µl of buffer. RNA yield and purity (OD 260/280) were assessed with a PicoDrop spectrophotometer (Picodrop Limited). Extracted RNA was either used directly for cDNA synthesis or stored at −80°C.

Statistical analyses

Statistical analyses were conducted assuming a matched design, as tumor and control samples originated from the same patients (vulvar carcinoma tissue vs. distant non-malignant vulvar tissue). The distribution of miRNA expression was examined before group comparisons. Normality was assessed with the Shapiro-Wilk test and homogeneity of variance with Levene's test. miRNA expression was right-skewed and departed significantly from normality in the larger subgroups (Shapiro-Wilk P<0.05 for the G2-3 grade group for all three miRNAs and for several FIGO subgroups), whereas variances were homogeneous (Levene's P>0.30 for all comparisons). As the normality assumption was not met, non-parametric tests were used throughout: the Mann-Whitney U test for two-group comparisons (tumor grade; FIGO I–III vs. IV), the Kruskal-Wallis test for comparisons across the four FIGO stages, and the Wilcoxon signed-rank test for the paired tumor-control comparison. Overall survival was analyzed with Kaplan-Meier estimates and the log-rank test. Statistical analyses were performed using the STATISTICA Application, version 13, TIBCO Software Inc. P<0.05 was considered to indicate a statistically significant difference.

Results

In the present study, no statistically significant differences in OS were observed with respect to disease stage according to the FIGO classification. According to current knowledge, patients with FIGO stage I disease demonstrate the highest survival rates, as illustrated in Fig. 1. The results further suggest an improved survival of patients with grade G1 compared with G2 or G3 tumors. Although statistical significance was not reached, an increased expression of miR-205 was associated with a higher FIGO stage in the histopathological evaluation. When grouping FIGO I–III together and comparing them with FIGO IV, a non-significant trend towards higher miR-205 expression in FIGO IV was observed (Mann-Whitney U; P=0.067; Fig. S1B). No clear associations were found between miR-223 (Fig. S2) or miR-590 (Fig. S3) expression and FIGO stage. Moreover, no tendency for an increased expression of miR-205 across different tumor grades was identified (Fig. S4). Although miR-223 expression appeared to increase slightly with higher tumor grade, this trend was not statistically significant (Fig. S5).

Figure 1. Overall survival according to FIGO stage. Kaplan-Meier analysis of overall survival stratified according to FIGO stage. Survival differences between groups were evaluated using the log-rank test. Patients alive at the last clinical follow-up were censored. The overall survival cohort consisted of 33 patients (1 patient passed away after surgery). Numbers at risk are displayed below the graph. Median survival times and 95% confidence intervals are shown where applicable. FIGO, International Federation of Gynecology and Obstetrics.

The analyses further revealed that, in direct comparisons between G1 tumors and the combined G2 + G3 category, a higher expression of miR-590 was consistently associated with greater cellular differentiation, that is, with the features typical of lower-grade disease (Fig. 2). This pattern supports the notion that miR-590 expression varies in a grade-dependent manner and may reflect underlying biological differences between well- and moderately/poorly differentiated lesions. Notably, the present study revealed that miR-590 expression was significantly lower in vulvar carcinoma compared with matched non-malignant vulvar tissue (Wilcoxon signed-rank test, P<0.001; Fig. 3). The absence of this miRNA in tissue was associated with a lower probability of a malignant tumor diagnosis, indicating that detectable miR-590 may help discriminate malignant from non-malignant specimens in this setting. Neither miR-205 nor miR-590 expression demonstrated an association with OS (log-rank P>0.05). A similar trend could nevertheless be appreciated, consistent with the behavior observed for miR-590, although it did not achieve statistical significance for these two markers. Finally, in the subset of patients with matched specimens (n=20), a lower miR-223 fold-change was significantly associated with an improved OS in the Kaplan-Meier analysis (log-rank P=0.013; median OS, 74 vs. 19 months in low vs. high fold-change groups), suggesting potential utility for risk stratification and outcome prediction in clinical practice (Fig. S6).

Figure 2. Association between miR-590 expression and tumor grade. Relative miR-590 expression according to histopathological grade (G1-G3). Data are presented as median with interquartile range. Group comparisons were performed using Mann-Whitney U test. Higher miR-590 expression was observed in well-differentiated tumors. Sample sizes for each group are indicated in the figure. miR, microRNA.

Figure 3. miR-590 expression in malignant and matched non-malignant vulvar tissue. Relative miR-590 expression in vulvar carcinoma vs. matched non-malignant vulvar tissue (n=22 matched pairs; log10 scale). Thin grey lines connect matched pairs. Boxes represent the IQR, the central line the median, and whiskers extend to values within 1.5×IQR; points are individual samples. The paired comparison used the Wilcoxon signed-rank test (P<0.001). miR-590 expression was significantly lower in carcinoma than in matched non-malignant tissue. miR, microRNA.

Discussion

In the present study, a comprehensive review of the literature on the role of miRNAs was provided in the Introduction. These molecules are involved in fundamental processes of tumorigenesis and may function as prognostic biomarkers. The present study conducted an analysis of miR-205, miR-223 and miR-590 expression among patients diagnosed with vulvar cancer compared with a control group. The present study aimed to demonstrate the association between miRNA expression and its effect on survival, the grading of tumors and stage according to the FIGO classification. Several limitations of the present study warrant consideration. First, the cohort size (n=38) reflects the rarity of vulvar carcinoma and limits statistical power, particularly for subgroup analyses. Second, the fold-change-based survival analysis was restricted to 20 patients with available matched specimens, yielding 10 events, sufficient for a single-variable Kaplan-Meier analysis (EPV=10), but not for multivariate Cox regression analysis. The prognostic significance of miR-223 should therefore be regarded as exploratory and hypothesis-generating. Third, the FIGO stage IV and G1 subgroups only comprised of 7 and 3 patients, respectively, precluding reliable conclusions from these comparisons. It should be noted that, due to the low incidence of this malignancy in the population, only a limited number of studies can be found in the available medical databases. A search of the available databases identified only seven original studies addressing miRNA expression in vulvar carcinoma. While miR-223 and miR-590 were selected based on findings from these studies, miR-205 was included due to its well-established role in tumor progression, angiogenesis, epithelial-mesenchymal transition and tumor microenvironment regulation in other malignancies. Comparison of key miRNA studies in vulvar cancer and positioning of the present study was presented in Table II. Therefore, validation in a prospective multicenter cohort remains essential.

Table II. Comparison of key miRNA studies in vulvar cancer and positioning of the present study.

Table II.

Comparison of key miRNA studies in vulvar cancer and positioning of the present study.

First author/s, year	Material/model	miRNA(s)	Expression trend	Functional effects	Target genes/pathways	Key limitations	Relevance to current study	(Refs.)
de Melo Maia et al, 2013	VSCC tissue	Multiple miRNAs	Dysregulated	Not functionally	Not defined	Exploratory profiling	Demonstrates need	(27)
			(profile-based)	assessed		only; no mechanistic	for targeted miRNA
						validation	studies
de Melo Maia et al, 2016	VSCC tissue +	miR-223-5p	Upregulated	↓ proliferation,	TP63 (p63)	Small cohort; limited	Supports dual role of	(19)
	SW962 cell line			↓ migration,		clinical correlation	miR-223; aligns with
				↑ invasion			our findings on
				(dual effect)			aggressiveness
Yang and Wu, 2016	VSCC tissue +	miR-590-5p	Upregulated	↑ proliferation,	TGFβRII/TGF-β	In vitro-dominant;	Confirms oncogenic	(16)
	A431 cells			↑ migration,	pathway	limited survival data	role of miR-590;
				↑ cell cycle			partially consistent
				progression			with our results
Yang and Guo, 2018	VSCC tissue +	miR-3147	Upregulated	↑ proliferation,	SMAD4	Limited cohort; no	Highlights role of	(29)
	cell line			↑ invasion		survival analysis	TGF-β-related
							pathways
Yang et al, 2019	VSCC cell line	miR-4712-5p	Upregulated	↑ proliferation,	PTEN/AKT	No patient validation	Supports importance	(30)
				↑ invasion	pathway		of PI3K/AKT signaling
Agostini et al, 2016	VSCC tissue	miR-30c, let-7a	Downregulated	Indirect tumor	HMGA2	Correlative; no	Indicates tumor	(31)
				suppressor role		functional assays	suppressor miRNAs in
							VSCC
Rymuza et al, 2025	Plasma (VSCC,	Multiple circulating	Upregulated in	Diagnostic	Not primary focus	No mechanistic	Supports liquid biopsy	(32)
	HSIL, dVIN)	miRNAs	VSCC	potential		insight	direction
Present study	VSCC tissue	miR-205,	miR-223 ↑	Clinical	Not primary focus;	Small cohort; lack of	Novel: survival
	(FFPE) + matched	miR-223,	(associated with	correlations with	putative: TP63,	functional validation	analysis + matched
	controls	miR-590	grade and OS);	differentiation,	TGFβRII, PTEN,		tissue + clinical
			miR-205 ↑ (trend	stage, survival	FOXA2-related		correlation
			with stage);		pathways
			miR-590 context-dependent

[i] miRNA, microRNA; VSCC, vulvar squamous cell carcinoma; SW962, SW962 vulvar squamous cell carcinoma cell line; TP63 (p63), tumor protein p63 (TP63); A431, A431 epidermoid carcinoma cell line; TGFβRII, transforming growth factor beta receptor II; TGF-β, transforming growth factor beta; SMAD4, SMAD family member 4; PTEN, phosphatase and tensin homolog; AKT, protein kinase B (AKT); HMGA2, high mobility group AT-hook 2; HSIL, high-grade squamous intraepithelial lesion; dVIN, differentiated vulvar intraepithelial neoplasia; FFPE, formalin-fixed, paraffin-embedded; OS, overall survival; FOXA2, forkhead box A2.

miR-223 has been implicated in a wide range of biological processes, including inflammation, fibrosis and tumorigenesis; however, its role in cancer appears to be highly context-dependent and, in some cases, paradoxical. Evidence from multiple tumor types indicates that miR-223 may exert both oncogenic and tumor-suppressive effects depending on the cellular context and molecular background. For instance, in pancreatic cancer, miR-223 overexpression has been shown to be associated with enhanced proliferation and invasion, whereas in cervical cancer it has been shown to exert antitumor effects through alternative regulatory pathways (15,18). This duality is also reflected in vulvar cancer. The only available study by de Melo Maia et al (19) demonstrated that miR-223-5p overexpression suppressed cell proliferation and migration, while simultaneously enhancing cellular invasiveness, suggesting a complex and potentially opposing role in tumor progression. Additionally, the inverse correlation between miR-223-5p expression and p63 levels, together with the association of low p63 expression with deeper tumor invasion, further supports the hypothesis that miR-223 may differentially regulate distinct aspects of tumor behavior (19). In the present study, a higher expression of miR-223 was associated with poorer tumor differentiation and a shorter overall survival, indicating a potential unfavorable prognostic impact. When interpreted in the context of existing data, these findings suggested that miR-223 may contribute to tumor progression not through a single dominant mechanism, but rather via a balance between antiproliferative and pro-invasive effects. This complexity may partially explain inconsistencies observed across different studies and highlights the need for careful interpretation of its biological role. To definitively characterize the net effect of miR-223 in VSCC, future studies are required to incorporate functional experimental models, including knockout and overexpression systems in VSCC cell lines, as well as pathway-oriented analyses. Such approaches would allow for a more precise delineation of its role in regulating proliferation, invasion and differentiation. From a clinical perspective, miR-223 may represent a promising biomarker of tumor aggressiveness, particularly in relation to differentiation status and patient survival. However, its potential translational application requires further validation in larger, independent cohorts, as well as assessment of its utility in risk stratification and as a minimally invasive biomarker (such as in liquid biopsy settings).

In various studies, miR-205 has been shown to influence critical processes, such as epithelial-mesenchymal transition, angiogenesis and cell proliferation (10–13,20–23). Specifically, in esophageal adenocarcinoma, a progressive decline in miR-205 expression has been shown to be associated with disease progression, suggesting its potential role as a biomarker for cancer progression (20). Moreover, the involvement of miR-205 in cancer-related pathways has been well-documented. It regulates various targets by binding to the 3′untranslated regions (3′UTRs) of mRNAs, thereby suppressing their translation and affecting signal transduction pathways critical for cancer cell survival and proliferation (22). For instance, miR-205 has been implicated in the regulation of genes, such as PTEN and other oncogenes, highlighting its multifaceted nature in cancer progression (23). In non-small cell lung cancer, elevated levels of miR-205 have been associated with an increased metastasis and poor clinical outcomes, further solidifying its role in oncogenesis (20,22). In the present study, an increased expression of miR-205 was also associated with a poorer prognosis, which was related to the diagnosis of more advanced disease among the patients.

miR-590 has garnered significant attention in recent cancer research due to its roles in various malignancies. Focusing particularly on its implications in vulvar cancer, studies suggest that miR-590 may function in complex regulatory mechanisms within diverse cancer contexts (16,24–26). The expression profile of miR-590 highlights its association with VSCC. One key study identified that miR-590-5p, investigated in the present study, exhibited oncogenic properties in VSCC, implicating its contribution to tumorigenesis (16). The research utilized miRNA expression profiling to elucidate the specific roles of miR-590-5p and suggested a potential regulatory mechanism that promotes tumor growth and metastasis through the modulation of various signaling pathways. The upregulation of miR-590-5p in VSCC is associated with promoting proliferation and invasion, with mechanistic emphasis on the downregulation of TGFβRII and the activation of downstream signaling that supports malignant behavior; this is consistent with the broader theme that miR-590-5p can modulate TGF-β and AKT pathways in vulvar carcinogenesis. It is noteworthy that while miR-590-5p appears to function as a driver in VSCC, its regulatory targets and pathways require further investigation, indicating a need for more exhaustive mapping of its functional implications in this type of cancer. Supporting the relevance of miR-590 in the broader spectrum of female malignancies, the comprehensive analysis conducted by Liolios et al (24) suggested that miRNAs, including miR-590, can participate in the complex networks of cancer development across different types of gynecological cancers. While research on miR-590 in vulvar cancer specifically has been limited, implications drawn from studies on other types of cancers reveal a consistent theme, the potential of miR-590 to drive cell proliferation, migration and invasion. The targeting of key proteins and pathways by miR-590 is noted, with evidence indicating that miR-590 is associated with enhanced proliferation by targeting critical regulators within the cell cycle (25,26). Another miRNA, miR-590-3p, is upregulated in epithelial ovarian cancer and directly targets the tumor suppressor FOXA2, reducing its mRNA and protein levels. This targeting promotes cancer cell proliferation, invasion and tumor growth, in part through a FOXA2-regulated pathway that includes versican upregulation when FOXA2 is suppressed (25). While these studies consistently link the miR-590-3p with FOXA-2 interaction, direct evidence that miR-590-5p regulates FOXA2 in VSCC is not established in the cited vulvar cancer literature.

In the unique context of vulvar cancer, investigations into the role of miR-590 have shed light on the potential for this miRNA to serve both as a biomarker for diagnosis and a therapeutic target. Current insights suggest that alterations in miR-590 levels could provide prognostic information, with preliminary data indicating that a higher expression is associated with more aggressive disease phenotypes (27). These results are consistent with the present study, which demonstrated that the absence of miR-590 expression was associated with a lower risk of malignant transformation. Importantly, the observed association between miR-590 expression and malignant transformation suggests that this miRNA may serve as a potential diagnostic biomarker capable of discriminating between malignant and non-malignant vulvar tissue. Moreover, treatment strategies aimed at modulating miR-590 levels may yield novel therapeutic avenues, particularly in enhancing the efficacy of existing therapies, as its regulatory functions appear to intersect with significant signaling pathways involved in cancer progression (28). Despite the existing data, limitations remain in elucidating the exact mechanisms through which miR-590 operates within the context of vulvar cancer. Further research is warranted to dissect the pathways affected by miR-590 and to explore its interactions with other miRNAs and signaling cascades in this malignancy. Such investigations could lead to the identification of combinatorial treatment protocols that effectively target miR-590 alongside traditional cancer therapies. In conclusion, miR-590 represents a promising avenue of research within the landscape of vulvar cancer. Its implicated functions in driving malignancy through distinct mechanisms underscore the critical need to further investigate its role as both a biomarker and a potential therapeutic target. Continued research may well position miR-590 as a key player in the future of targeted cancer therapies.

In conclusion, the present study highlights the potential clinical relevance of miR-205, miR-223 and miR-590 expression in relation to patient survival, clinical stage at diagnosis and histopathological grading. These findings suggest that selected miRNAs may contribute to risk stratification and reflect tumor aggressiveness in VSCC. However, given the rarity of this malignancy, the relatively limited cohort size remains a critical constraint, potentially affecting the statistical power and generalizability of the results. In this context, the establishment of a shared, global database for patients with vulvar cancer should be strongly considered, as it would facilitate larger-scale, multicenter analyses and the more robust validation of miRNA-based biomarkers. From a translational perspective, the findings presented in the present study opened potential avenues for future clinical applications. In particular, miR-223 may represent a promising candidate for minimally invasive diagnostics, for example within liquid biopsy-based approaches, given its previously demonstrated high sensitivity in other malignancies such as colorectal cancer. Additionally, miR-205, due to its involvement in the regulation of the tumor microenvironment, may constitute a potential target for the development of novel therapeutic strategies, including immunotherapy, miRNA-based or nanomedicine-driven interventions. Nevertheless, these applications require further experimental validation and prospective clinical studies before their implementation in routine clinical practice can be considered.

Supplementary Material

Supporting Data

Acknowledgements

The authors would like to express their deepest gratitude to Dr Tomasz Krawczyk (Department of Clinical Pathology, Polish Mother's Memorial Hospital-Research Institute, Lodz, Poland) for his invaluable contributions to the conception and development of the present study. His expertise, dedication, and thoughtful insights markedly shaped the direction of this study. Dr Krawczyk passed away prior to the completion and publication of this article and the authors honor his memory with great respect and appreciation.

Funding

The present study was funded by an internal funding grant no. 17GW/2021 of Polish Mother's Memorial Hospital-Research Institute, Lodz, Poland.

Availability of data and materials

The data generated in the present study may be requested from the corresponding author.

Authors' contributions

EL was involved in the conceptualization of the present study, as well as in data collection and in the writing, revising and editing of the manuscript. ESM was involved in the genetic assays. BT was involved in the statistical analysis and in the preparation of the figures. HR, AM and MW were involved in the conception of the study and in scientific supervision. OM was involved in data collection. JB was involved in the conception and design of the study. All authors read and approved the final manuscript. EL and MW confirm the authenticity of all the raw data.

Ethics approval and consent to participate

The study protocol was approved by the Bioethical Committee of the Polish Mother's Memorial Hospital, Lodz, Poland (approval no. 49/2021). Written informed consent was obtained from each participant. All experimental methods in the study abide by the Declaration of Helsinki.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References