Solanum lycopersicum and Olea europaea microRNAs as Epithelial-Mesenchymal-Transition regulators in prostate cancer

Solanum lycopersicum and Olea europaea microRNAs as Epithelial-Mesenchymal-Transition regulators in prostate cancer | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Solanum lycopersicum and Olea europaea microRNAs as Epithelial-Mesenchymal-Transition regulators in prostate cancer Alessandra Minchella, Francesca Corsi, Pier Giorgio Natali, Vittorio Colizzi, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8743228/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Prostate cancer (PC) is the second most common malignancy among men worldwide. Treatments for metastatic PC, especially in the castration-resistant stage, often yield suboptimal outcomes and rarely achieve a cure. This underscores the urgent need for improved preventive and therapeutic strategies. In recent years, plant-derived microRNAs (miRs) have emerged as potential epigenetic regulators across kingdoms, potentially influencing pathways involved in tumour progression. Within a translational medicine framework, we evaluated a newly developed tomato-based food supplement composed of on 98% heated, spray dried whole tomato ( Solanum lycopersicum ) and 2% olive wastewater ( Olea europaea ), both key nutrients of the Mediterranean diet. Here, we investigate the anti-tumoral potential of miRs extracted from a resuspension of the supplement (called TOFS), with particular emphasis on their modulation of epithelial–mesenchymal transition (EMT), using the prostate cancer PC3 cell line as an in vitro model. TOFS miRs were quantified and analysed by NGS-based analysis, identifying miRNAs from 25 families with known human targets involved in tumorigenesis. The regulation by miRs of EMT-related factors, including TGF-β1, Snail, β-catenin, and E-cadherin, was assessed as indicators of cell–cell adhesion, cytoskeletal stability, and intercellular communication using ELISA and Western blot analyses. The results indicate that TOFS miRs modulate key cellular processes in PC3 cells, particularly their migratory activity. This finding further prove the capability of TOFS to modulate experimental prostate carcinogenesis and the potential to provide clinical benefits in benign prostatic hyperplasia in humans. microRNA1 Solanum lycopersicum 2 Olea europaea 3 EMT4 prostate cancer5 Cross-kingdom interaction6 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Prostate cancer (PC) is the second most common cancer in aging men and the fifth leading cause of cancer-related deaths worldwide (1). In 2020, approximately 1.4 million new cases of PC were diagnosed, resulting in nearly 375,000 deaths (2). The tumour usually progresses with limited aggressiveness and a paucity of initial symptoms (3). While a large fraction of tumours, especially those occurring in old age, require conservative treatment, more aggressive forms, such as castration-resistant PC, can spread rapidly to lymph nodes, bones, and liver, requiring long and multimodal therapy and posing a significant global health challenge and economic burden (4). Natural agents from plants and their fruits play a pivotal role in cancer prevention and therapy (5); phyto-nutrients from extracts or purified compounds, are increasingly used to treat prostate benign hyperplasia, owing to their antioxidant, anti-inflammatory, and anti-cancer activities (6). A population-based study demonstrated that diet can have an antioxidant and anti-inflammatory effect inversely associated with prostate cancer aggressiveness (7). Tomatoes, as well as tomato products, are an important source of anti-inflammatory compounds with antiandrogenic properties (8,9). In tomatoes, natural synergy, cooking, and sustainable production make biofortification a nutrition-sensitive alternative to pill-based supplementation (10). Similarly, olive oil can be enriched with polyphenols recovered from olive-mill wastewater, valorising by-products to improve health benefits and circular sustainability (11). Biofortified foods enhance nutrients directly in the food matrix offering better bioavailability, safety, and dietary integration than isolated phyto-nutrients or supplements. Interestingly, the combination of biofortified tomato and olive wastewater has been showed to contribute to reduced cardiovascular risk and enhanced immune response and to have antibacterial, antiviral and anti-cancer properties (12,13). A newly developed biofortified tomato-olive supplement made of 98% spray dried whole tomato ( Solanum lycopersicum ) and 2% olive wastewater ( Olea europaea) can inhibit prostate carcinogenesis in vivo in mouse model, by regulating major metabolic pathways involved in the expression of a wide range of oncogenic genes (14,15) (patent No. EP2851080A1). However, the health benefits of the supplement’s components are dose-dependent and their synergic interaction is still under evaluation (16,17). Among the bioactive compounds, plant-derived microRNAs (miRs) can be found, representing a unique class of conserved small non-coding RNAs (18–24 nucleotides) that influence key biological processes (18,19), regulating gene expression post-transcriptionally by repressing translation and promoting mRNA degradation through interactions with their 3' untranslated regions (3'UTR) (20). They are conserved across all species and can act as regulatory molecules beyond species boundaries. Notably, plant-derived miRs can be transferred to humans through dietary intake, exerting regulatory effects on human gene expression via cross-kingdom interactions (21–24). Indeed, Minutolo et al .(23) showed that miRs extracted from Olea europaea drupes have anti-proliferative and anti-cancer effects in human cells, in line with a patented strategy exploiting plant-derived microRNAs for cancer prevention and treatment (EU patent No. EP3216869A1). The epithelial-mesenchymal transition (EMT) is an essential mechanism driving epigenetic (25) and metabolic (26) reprogramming of carcinoma cells, including PC (27), leading to a mesenchymal phenotype associated with cancer invasiveness and metastasis spreading (28). EMT cause progressive loss of epithelial characteristics, including cell polarity, proliferation control, and differentiation, resulting in increased cell motility, invasiveness, and resistance to therapy-induced apoptosis (29,30). These processes intertwine with the signalling pathway of the transforming growth factor-β1 (TGF-β1), which is known to be overexpressed in a variety of cancers to induce EMT, including colon cancer among others (31). TGF-β1 targets Snail, a protein that directly inhibit E-cadherin transcription by binding its promoter region (32). The decline in E-cadherin levels is a notable hallmark of EMT; indeed, E-cadherin creates adherens junctions for cell-cell adhesion, stabilized by β-catenin. The E-cadherin/β-catenin complex is therefore critical in preserving epithelial integrity. In this context, TGF-β1 triggers the dissociation of β-catenin and its stabilization in the cytoplasm, enabling its nuclear import, where it may influence cellular processes such as proliferation, migration, and invasion (33) . In this study, we performed an in vitro analysis on castration-resistant prostate cancer cells, supported by an in-depth bioinformatic investigation to identify microRNA components of a water resuspension of the biofortified tomato and olive-based supplement (called TOFS) to evaluate its anti-prostate tumour activity by downregulating EMT-mediated progression. Materials and methods Extraction of the miRs pools from water-resuspended tomato and olive food supplement (TOFS) MiRs were obtained from a water resuspension of a food supplement (TOFS) registered with the Italian Ministry of Health (EU patent No. EP2851080A1) made of 98% spray-dried whole tomatoes and 2% olive mill wastewater and kindly provided by Janus Pharma (Rome, Italy). A total of 6,5 g of supplement powder was dissolved in 100 mL of distilled water, yielding a final concentration of 65 mg/mL. The TOFS stock solution was centrifuged at 100 × g, and the supernatant was recovered and filtered through a 0.45 µm filter unit (Minisart®) to eliminate debris. The resulting aliquots were stored at -20°C until use. TOFS stock solution was diluted to obtain a final concentration of 1 mg/ml, 5 mg/ml and 10 mg/ml and MiRs were extracted using the GenUP Micro RNA Kit (BiotechRabbit, Germany), according to the manufacturer’s instructions. TOFS miRs were quantified with a NanoDrop™ Lite Spectrophotometer (Thermo Fisher Scientific, United States), by measuring absorbance at 260–280 nm. TOFS miRs characterization Library preparation of the TOFS miRs was performed using the QIAseq miRNA library kit (Qiagen, Hilden, Germany). All samples were sequenced in 75 single-end on an Illumina NextSeq500 platform according to QIAseq miRNA Library Kit Handbook for precision small RNA library prep for Illumina ® NGS systems. Raw sequencing read quality was assessed using FastQC v0.11.9(34) and MultiQC v1.10.1(35). Adapter trimming was performed with cutadapt v3.4 (36), and a custom script was subsequently used for read deduplication based on Unique Molecular Identifiers (UMIs). The processed reads were aligned using SHRiMP v2.2.3(37)against reference hairpin databases: miRBase (release 22.1) (38) for Solanum lycopersicum (Sly) and the Olive Genome Consortium database for Olea europaea (Oeu). To quantify the expression of mature miRNAs, the BEDTools suite v2.30.0 was employed to identify overlapping mappings between the aligned reads and the corresponding mature sequences. For S. lycopersicum , mature miRNA coordinates were obtained from the miRBase annotation, whereas for O. europaea , the reference set consisted of 136 conserved miRNAs and putative novel miRNAs identified by Yanik et al. (2013) (39). miRNA target prediction We employed IntaRNA3 (40) to investigate whether S. lycopersicum miRNAs target EMT pathway gene transcripts. By predicting RNA–RNA interactions through thermodynamic stability, IntaRNA3 provides the high sensitivity required for modeling complex miRNA–mRNA. IntaRNA3 calculates interaction energies between RNA sequences, where lower energy scores indicate more stable and likely interactions. In our analysis, we input sequences of known S. lycopersicum miRs and the 3' untranslated regions (3' UTRs) of human EMT pathway mRNAs into IntaRNA3. To minimize false positives, we applied a stringent filter, considering only interactions with a free energy (ΔG) below − 15 kcal/mol as significant. This approach allowed us to focus on the most thermodynamically stable and biologically relevant potential interactions, suggesting a possible regulatory mechanism where tomato miRNAs could target human EMT pathway transcripts for translational repression or degradation. Functional enrichment analysis To further investigate the biological relevance of the predicted miRNA-mRNA target gene interactions, the list of potentially regulated transcript identified by IntaRNA3 was subjected to Functional Enrichment Analysis. This analysis aims to identify over-represented biological pathways, functions, or processes within a gene list, providing insights into the potential functional impact of miRNA-mediated regulation. We utilized two distinct tools for this purpose: Metascape and FunRich. Metascape is a comprehensive web-based platform that leverages multiple gene annotation databases and pathway repositories to perform Gene Ontology (GO) enrichment, pathway enrichment analysis, and protein-protein interaction network analysis. It statistically determines significantly enriched terms within the input gene list compared to a background set. FunRich, a standalone software tool, also performs functional enrichment analysis, focusing on gene set enrichment from various databases. It provides statistical measures to assess the significance of enriched functional terms and pathways, offering complementary insights into the biological themes associated with the potentially miRNA-regulated EMT genes. By using both Metascape and FunRich, we aimed to obtain a robust and comprehensive functional characterization of the genes predicted to be targeted by S. lycopersicum miRNAs. PC3 cell culture and transfection Human prostate cancer PC3 cells, a bone metastasis of grade IV prostatic adenocarcinoma (ATCC, CRL-1435), were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Invitrogen, USA). The culture medium was supplemented with 10% fetal bovine serum (FBS, Invitrogen, Germany), 2mM glutamine (HyCloneTM, UK), 100 U/mL penicillin and 100 U/mL streptomycin (HyCloneTM, UK). The cells were maintained at 37°C in a humidified incubator with 5% CO 2 to generate a confluent monolayer and were routinely split by trypsinization with Trypsin-EDTA (Euroclone, IT). Experiments were performed with cell viability consistently above 98%. PC3 cells were transfected with TOFS miRs, present in 1, 5 and 10 mg of TOFS solution for 24, 48 and 72 hours using Lipofectamine™ 2000 (Hi-Fect, HF, Invitrogen, USA), according to the manufacturer’s instructions. Wound healing assay The ability of PC3 cells transfected with miRs to migrate was evaluated using a wound healing assay. Cell migration, indeed, is strictly correlated to activation of the EMT process (41). PC3 cells were seeded at a density of 0.15 x 10 6 cells/ml in RPMI medium. A confluent monolayer of cells was obtained after 24 hours at 37°C in 5% CO 2 . Cells were then transfected as describe above with TOFS miRs, and scratch wounds were created mechanically using a sterile pipette tip. PC3 cell migration was observed at 24, 48 and 72 hours post-transfection, and images were acquired with a microscope [NIKON, (ECLIPSE Ts2) coupled to a camera alexasoft TP1080HDMI]. The migration rate was measured by Image J and expressed as fold change (Treated/Untreated) of the cell-free area. TGF-β1 evaluation Supernatants from the wound healing assays were collected and TGF-β1 release was investigated by using TGF-β1 Duoset ELISA Kits (R&D Systems Inc) according to manufacturer’s instruction. The optical density was measured at 450 nm (reference 540 nm) using the Infinite®200 PRO (Tecan Trading AG). Data analysis was carried out using GraphPad Prism v8.4.3 software, and cytokine concentration was calculated based on the standard curve. Western blot analysis of β-catenin, E-cadherin, Snail Aliquots of 1 × 10 6 cells, subjected to various experimental conditions, were lysed and processed for western blot analysis according to standard protocol. The primary antibodies used were mouse monoclonal antibodies directed against β-catenin (13-8400), E-cadherin (13-1700), Snail (MA5-15791), and GAPDH (Abcam: 9484) as a loading control. Antibodies were prepared according to the manufacturer's instruction. The secondary antibody used was HRP-conjugated goat anti-mouse IgG (Invitrogen, Cat. No. A28177). Proteins were detected with the ECL (Millipore, WBKLS0500) and proteins’ specific signals were quantified by densitometry using ImageJ software. β-catenin immunofluorescence Transfected cells were grown in 96-well plates and fixed with 4% paraformaldehyde for 15 min. Immunofluorescence staining was performed at room temperature (RT) in PBS 0,2% Triton X-100 and PBS with 1% bovine serum albumin (BSA), as permeabilizing and blocking agents, respectively. Cells were incubated for 1 hour with a primary antibody against β-catenin (13-8400). After 3 washing, cells were incubated for 1 h with the Tetramethyl Rhodamine Isothiocyanate (TRITC)-conjugated secondary antibody (T2402, Sigma-Aldrich, St. Louis, MO, USA) and then stained with DAPI (2 µg/mL) for nuclei detection. Images were captured using a ZEISS Axio Observer microscope. Unbiased staining intensity was estimated after background signal elimination through pixel fluorescence analysis according to Carl Zeiss Microscopy GmbH’s ZEN software (version 3.0, Jena, Germany). At least four images for each sample were analysed to obtain the relative frequency distribution of β-catenin fluorescence intensity. Statistical analysis Data are presented as mean values ± standard deviation (SD). All experiments were performed at least in triplicate (biologically independent measurements). Significant differences are shown as p- value: p < 0.05 (*), p < 0.01 (**), p < 0.001 (***) or p < 0.0001 (****). A nonparametric two-way post-hoc ANOVA corrected by t-test was performed. Results A biofortified food supplement (patent No. EP2851080A1) based on 98% spray-dried whole tomatoes and 2% olive mill wastewater (TOFS) was evaluated for the presence of miRNAs. The identified miRs were characterized for their antitumor effect at first in silico and then experimentally on PC3 cells, a castration-resistant high-grade PC cell line derived from bone metastases. Sequencing and characterisation of the miRs present in the TOFS A total of 78 miRs extracted from TOFS solution were successfully sequenced, including 64 from O. europaea ( oeu -miRs) and 14 from S. lycopersicum ( sly- miRs) (Fig. 1 A). As shown in Fig. 1 B, 69 of the 78 (87%) miRs identified by sequencing belonged to well-known plant miRs families. The most represented TOFS miRs belonging to plant miRs families were miR169, miR166, miR395, and miR319, in which 17, 11, 8, and 9 miRs were identified, respectively. Most of these were oeu -miRs, for instance from the miR169 family, 14 were oeu -miRs ( oeu -miRs 169 d-m, r, s, v, w) and three were sly- miRs ( sly -miRs 169b and d), while in the miR166, miR395, and miR319 families only oeu -miRs were found ( oeu -miR166 a-f, h, I, j, k, q; oeu -miR 395 b-j; oeu -miRs 319 a-h). The other plant miRs families (159, miR167, miR171, miR172, miR394, miR396, miR399, miR403) were represented by 1 to 4 oeu- miRs (Fig. 1 B and Table S1 ), with the exception of the miR168 family, in which we observed the presence of miRs belonging to both species (sly -miRs 168a and b-5p and oeu -miRs 168a and b). The sequencing highlights the presence of 8 other sly -miRs ( sly -miRs10529, sly -miRs10534, sly -miRs 10542, sly -miRs 1918, sly -miRs 482, sly -miRs 5300, sly -miRs7981, sly -miRs9478) that do not belong to any of the families described above ( Table S1 ). Quantitative Unique Molecular Identifier (UMI)-based analysis reported in Table 1, revealed that among sly -miR10529 and sly -miR1918 were the most abundant, with approximately 75,400 and 39,000 UMIs, respectively; the other miRs were expressed with UMIs range 7,800-2,600. The most abundant oeu -miRs were miR168a and miR168b, with approximately 98,800 and 93,600 UMIs, respectively, while for the others oeu -miRs different levels of expression ranged from 10,400 to 2,600 were observed ( Table S1 ). Since UMIs correct for PCR amplification bias, these values provided a reliable estimate of the true relative abundance of the respective miRNAs. These results provide valuable insights into the presence in the TOFS of several miRs belonging to known plant species, which may have a regulatory role in humans. Computational prediction of TOFS miRs targeting human genes, and network analysis of the processes involved in EMT pathway The IntaRNA3 tool, an advanced bioinformatics algorithm for rapid and accurate prediction of RNA-RNA interactions (primarily between small regulatory RNAs and their target mRNAs) was employed to evaluate the likelihood of interactions between TOFS miRs and genes involved in EMT process. As shown in Fig. 2 A and Table S2 , 56 out of the 78 identified TOFS miRs were predicted to bind to 68 mRNAs associated with EMT. Notably, sly-miR482d-5p was able to regulate 65 of these 68 genes, whereas the other miRs exhibited a range of target gene numbers from 17 down to 1 (see Table S2 and Fig. 2 A). Remarkably, SMAD family member 2 (SMAD2) was highly regulated, being targeted by thirty-five TOFS miRs; DSC2, a transmembrane cell anchoring protein, by twenty-seven; N-cadherin (CDH2) by twenty-two; ZEB1 by twenty; and seventeen TOFS miRs were predicted to target VPS13A (Fig. 2 B). Three out of five top target genes are upregulated in the TGF-β1-induced EMT process (42); therefore, their downregulation mediated by plant microRNAs could potentially reverse EMT. Considering the key role of EMT in both PC metastatic progression and therapy resistance, we exploited the online tool Metascape (43) to investigate the possible antitumor relevance of TOFS miRs within EMT-related networks. Network analysis revealed a highly interconnected and complex set of biological pathways involved in EMT (Fig. 3 A-B). The constructed networks highlight key processes such as EMT in colorectal cancer, proteoglycans in cancer, tube morphogenesis, extracellular matrix organization, and cell population proliferation. Additional pathways detected included enzyme-linked receptor protein signalling, Hippo signaling, focal adhesion, and cell differentiation, underscoring the multifactorial and tightly regulated nature of EMT. The diverse biological processes identified, including neural crest differentiation, response to wounding, and cell-cell communication, demonstrate that EMT is integrated with multiple cellular functions extending beyond classical tumour progression pathways. Complementing the network data, enrichment analysis quantified the statistical significance of these pathways in the context of EMT gene modulation (Fig. 4 A-B). The results show strong enrichment (-log 10 p- values) for hallmark EMT pathways, such as epithelial–mesenchymal transition in colorectal cancer and related cellular processes including extracellular matrix organization and proteoglycan function. This enrichment pattern was particularly significant when considering TOFS miR target genes, reinforcing the biological relevance of these miRs in modulating EMT. Notably, pathways related to signaling mechanisms (e.g., enzyme-linked receptor protein signalling, Hippo pathway) and cell–cell communication were also significantly enriched, indicating that TOFS miRs potentially orchestrate complex regulatory networks governing EMT phenotypes. Together, the network and enrichment findings reveal that the molecular landscape of EMT is characterized by extensive cross-talk among multiple biological pathways. These data support a model where TOFS miRs influence EMT by targeting a broad range of interconnected pathways critical for cellular plasticity, adhesion, and differentiation, which are essential for both physiological and pathological EMT processes. The analysis revealed that TOFS miRs may regulate several genes associated with EMT, influencing pivotal EMT-related cellular processes. Regulatory effect of TOFS miRs on PC3 cell migration To investigate the impact of TOFS miRs on PC3 cell migration, a wound-healing assay was performed at 0, 24, 48, and 72 h after administration of 1, 5 and 10 mg of TOFS miRs. Twenty-four hours after treatments, similar growth and migration are observed in each condition. Compared to control cells, which showed a clear wound closure at 48 h, cells transfected displayed a significantly over two-fold reduced migratory rate which was inversely proportional to treatment concentrations (Fig. 5 A and B ), highlighting the anti-migratory effect of the plant miRs. In particular, a dose of 1 mg of miRs resulted the most efficacious at all time points, with a 2.5-fold increase in cell-free area at 48 h compared to the control (Fig. 5 B). At 72 hours, the cell-free area was significantly higher only for cells treated with 1 and 10 mg of TOFS miRs compared to the control. These data may suggest a potential regulatory effect on the migratory capacity of PC3 cells. During proliferation and migration, cells typically release transforming growth factor beta (TGF-β1 (44). We therefore measured TGF-β1 levels in PC3 supernatants obtained from the wound healing experiments by ELISA assay. Expectedly, TGF-β1 expression showed a significant increase over time. Specifically, expression levels at 48 and 72 hours were significantly higher compared to 24 hours (p < 0.01 (**) and p < 0.0001 (****) (Fig. 5 C). At 48 h post-transfection, a significant decrease in TGF-β1 expression level was observed across the three TOFS miRs treatments compared to the control; at 72 h, instead, a significant reduction persisted only in cells treated with 1 mg/mL miRs (Fig. 5 C). Effects of TOFS miRs on the EMT-markers E-cadherin, β-catenin, and Snail in PC3 cells Considering the anti-migratory effect of the TOFS miRs displayed in the wound-healing assay, the expression levels of different proteins involved in EMT, including E-cadherin, β-catenin and Snail, were investigated. E-cadherin, a transmembrane protein that plays a crucial role in calcium-dependent cell-cell adhesion, is usually down-modulated in cancer compared to normal conditions. The treatment with TOFS miRs induced a significant increase in E-cadherin expression in PC3 at 48 and 72 h (Figs. 6 A, B), suggesting a restoration of epithelial characteristics. The expression of Snail, a key EMT transcription factor which negatively regulates E-cadherin, was then investigated. Although not statistically significant, Snail showed a slight decrease in its expression at 24 h after TOFS administration (Figs. 6 A and C ). Finally, we evaluated the expression of β-catenin, an adhesion junction protein involved in cell-cell connections, whose upregulation/accumulation is typically implicated in cancer progression (45). The expression of this protein was modulated by TOFS miRs treatments, showing a significant downregulation at 48 and 72 h compared with the control (Figs. 6 A and D ). Western blot analysis was quantified by densitometric analysis. The data were expressed as OD-Bkg/mm 2 , the background (Bkg) of the picture was subtracted, and each sample was normalised to the respective GAPDH value. The results show the mean of three independent biological experiments, *p < 0.05,**p < 0.01,***p < 0.001 and ****p < 0.0001. During EMT, β-catenin is not only overexpressed, but is also typically aberrantly translocated from the cell membrane to the nucleus. This process is closely associated with the activation of the Wnt/β-catenin signalling pathway, which plays a pivotal role in the regulation of cell proliferation, migration, and survival (46). We therefore investigate β-catenin subcellular localization. Cells pre-treated with 2 ng/ml TGF-β1 stimulus to induce EMT, were then administered with TOFS miRs (Fig. 7 A). At 48 h post-transfection, TGF-β1-treated cells exhibited nuclear β-catenin, as per EMT process, whilst TOFS-treated cells showed β-catenin membrane re-localization (Fig. 7 B). Discussion Prostate cancer (PC) is the leading cause of cancer-related death among middle-aged and older men worldwide. Intense multidisciplinary research is ongoing today to develop more effective preventive strategies to delay or mitigate disease severity, as well as integrated therapy to improve outcomes in patients with aggressive PC (47). Epidemiological evidence has highlighted that the risk of developing PC is significantly increased with the consumption of a typical “Western-style” diet, characterized by high intakes of red and processed meats as well as white bread, fried fish, high-fat milk and chips(47). On the other hand, adherence to the Mediterranean diet, which is rich in antioxidant compounds derived from olive oil and tomatoes, has been shown to be associated to reduced PC incidence, slowing Gleason grade progression in patients with low-grade prostate cancer (48–51). The antioxidant and anti-inflammatory properties of the food included in the Mediterranean diet are linked to prostate health, however, the extent of the benefits depends on the specific compounds and how the body processes them, which affects their bioavailability. Biofortified functional food is known to increase food bioavailability; indeed, a newly developed tomato and olive biofortified supplement has shown potent antioxidant activity and the capability to downregulate the pathways involved in the progression of prostate tumours (15). Moreover, the biofortified supplement investigated, is richer in nutrients than raw and cooked market products (52), its intake could therefore facilitate compliance with the “Prostate Dietary Index”, which has been shown to be associated with a 18 % lowerrisk of prostate cancer incidence (53). Plant miRs can modulate the post-transcriptional regulation of human mRNA as human miRs do, in a process known as cross-kingdom interaction. This regulatory ability has generated significant interest due to its potential to develop new therapeutic agents to treat diseases associated with miRs dysregulation, including cancer (54–57). In this study, we demonstrate that TOFS contains miRs potentially capable to modulate gene expression. A bio-informatic analysis reveals that 56 TOFS miRs modulate the expression of 65 genes involved in EMT, a mandatory step in gaining the metastatic phenotype(27). While the field is still emerging and somewhat controversial, experimental evidence indicates that plant-derived miRNAs ingested through the diet can survive gastrointestinal digestion, enter circulation, and modulate the expression of mammalian oncogenes, tumour suppressors, and other cancer-related transcripts, suggesting potential implications in cancer biology and prevention(58). Indeed, it is known that plant-derived miRs can influence mammalian gene expression in a cross-kingdom manner (23). We detected 78 miRNAs belonging to 25 families several of which belonged to highly conserved families across various species; overall, most of the identified miRs originated from O. europaea and, among these, oeu -miR156, oeu -miR159, oeu -miR166, and oeu -miR169 were the most represented. Plant miR159 has been previously identified in human sera and its level has been inversely associated with breast cancer progression and incidence (59). Among the EMT-related genes most frequently recognised by the TOFS miRs were CDH2, SMAD2 and ZEB1, which are typically upregulated during the EMT process. These genes appear to be potential targets of TOFS miRs, which could then compensate for the reduced levels of endogenous human miRs (60) that usually regulate their expression. We evaluated the ability of PC3 cells transfected with miRs to migrate through a wound healing assay. Cell migration, indeed, is strictly correlated to activation of the EMT process (41). We observed that miRs treatment induces a decrease in the cells' migratory capacity, allowing us to hypothesize that miRs treatment could counteract EMT. Our EMT analysis focused on evaluating the level of expression of TGF-β1. The significant reduction in TGF-β1 observed in PC3 cells induced by TOFS miRs, has a powerful impact on EMT dynamics since it significantly alter TGF-β1 pathway, leading a slight decrease in the expression of transcription factor Snail protein and β-catenin, which results in an increase of E-cadherin expression, resulting in restored cell-to-cell adhesion and reduced migration. The subcellular distribution of β-catenin was next evaluated in PC3 cells forced to undergo EMT via TGF-β1 treatment. As expected, β-catenin localized mainly in the cytoplasm and nucleus with TGF-β1 treatment but miRs treatment promoted a re-localization to the membrane post treatment. These data confirm the ability of TOFS miRs to restore an epithelial phenotype. Overall, the results confirm the ability of TOFS miRs to down regulated cell migration in PC3 cells by targeting the expression of key proteins involved in EMT. The use of a supplement made of biofortified tomato obtained with a prolonged patented heating process together with the implementation of an extraction protocol to recover microRNAs from TOFS, outlines a scenario of dual economic and strategic advantage. On one hand, the utilization of edible plants for the production of therapeutic small RNAs represents a ground-breaking and economical alternative to the currently expensive chemical synthesis required for artificial miRNAs(23). On the other hand, this practice is deeply rooted in the circular economy and waste-to-resource models, converting abundant agro-industrial residues, such as olive mill wastewater and pomace, from environmental liabilities requiring costly disposal into valuable raw materials for the nutraceutical industry (61). Consequently, this approach not only drastically reduces the production costs of molecules capable of regulating human gene expression, but also generates new revenue streams for producers, effectively closing the loop of two of the most globally widespread agricultural chains (61). From a translational perspective, the ability of TOFS-derived miRs to restore epithelial markers and suppress EMT-associated signalling suggests a potential role in limiting prostate cancer cell invasiveness and metastatic potential. While not intended as a standalone therapeutic approach, such diet-derived miRs may represent a promising adjunct strategy to complement existing treatments aimed at controlling disease progression. Conclusions Through a multiparametric analysis, the present study demonstrates that a newly available food supplement of well-defined formulation based on Solanum lycopersicum and Olea europaea contains plant-derived miRs capable of modulating PC3 cells behaviour. Specifically, TOFS-derived miRs interfere with the expression of key EMT-related proteins, re-establishing a non-cancerogenic epithelial phenotype and suppressing features associated with metastatic potential including cell migratory activity. Beyond the observed effects on EMT regulation, the miRs identified in TOFS may act in concert with other bioactive micronutrients naturally present in the supplement, including highly bioavailable lycopene, quercetin, narigenin, and verbascoside, thereby contributing to a broader protective molecular environment that supports prostate health. Importantly, these findings support the concept that biofortified functional foods can act not only as sources of antioxidant compounds but also as carriers of biologically active plant-derived miRs with cross-kingdom regulatory potential. While their bioavailability and long-term effects in humans require further investigation, our data provide mechanistic in vitro evidence that dietary miRs can modulate cancer-relevant pathways. From a translational and preventive perspective, TOFS-derived miRs are not intended to replace established prostate cancer therapies, but may represent a complementary approach to limit disease progression or tumour aggressiveness, particularly in early-stage disease. Future studies on TOFS will be needed to validate these effects in vivo and to assess their clinical relevance. Indeed, previous findings from epidemiological trials already raised awareness that processed tomato products were associated with a 30–40% reduction of PC risk (49). Finally, the production of TOFS within a circular economy framework, leveraging agro-industrial by-products such as olive mill wastewater, highlights a sustainable and economically viable strategy for developing nutraceuticals with added biological value. Abbreviations 3'UTR 3' untranslated regions Bkg Background BSA Bovine Serum Albumin CDH2 N-cadherin EMT Epithelial-Mesenchymal Transition FBS Fetal Bovine Serum GO Gene Ontology HF Lipofectamine™ 2000 MiRs MicroRNAs Oeu Olea europaea Oeu miRs O. europaea miRs PC Prostate Cancer RPMI Roswell Park Memorial Institute RT Room Temperature SD Standard Deviation Sly Solanum lycopersicum Sly miRs S. lycopersicum miRs SMAD2 SMAD family member 2 TGF-β1 Transforming Growth Factor-β1 TOFS Tomato and Olive Food Supplement TRITC Tetramethyl Rhodamine Isothiocyanate UMI Unique Molecular Identifiers Declarations Ethics approval and consent to participate Not applicable Consent for publication Not applicable Competing interests PGN has a professional affiliation with Janus Pharma S.r.l. (Rome, Italy). The other authors declare no conflicts of interest. Funding This work was supported by the CANVAS project under Grant Agreement No. 101079510. Author Contribution Conceptualization and study design: CM, MP, PGN, VC; Wet-lab experiments and sample preparation: AM, FC, CT. NGS Sequencing: SF. Bioinformatic and biostatistical analyses: FI, AM; Validation: MP, CM, PGN, VC. Writing—original draft preparation, AM. Writing—review and editing: all authors have read, contributed equally to the revision, and approved the final version of the manuscript. Acknowledgement PGN acknowledges partial support from the Federico Calabresi Foundation (Rome, Italy). Data Availability The datasets analysed during the current study are included in this published article and its additional file, and are also available in the Zenodo repository [ [10.5281/zenodo.18387556](https:/doi.org/10.5281/zenodo.18387556) ]. References Schafer EJ, Laversanne M, Sung H, Soerjomataram I, Briganti A, Dahut W, et al. Recent Patterns and Trends in Global Prostate Cancer Incidence and Mortality: An Update. Eur Urol. 2025 Mar;87(3):302–13. 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Cell Res. 2015 Apr 27;25(4):521–4. Enaime G, Dababat S, Wichern M, Lübken M. Olive mill wastes: from wastes to resources. Environmental Science and Pollution Research. 2024 Feb 26;31(14):20853–80. Additional Declarations No competing interests reported. Supplementary Files Additionalfile1.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8743228","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":585090607,"identity":"848ac0ca-3ec2-4c1b-9bf1-c0b3f166b62e","order_by":0,"name":"Alessandra Minchella","email":"","orcid":"","institution":"University of Rome Tor Vergata","correspondingAuthor":false,"prefix":"","firstName":"Alessandra","middleName":"","lastName":"Minchella","suffix":""},{"id":585090608,"identity":"e9b140de-573f-49d2-b561-3874256ffca3","order_by":1,"name":"Francesca Corsi","email":"","orcid":"","institution":"University of Rome Tor Vergata","correspondingAuthor":false,"prefix":"","firstName":"Francesca","middleName":"","lastName":"Corsi","suffix":""},{"id":585090609,"identity":"c99ca7b6-6403-4188-9f75-ee40d722a31f","order_by":2,"name":"Pier Giorgio Natali","email":"","orcid":"","institution":"Mediterranean Taskforce for Cancer Control","correspondingAuthor":false,"prefix":"","firstName":"Pier","middleName":"Giorgio","lastName":"Natali","suffix":""},{"id":585090610,"identity":"1d6ac315-c915-4131-870b-45a47db4e045","order_by":3,"name":"Vittorio Colizzi","email":"","orcid":"","institution":"Complex Hospitalo-Universitaire Bon Samaritain","correspondingAuthor":false,"prefix":"","firstName":"Vittorio","middleName":"","lastName":"Colizzi","suffix":""},{"id":585090612,"identity":"662ec147-e27c-442e-b196-15c53c13af4c","order_by":4,"name":"Sofia Fucile","email":"","orcid":"","institution":"Personal Genomics","correspondingAuthor":false,"prefix":"","firstName":"Sofia","middleName":"","lastName":"Fucile","suffix":""},{"id":585090613,"identity":"4bc80825-249f-4e99-afac-87417319e3fb","order_by":5,"name":"Costanza Tacoli","email":"","orcid":"","institution":"University of Rome Tor Vergata","correspondingAuthor":false,"prefix":"","firstName":"Costanza","middleName":"","lastName":"Tacoli","suffix":""},{"id":585090615,"identity":"290e8dbb-1c58-43cd-a9f9-f3d938de161e","order_by":6,"name":"Federico Iacovelli","email":"","orcid":"","institution":"University of Rome Tor Vergata","correspondingAuthor":false,"prefix":"","firstName":"Federico","middleName":"","lastName":"Iacovelli","suffix":""},{"id":585090616,"identity":"35cfd6e5-eaff-46bf-9d64-9049545f6163","order_by":7,"name":"Marina Potestà","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBklEQVRIiWNgGAWjYJCCA0hsGwYGZiiDjaAWNjArDaYljYENnx4kLYdh3MMMuKzRbT/78HBBDUMev3zz4c8fas4nbmfnffjoRg0zA598A1YtZmfSDQ7POMZQLNnGliZx4NjtxJ3N7MbGOcfYcDrM7EAaw2EeNobEDcd4zBgOsN1O3HCYjU06h40Ht5bzz4Ba/oG08H/+cODfOaiWfxK4tdwA2sLbBraFQeJg2wGIltw2AzxagLbw9kkA/ZJmJnG2L9kYqIXZOLcvgYeNLQGHw9KYP/N8s8njZz78+EPFNzvZDeePMT7O+fZfTr75AHZrIEAC00AefOpBALsbRsEoGAWjYBSAAAAH81oTLOqMcQAAAABJRU5ErkJggg==","orcid":"","institution":"University of Rome Tor Vergata","correspondingAuthor":true,"prefix":"","firstName":"Marina","middleName":"","lastName":"Potestà","suffix":""},{"id":585090617,"identity":"cc963ed4-464f-4642-93e6-97320750fc3a","order_by":8,"name":"Carla Montesano","email":"","orcid":"","institution":"University of Rome Tor Vergata","correspondingAuthor":false,"prefix":"","firstName":"Carla","middleName":"","lastName":"Montesano","suffix":""}],"badges":[],"createdAt":"2026-01-30 16:23:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8743228/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8743228/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":101948647,"identity":"4494d7fd-bff5-41cb-9886-4679995d976b","added_by":"auto","created_at":"2026-02-05 10:16:01","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":186376,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterization of TOFS miRs. \u003c/strong\u003eA) PieChart of the relative percentage of \u003cem\u003esly\u003c/em\u003e-miRs and \u003cem\u003eoeu\u003c/em\u003e-miRs present in the TOFS. B) Histogram showing the number of miRs (y-axis) for each known miRNA family (x-axis), identified in \u003cem\u003eS. lycopersicum\u003c/em\u003e (black bar) and \u003cem\u003eO. europaea \u003c/em\u003e(grey bar)\u003cem\u003e.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8743228/v1/ebd7bd47c12521b2a376b8ff.png"},{"id":102397310,"identity":"74c38d80-e2f4-4a1e-98f2-273c4cbfb82b","added_by":"auto","created_at":"2026-02-11 10:15:19","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":403879,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterization of TOFS miRs. \u003c/strong\u003eA) PieChart of the relative percentage of \u003cem\u003esly\u003c/em\u003e-miRs and \u003cem\u003eoeu\u003c/em\u003e-miRs present in the TOFS. B) Histogram showing the number of miRs (y-axis) for each known miRNA family (x-axis), identified in \u003cem\u003eS. lycopersicum\u003c/em\u003e (black bar) and \u003cem\u003eO. europaea \u003c/em\u003e(grey bar)\u003cem\u003e.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8743228/v1/c73533a7caf89a3998a137be.png"},{"id":101948648,"identity":"47426048-0d60-42fd-82f8-60165cf13178","added_by":"auto","created_at":"2026-02-05 10:16:01","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":504229,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEnrichment network analysis for EMT.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e Networks layout of the clusters generated with the list of the genes associated with the EMT and the EMT-related tumorigenesis process. \u003cstrong\u003e(B)\u003c/strong\u003e Networks layout of the clusters generated with the list of genes regulated by TOFS miRs. Gene clusters are color-coded and ranked by decreasing representation. Each circle node represents one enriched term; its size is proportional to the number of input genes falling into that term, and its colour represents the cluster identity (i.e. nodes of the same colour belong to the same cluster). All similar terms with a Kappa similarity score \u0026gt; 0.3 are connected by edges (the thicker the edge higher the similarity). One term from each cluster has been as a label. Edge thickness correlates directly with the similarity score between enriched terms: thicker lines indicate stronger connections (typically reflecting a higher number of shared genes), whereas thinner lines indicate lower overlap. Created by Metascape [\u003ca href=\"http://metascape.org/\"\u003ehttp://metascape.org\u003c/a\u003e].\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8743228/v1/d56d2d388d31f28502111ca5.png"},{"id":102298590,"identity":"9fb2da59-38cb-412e-94ad-f12b908d847a","added_by":"auto","created_at":"2026-02-10 10:51:06","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":627891,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBiological processes enrichment analysis of putative TOFS miRs regulated genes\u003c/strong\u003e. \u003cstrong\u003e(A)\u003c/strong\u003e Enrichment analysis of the genes associated with EMT and the EMT-related tumorigenesis process. \u003cstrong\u003e(B)\u003c/strong\u003e Enrichment analysis related to the modulated genes by the most conserved TOFS miRs. Up to the top 20 enriched clusters are shown coloured by \u003cem\u003ep\u003c/em\u003e-values. All the statistically enriched terms were identified (GO/KEGG terms, canonical pathways, hall mark gene sets, etc.), accumulative hypergeometric \u003cem\u003ep\u003c/em\u003e-values and enrichment factors were calculated and used for filtering. Then 0.3 kappa score was applied as the threshold to divide the tree into term clusters. Created by Metascape [\u003ca href=\"http://metascape.org/\"\u003ehttp://metascape.org\u003c/a\u003e].\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8743228/v1/65f4f0941228cd2c62da8e5b.png"},{"id":102404233,"identity":"95a2e9e5-2a73-42a4-a777-4244abc6eddc","added_by":"auto","created_at":"2026-02-11 11:03:35","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":552363,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTime- and dose-dependent effects of TOFS miRs on PC3 cell migration. \u003c/strong\u003eWound healing assay with 1, 5, and 10 mg of TOFS miRs at 0, 24, 48, and 72 h post-treatment. A) Representative microscopy images (4X) showing PC3 cell migration at each time point for all the treatments (HF). B) Histogram of the fold change of the percentage of cell-free area at each time point. All data was derived from at least three independent biological experiments (*p\u0026lt;0.05). The percentage of cell-free area was assessed by ImageJ software. C) Representative histogram of TGF- β1 expression in PC3 cells subject to wound healing assay. The results show the mean of three independent biological experiments, *p\u0026lt;0.05,**p\u0026lt;0.01,***p\u0026lt;0.001 and ****p\u0026lt;0.0001. Two way ANOVA and a Tukey multiple comparison test were used.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8743228/v1/966db00433d8468207882b38.png"},{"id":102295061,"identity":"328ca133-d286-469e-8a05-ff3067155daa","added_by":"auto","created_at":"2026-02-10 10:08:17","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":355933,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of TOFS miRs on EMT protein related expression.\u003c/strong\u003e A) One representative Western blot image of E-cadherin, β-catenin, Snail and GAPDH proteins modulation 24, 48 and 72 hours after 1, 5 and 10 mg of TOFS miRs transfection, compared to HF treatment. B-D) In the graphs were reported: E-cadherin (panel B), Snail protein (panel C) and β-catenin (panel D) levels under treatments.\u003c/p\u003e\n\u003cp\u003eWestern blot analysis was quantified by densitometric analysis. The data were expressed as OD-Bkg/mm\u003csup\u003e2\u003c/sup\u003e, the background (Bkg) of the picture was subtracted, and each sample was normalised to the respective GAPDH value. The results show the mean of three independent biological experiments, *p\u0026lt;0.05,**p\u0026lt;0.01,***p\u0026lt;0.001 and ****p\u0026lt;0.0001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8743228/v1/6ec8a1650416afa4913db856.png"},{"id":101948651,"identity":"7e3dfe4d-bfa9-4d6d-b208-6e5f457be449","added_by":"auto","created_at":"2026-02-05 10:16:01","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":402560,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eβ-catenin localization in PC3 undergoing TGF-β1-induced EMT. \u003c/strong\u003e(A)\u003cstrong\u003e \u003c/strong\u003ePhase-contrast images of control and TGF- β1-treated PC3. Scale bars = 50 µm. (B) Fluorescence microscope images of β-catenin localization in PC3 cells undergoing TGF-β1-induced EMT, with or without TOFS miRs treatments at 48 h. β-catenin (rhodamine/green) and cell nuclei (blue). Scale bars = 50 µm. The images are representative of three independent experiments.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8743228/v1/9aeec97273f0739185448493.png"},{"id":102652138,"identity":"f0e250e4-e2e8-4f95-b201-684b6fbb4bb8","added_by":"auto","created_at":"2026-02-14 07:10:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4312369,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8743228/v1/5da796bb-dc32-4370-addd-42ef8b288b6d.pdf"},{"id":102298524,"identity":"2f5515c3-e474-4992-b052-286bbe99a908","added_by":"auto","created_at":"2026-02-10 10:42:57","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":40333,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8743228/v1/3c1fb85c5f9fe7ff6db1ab7f.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Solanum lycopersicum and Olea europaea microRNAs as Epithelial-Mesenchymal-Transition regulators in prostate cancer","fulltext":[{"header":"Introduction","content":"\u003cp\u003eProstate cancer (PC) is the second most common cancer in aging men and the fifth leading cause of cancer-related deaths worldwide (1). In 2020, approximately 1.4\u0026nbsp;million new cases of PC were diagnosed, resulting in nearly 375,000 deaths (2). The tumour usually progresses with limited aggressiveness and a paucity of initial symptoms (3). While a large fraction of tumours, especially those occurring in old age, require conservative treatment, more aggressive forms, such as castration-resistant PC, can spread rapidly to lymph nodes, bones, and liver, requiring long and multimodal therapy and posing a significant global health challenge and economic burden (4).\u003c/p\u003e \u003cp\u003eNatural agents from plants and their fruits play a pivotal role in cancer prevention and therapy (5); phyto-nutrients from extracts or purified compounds, are increasingly used to treat prostate benign hyperplasia, owing to their antioxidant, anti-inflammatory, and anti-cancer activities (6). A population-based study demonstrated that diet can have an antioxidant and anti-inflammatory effect inversely associated with prostate cancer aggressiveness (7).\u003c/p\u003e \u003cp\u003eTomatoes, as well as tomato products, are an important source of anti-inflammatory compounds with antiandrogenic properties (8,9). In tomatoes, natural synergy, cooking, and sustainable production make biofortification a nutrition-sensitive alternative to pill-based supplementation (10). Similarly, olive oil can be enriched with polyphenols recovered from olive-mill wastewater, valorising by-products to improve health benefits and circular sustainability (11). Biofortified foods enhance nutrients directly in the food matrix offering better bioavailability, safety, and dietary integration than isolated phyto-nutrients or supplements. Interestingly, the combination of biofortified tomato and olive wastewater has been showed to contribute to reduced cardiovascular risk and enhanced immune response and to have antibacterial, antiviral and anti-cancer properties (12,13). A newly developed biofortified tomato-olive supplement made of 98% spray dried whole tomato (\u003cem\u003eSolanum lycopersicum\u003c/em\u003e) and 2% olive wastewater (\u003cem\u003eOlea europaea)\u003c/em\u003e can inhibit prostate carcinogenesis \u003cem\u003ein vivo\u003c/em\u003e in mouse model, by regulating major metabolic pathways involved in the expression of a wide range of oncogenic genes (14,15) (patent No. EP2851080A1).\u003c/p\u003e \u003cp\u003eHowever, the health benefits of the supplement\u0026rsquo;s components are dose-dependent and their synergic interaction is still under evaluation (16,17). Among the bioactive compounds, plant-derived microRNAs (miRs) can be found, representing a unique class of conserved small non-coding RNAs (18\u0026ndash;24 nucleotides) that influence key biological processes (18,19), regulating gene expression post-transcriptionally by repressing translation and promoting mRNA degradation through interactions with their 3' untranslated regions (3'UTR) (20). They are conserved across all species and can act as regulatory molecules beyond species boundaries. Notably, plant-derived miRs can be transferred to humans through dietary intake, exerting regulatory effects on human gene expression \u003cem\u003evia\u003c/em\u003e cross-kingdom interactions (21\u0026ndash;24). Indeed, Minutolo \u003cem\u003eet al\u003c/em\u003e.(23) showed that miRs extracted from \u003cem\u003eOlea europaea\u003c/em\u003e drupes have anti-proliferative and anti-cancer effects in human cells, in line with a patented strategy exploiting plant-derived microRNAs for cancer prevention and treatment (EU patent No. EP3216869A1).\u003c/p\u003e \u003cp\u003eThe epithelial-mesenchymal transition (EMT) is an essential mechanism driving epigenetic (25) and metabolic (26) reprogramming of carcinoma cells, including PC (27), leading to a mesenchymal phenotype associated with cancer invasiveness and metastasis spreading (28). EMT cause progressive loss of epithelial characteristics, including cell polarity, proliferation control, and differentiation, resulting in increased cell motility, invasiveness, and resistance to therapy-induced apoptosis (29,30). These processes intertwine with the signalling pathway of the transforming growth factor-β1 (TGF-β1), which is known to be overexpressed in a variety of cancers to induce EMT, including colon cancer among others (31). TGF-β1 targets Snail, a protein that directly inhibit E-cadherin transcription by binding its promoter region (32). The decline in E-cadherin levels is a notable hallmark of EMT; indeed, E-cadherin creates adherens junctions for cell-cell adhesion, stabilized by β-catenin. The E-cadherin/β-catenin complex is therefore critical in preserving epithelial integrity. In this context, TGF-β1 triggers the dissociation of β-catenin and its stabilization in the cytoplasm, enabling its nuclear import, where it may influence cellular processes such as proliferation, migration, and invasion (33) .\u003c/p\u003e \u003cp\u003eIn this study, we performed an \u003cem\u003ein vitro\u003c/em\u003e analysis on castration-resistant prostate cancer cells, supported by an in-depth bioinformatic investigation to identify microRNA components of a water resuspension of the biofortified tomato and olive-based supplement (called TOFS) to evaluate its anti-prostate tumour activity by downregulating EMT-mediated progression.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eExtraction of the miRs pools from water-resuspended tomato and olive food supplement (TOFS)\u003c/h2\u003e \u003cp\u003eMiRs were obtained from a water resuspension of a food supplement (TOFS) registered with the Italian Ministry of Health (EU patent No. EP2851080A1) made of 98% spray-dried whole tomatoes and 2% olive mill wastewater and kindly provided by Janus Pharma (Rome, Italy). A total of 6,5 g of supplement powder was dissolved in 100 mL of distilled water, yielding a final concentration of 65 mg/mL. The TOFS stock solution was centrifuged at 100 \u0026times; g, and the supernatant was recovered and filtered through a 0.45 \u0026micro;m filter unit (Minisart\u0026reg;) to eliminate debris. The resulting aliquots were stored at -20\u0026deg;C until use.\u003c/p\u003e \u003cp\u003eTOFS stock solution was diluted to obtain a final concentration of 1 mg/ml, 5 mg/ml and 10 mg/ml and MiRs were extracted using the GenUP Micro RNA Kit (BiotechRabbit, Germany), according to the manufacturer\u0026rsquo;s instructions. TOFS miRs were quantified with a NanoDrop\u0026trade; Lite Spectrophotometer (Thermo Fisher Scientific, United States), by measuring absorbance at 260\u0026ndash;280 nm.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eTOFS miRs characterization\u003c/h3\u003e\n\u003cp\u003eLibrary preparation of the TOFS miRs was performed using the QIAseq miRNA library kit (Qiagen, Hilden, Germany). All samples were sequenced in 75 single-end on an Illumina NextSeq500 platform according to QIAseq miRNA Library Kit Handbook for precision small RNA library prep for Illumina \u0026reg;\u003c/p\u003e \u003cp\u003eNGS systems.\u003c/p\u003e \u003cp\u003eRaw sequencing read quality was assessed using FastQC v0.11.9(34) and MultiQC v1.10.1(35). Adapter trimming was performed with cutadapt v3.4 (36), and a custom script was subsequently used for read deduplication based on Unique Molecular Identifiers (UMIs). The processed reads were aligned using SHRiMP v2.2.3(37)against reference hairpin databases: miRBase (release 22.1) (38) for \u003cem\u003eSolanum lycopersicum\u003c/em\u003e (Sly) and the Olive Genome Consortium database for \u003cem\u003eOlea europaea\u003c/em\u003e (Oeu). To quantify the expression of mature miRNAs, the BEDTools suite v2.30.0 was employed to identify overlapping mappings between the aligned reads and the corresponding mature sequences. For \u003cem\u003eS. lycopersicum\u003c/em\u003e, mature miRNA coordinates were obtained from the miRBase annotation, whereas for \u003cem\u003eO. europaea\u003c/em\u003e, the reference set consisted of 136 conserved miRNAs and putative novel miRNAs identified by Yanik et al. (2013) (39).\u003c/p\u003e\n\u003ch3\u003emiRNA target prediction\u003c/h3\u003e\n\u003cp\u003eWe employed IntaRNA3 (40) to investigate whether \u003cem\u003eS. lycopersicum\u003c/em\u003e miRNAs target EMT pathway gene transcripts. By predicting RNA\u0026ndash;RNA interactions through thermodynamic stability, IntaRNA3 provides the high sensitivity required for modeling complex miRNA\u0026ndash;mRNA. IntaRNA3 calculates interaction energies between RNA sequences, where lower energy scores indicate more stable and likely interactions. In our analysis, we input sequences of known \u003cem\u003eS. lycopersicum\u003c/em\u003e miRs and the 3' untranslated regions (3' UTRs) of human EMT pathway mRNAs into IntaRNA3. To minimize false positives, we applied a stringent filter, considering only interactions with a free energy (ΔG) below \u0026minus;\u0026thinsp;15 kcal/mol as significant. This approach allowed us to focus on the most thermodynamically stable and biologically relevant potential interactions, suggesting a possible regulatory mechanism where tomato miRNAs could target human EMT pathway transcripts for translational repression or degradation.\u003c/p\u003e\n\u003ch3\u003eFunctional enrichment analysis\u003c/h3\u003e\n\u003cp\u003eTo further investigate the biological relevance of the predicted miRNA-mRNA target gene interactions, the list of potentially regulated transcript identified by IntaRNA3 was subjected to Functional Enrichment Analysis. This analysis aims to identify over-represented biological pathways, functions, or processes within a gene list, providing insights into the potential functional impact of miRNA-mediated regulation. We utilized two distinct tools for this purpose: Metascape and FunRich. Metascape is a comprehensive web-based platform that leverages multiple gene annotation databases and pathway repositories to perform Gene Ontology (GO) enrichment, pathway enrichment analysis, and protein-protein interaction network analysis. It statistically determines significantly enriched terms within the input gene list compared to a background set. FunRich, a standalone software tool, also performs functional enrichment analysis, focusing on gene set enrichment from various databases. It provides statistical measures to assess the significance of enriched functional terms and pathways, offering complementary insights into the biological themes associated with the potentially miRNA-regulated EMT genes. By using both Metascape and FunRich, we aimed to obtain a robust and comprehensive functional characterization of the genes predicted to be targeted by \u003cem\u003eS. lycopersicum\u003c/em\u003e miRNAs.\u003c/p\u003e\n\u003ch3\u003ePC3 cell culture and transfection\u003c/h3\u003e\n\u003cp\u003eHuman prostate cancer PC3 cells, a bone metastasis of grade IV prostatic adenocarcinoma (ATCC, CRL-1435), were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Invitrogen, USA). The culture medium was supplemented with 10% fetal bovine serum (FBS, Invitrogen, Germany), 2mM glutamine (HyCloneTM, UK), 100 U/mL penicillin and 100 U/mL streptomycin (HyCloneTM, UK). The cells were maintained at 37\u0026deg;C in a humidified incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e to generate a confluent monolayer and were routinely split by trypsinization with Trypsin-EDTA (Euroclone, IT). Experiments were performed with cell viability consistently above 98%. PC3 cells were transfected with TOFS miRs, present in 1, 5 and 10 mg of TOFS solution for 24, 48 and 72 hours using Lipofectamine\u0026trade; 2000 (Hi-Fect, HF, Invitrogen, USA), according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eWound healing assay\u003c/h2\u003e \u003cp\u003eThe ability of PC3 cells transfected with miRs to migrate was evaluated using a wound healing assay. Cell migration, indeed, is strictly correlated to activation of the EMT process (41). PC3 cells were seeded at a density of 0.15 x 10\u003csup\u003e6\u003c/sup\u003e cells/ml in RPMI medium. A confluent monolayer of cells was obtained after 24 hours at 37\u0026deg;C in 5% CO\u003csub\u003e2\u003c/sub\u003e. Cells were then transfected as describe above with TOFS miRs, and scratch wounds were created mechanically using a sterile pipette tip. PC3 cell migration was observed at 24, 48 and 72 hours post-transfection, and images were acquired with a microscope [NIKON, (ECLIPSE Ts2) coupled to a camera alexasoft TP1080HDMI].\u003c/p\u003e \u003cp\u003eThe migration rate was measured by Image J and expressed as fold change (Treated/Untreated) of the cell-free area.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eTGF-β1 evaluation\u003c/h3\u003e\n\u003cp\u003eSupernatants from the wound healing assays were collected and TGF-β1 release was investigated by using TGF-β1 Duoset ELISA Kits (R\u0026amp;D Systems Inc) according to manufacturer\u0026rsquo;s instruction. The optical density was measured at 450 nm (reference 540 nm) using the Infinite\u0026reg;200 PRO (Tecan Trading AG). Data analysis was carried out using GraphPad Prism v8.4.3 software, and cytokine concentration was calculated based on the standard curve.\u003c/p\u003e\n\u003ch3\u003eWestern blot analysis of β-catenin, E-cadherin, Snail\u003c/h3\u003e\n\u003cp\u003eAliquots of 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells, subjected to various experimental conditions, were lysed and processed for western blot analysis according to standard protocol. The primary antibodies used were mouse monoclonal antibodies directed against β-catenin (13-8400), E-cadherin (13-1700), Snail (MA5-15791), and GAPDH (Abcam: 9484) as a loading control. Antibodies were prepared according to the manufacturer's instruction. The secondary antibody used was HRP-conjugated goat anti-mouse IgG (Invitrogen, Cat. No. A28177). Proteins were detected with the ECL (Millipore, WBKLS0500) and proteins\u0026rsquo; specific signals were quantified by densitometry using ImageJ software.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eβ-catenin immunofluorescence\u003c/h2\u003e \u003cp\u003eTransfected cells were grown in 96-well plates and fixed with 4% paraformaldehyde for 15 min. Immunofluorescence staining was performed at room temperature (RT) in PBS 0,2% Triton X-100 and PBS with 1% bovine serum albumin (BSA), as permeabilizing and blocking agents, respectively. Cells were incubated for 1 hour with a primary antibody against β-catenin (13-8400). After 3 washing, cells were incubated for 1 h with the Tetramethyl Rhodamine Isothiocyanate (TRITC)-conjugated secondary antibody (T2402, Sigma-Aldrich, St. Louis, MO, USA) and then stained with DAPI (2 \u0026micro;g/mL) for nuclei detection. Images were captured using a ZEISS Axio Observer microscope. Unbiased staining intensity was estimated after background signal elimination through pixel fluorescence analysis according to Carl Zeiss Microscopy GmbH\u0026rsquo;s ZEN software (version 3.0, Jena, Germany). At least four images for each sample were analysed to obtain the relative frequency distribution of β-catenin fluorescence intensity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData are presented as mean values\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). All experiments were performed at least in triplicate (biologically independent measurements). Significant differences are shown as \u003cem\u003ep-\u003c/em\u003evalue: p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 (*), p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 (**), p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 (***) or p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 (****). A nonparametric two-way post-hoc ANOVA corrected by t-test was performed.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eA biofortified food supplement (patent No. EP2851080A1) based on 98% spray-dried whole tomatoes and 2% olive mill wastewater (TOFS) was evaluated for the presence of miRNAs. The identified miRs were characterized for their antitumor effect at first \u003cem\u003ein silico\u003c/em\u003e and then experimentally on PC3 cells, a castration-resistant high-grade PC cell line derived from bone metastases.\u003c/p\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eSequencing and characterisation of the miRs present in the TOFS\u003c/h2\u003e \u003cp\u003eA total of 78 miRs extracted from TOFS solution were successfully sequenced, including 64 from \u003cem\u003eO. europaea\u003c/em\u003e (\u003cem\u003eoeu\u003c/em\u003e-miRs) and 14 from \u003cem\u003eS. lycopersicum\u003c/em\u003e (\u003cem\u003esly-\u003c/em\u003emiRs) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, 69 of the 78 (87%) miRs identified by sequencing belonged to well-known plant miRs families. The most represented TOFS miRs belonging to plant miRs families were miR169, miR166, miR395, and miR319, in which 17, 11, 8, and 9 miRs were identified, respectively.\u003c/p\u003e \u003cp\u003eMost of these were \u003cem\u003eoeu\u003c/em\u003e-miRs, for instance from the miR169 family, 14 were \u003cem\u003eoeu\u003c/em\u003e-miRs (\u003cem\u003eoeu\u003c/em\u003e-miRs 169 d-m, r, s, v, w) and three were \u003cem\u003esly-\u003c/em\u003emiRs (\u003cem\u003esly\u003c/em\u003e-miRs 169b and d), while in the miR166, miR395, and miR319 families only \u003cem\u003eoeu\u003c/em\u003e-miRs were found (\u003cem\u003eoeu\u003c/em\u003e-miR166 a-f, h, I, j, k, q; \u003cem\u003eoeu\u003c/em\u003e-miR 395 b-j; \u003cem\u003eoeu\u003c/em\u003e-miRs 319 a-h).\u003c/p\u003e \u003cp\u003eThe other plant miRs families (159, miR167, miR171, miR172, miR394, miR396, miR399, miR403) were represented by 1 to 4 \u003cem\u003eoeu-\u003c/em\u003emiRs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB \u003cb\u003eand Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e), with the exception of the miR168 family, in which we observed the presence of miRs belonging to both species \u003cem\u003e(sly\u003c/em\u003e-miRs 168a and b-5p and \u003cem\u003eoeu\u003c/em\u003e-miRs 168a and b).\u003c/p\u003e \u003cp\u003eThe sequencing highlights the presence of 8 other \u003cem\u003esly\u003c/em\u003e-miRs (\u003cem\u003esly\u003c/em\u003e-miRs10529, \u003cem\u003esly\u003c/em\u003e-miRs10534, \u003cem\u003esly\u003c/em\u003e-miRs 10542, \u003cem\u003esly\u003c/em\u003e-miRs 1918, \u003cem\u003esly\u003c/em\u003e-miRs 482, \u003cem\u003esly\u003c/em\u003e-miRs 5300, \u003cem\u003esly\u003c/em\u003e-miRs7981, \u003cem\u003esly\u003c/em\u003e-miRs9478) that do not belong to any of the families described above (\u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eQuantitative Unique Molecular Identifier (UMI)-based analysis reported in Table\u0026nbsp;1, revealed that among \u003cem\u003esly\u003c/em\u003e-miR10529 and \u003cem\u003esly\u003c/em\u003e-miR1918 were the most abundant, with approximately 75,400 and 39,000 UMIs, respectively; the other miRs were expressed with UMIs range 7,800-2,600.\u003c/p\u003e \u003cp\u003eThe most abundant \u003cem\u003eoeu\u003c/em\u003e-miRs were miR168a and miR168b, with approximately 98,800 and 93,600 UMIs, respectively, while for the others \u003cem\u003eoeu\u003c/em\u003e-miRs different levels of expression ranged from 10,400 to 2,600 were observed (\u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eSince UMIs correct for PCR amplification bias, these values provided a reliable estimate of the true relative abundance of the respective miRNAs.\u003c/p\u003e \u003cp\u003eThese results provide valuable insights into the presence in the TOFS of several miRs belonging to known plant species, which may have a regulatory role in humans.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eComputational prediction of TOFS miRs targeting human genes, and network analysis of the processes involved in EMT pathway\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe IntaRNA3 tool, an advanced bioinformatics algorithm for rapid and accurate prediction of RNA-RNA interactions (primarily between small regulatory RNAs and their target mRNAs) was employed to evaluate the likelihood of interactions between TOFS miRs and genes involved in EMT process. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and \u003cb\u003eTable S2\u003c/b\u003e, 56 out of the 78 identified TOFS miRs were predicted to bind to 68 mRNAs associated with EMT. Notably, \u003cem\u003esly-miR482d-5p\u003c/em\u003e was able to regulate 65 of these 68 genes, whereas the other miRs exhibited a range of target gene numbers from 17 down to 1 (see \u003cb\u003eTable S2\u003c/b\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Remarkably, SMAD family member 2 (SMAD2) was highly regulated, being targeted by thirty-five TOFS miRs; DSC2, a transmembrane cell anchoring protein, by twenty-seven; N-cadherin (CDH2) by twenty-two; ZEB1 by twenty; and seventeen TOFS miRs were predicted to target VPS13A (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Three out of five top target genes are upregulated in the TGF-β1-induced EMT process (42); therefore, their downregulation mediated by plant microRNAs could potentially reverse EMT.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eConsidering the key role of EMT in both PC metastatic progression and therapy resistance, we exploited the online tool Metascape (43) to investigate the possible antitumor relevance of TOFS miRs within EMT-related networks.\u003c/p\u003e \u003cp\u003eNetwork analysis revealed a highly interconnected and complex set of biological pathways involved in EMT (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-B). The constructed networks highlight key processes such as EMT in colorectal cancer, proteoglycans in cancer, tube morphogenesis, extracellular matrix organization, and cell population proliferation. Additional pathways detected included enzyme-linked receptor protein signalling, Hippo signaling, focal adhesion, and cell differentiation, underscoring the multifactorial and tightly regulated nature of EMT. The diverse biological processes identified, including neural crest differentiation, response to wounding, and cell-cell communication, demonstrate that EMT is integrated with multiple cellular functions extending beyond classical tumour progression pathways.\u003c/p\u003e \u003cp\u003eComplementing the network data, enrichment analysis quantified the statistical significance of these pathways in the context of EMT gene modulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-B). The results show strong enrichment (-log\u003csub\u003e10\u003c/sub\u003e \u003cem\u003ep-\u003c/em\u003evalues) for hallmark EMT pathways, such as epithelial\u0026ndash;mesenchymal transition in colorectal cancer and related cellular processes including extracellular matrix organization and proteoglycan function. This enrichment pattern was particularly significant when considering TOFS miR target genes, reinforcing the biological relevance of these miRs in modulating EMT. Notably, pathways related to signaling mechanisms (e.g., enzyme-linked receptor protein signalling, Hippo pathway) and cell\u0026ndash;cell communication were also significantly enriched, indicating that TOFS miRs potentially orchestrate complex regulatory networks governing EMT phenotypes.\u003c/p\u003e \u003cp\u003eTogether, the network and enrichment findings reveal that the molecular landscape of EMT is characterized by extensive cross-talk among multiple biological pathways. These data support a model where TOFS miRs influence EMT by targeting a broad range of interconnected pathways critical for cellular plasticity, adhesion, and differentiation, which are essential for both physiological and pathological EMT processes.\u003c/p\u003e \u003cp\u003eThe analysis revealed that TOFS miRs may regulate several genes associated with EMT, influencing pivotal EMT-related cellular processes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eRegulatory effect of TOFS miRs on PC3 cell migration\u003c/h2\u003e \u003cp\u003eTo investigate the impact of TOFS miRs on PC3 cell migration, a wound-healing assay was performed at 0, 24, 48, and 72 h after administration of 1, 5 and 10 mg of TOFS miRs. Twenty-four hours after treatments, similar growth and migration are observed in each condition. Compared to control cells, which showed a clear wound closure at 48 h, cells transfected displayed a significantly over two-fold reduced migratory rate which was inversely proportional to treatment concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA \u003cb\u003eand B\u003c/b\u003e), highlighting the anti-migratory effect of the plant miRs. In particular, a dose of 1 mg of miRs resulted the most efficacious at all time points, with a 2.5-fold increase in cell-free area at 48 h compared to the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). At 72 hours, the cell-free area was significantly higher only for cells treated with 1 and 10 mg of TOFS miRs compared to the control.\u003c/p\u003e \u003cp\u003eThese data may suggest a potential regulatory effect on the migratory capacity of PC3 cells.\u003c/p\u003e \u003cp\u003eDuring proliferation and migration, cells typically release transforming growth factor beta (TGF-β1 (44). We therefore measured TGF-β1 levels in PC3 supernatants obtained from the wound healing experiments by ELISA assay. Expectedly, TGF-β1 expression showed a significant increase over time. Specifically, expression levels at 48 and 72 hours were significantly higher compared to 24 hours (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 (**) and p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 (****) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). At 48 h post-transfection, a significant decrease in TGF-β1 expression level was observed across the three TOFS miRs treatments compared to the control; at 72 h, instead, a significant reduction persisted only in cells treated with 1 mg/mL miRs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eEffects of TOFS miRs on the EMT-markers E-cadherin, β-catenin, and Snail in PC3 cells\u003c/h2\u003e \u003cp\u003eConsidering the anti-migratory effect of the TOFS miRs displayed in the wound-healing assay, the expression levels of different proteins involved in EMT, including E-cadherin, β-catenin and Snail, were investigated.\u003c/p\u003e \u003cp\u003eE-cadherin, a transmembrane protein that plays a crucial role in calcium-dependent cell-cell adhesion, is usually down-modulated in cancer compared to normal conditions. The treatment with TOFS miRs induced a significant increase in E-cadherin expression in PC3 at 48 and 72 h (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, B), suggesting a restoration of epithelial characteristics. The expression of Snail, a key EMT transcription factor which negatively regulates E-cadherin, was then investigated. Although not statistically significant, Snail showed a slight decrease in its expression at 24 h after TOFS administration (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA \u003cb\u003eand C\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eFinally, we evaluated the expression of β-catenin, an adhesion junction protein involved in cell-cell connections, whose upregulation/accumulation is typically implicated in cancer progression (45). The expression of this protein was modulated by TOFS miRs treatments, showing a significant downregulation at 48 and 72 h compared with the control (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA \u003cb\u003eand D\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWestern blot analysis was quantified by densitometric analysis. The data were expressed as OD-Bkg/mm\u003csup\u003e2\u003c/sup\u003e, the background (Bkg) of the picture was subtracted, and each sample was normalised to the respective GAPDH value. The results show the mean of three independent biological experiments, *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05,**p\u0026thinsp;\u0026lt;\u0026thinsp;0.01,***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 and ****p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001.\u003c/p\u003e \u003cp\u003eDuring EMT, β-catenin is not only overexpressed, but is also typically aberrantly translocated from the cell membrane to the nucleus. This process is closely associated with the activation of the Wnt/β-catenin signalling pathway, which plays a pivotal role in the regulation of cell proliferation, migration, and survival (46). We therefore investigate β-catenin subcellular localization. Cells pre-treated with 2 ng/ml TGF-β1 stimulus to induce EMT, were then administered with TOFS miRs (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). At 48 h post-transfection, TGF-β1-treated cells exhibited nuclear β-catenin, as per EMT process, whilst TOFS-treated cells showed β-catenin membrane re-localization (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eProstate cancer (PC) is the leading cause of cancer-related death among middle-aged and older men worldwide. Intense multidisciplinary research is ongoing today to develop more effective preventive strategies to delay or mitigate disease severity, as well as integrated therapy to improve outcomes in patients with aggressive PC (47). Epidemiological evidence has highlighted that the risk of developing PC is significantly increased with the consumption of a typical \u0026ldquo;Western-style\u0026rdquo; diet, characterized by high intakes of red and processed meats as well as white bread, fried fish, high-fat milk and chips(47). On the other hand, adherence to the Mediterranean diet, which is rich in antioxidant compounds derived from olive oil and tomatoes, has been shown to be associated to reduced PC incidence, slowing Gleason grade progression in patients with low-grade prostate cancer (48\u0026ndash;51).\u003c/p\u003e \u003cp\u003eThe antioxidant and anti-inflammatory properties of the food included in the Mediterranean diet are linked to prostate health, however, the extent of the benefits depends on the specific compounds and how the body processes them, which affects their bioavailability.\u003c/p\u003e \u003cp\u003eBiofortified functional food is known to increase food bioavailability; indeed, a newly developed tomato and olive biofortified supplement has shown potent antioxidant activity and the capability to downregulate the pathways involved in the progression of prostate tumours (15). Moreover, the biofortified supplement investigated, is richer in nutrients than raw and cooked market products (52), its intake could therefore facilitate compliance with the \u0026ldquo;Prostate Dietary Index\u0026rdquo;, which has been shown to be associated with a 18 % lowerrisk of prostate cancer incidence (53).\u003c/p\u003e \u003cp\u003ePlant miRs can modulate the post-transcriptional regulation of human mRNA as human miRs do, in a process known as cross-kingdom interaction. This regulatory ability has generated significant interest due to its potential to develop new therapeutic agents to treat diseases associated with miRs dysregulation, including cancer (54\u0026ndash;57).\u003c/p\u003e \u003cp\u003eIn this study, we demonstrate that TOFS contains miRs potentially capable to modulate gene expression. A bio-informatic analysis reveals that 56 TOFS miRs modulate the expression of 65 genes involved in EMT, a mandatory step in gaining the metastatic phenotype(27). While the field is still emerging and somewhat controversial, experimental evidence indicates that plant-derived miRNAs ingested through the diet can survive gastrointestinal digestion, enter circulation, and modulate the expression of mammalian oncogenes, tumour suppressors, and other cancer-related transcripts, suggesting potential implications in cancer biology and prevention(58).\u003c/p\u003e \u003cp\u003eIndeed, it is known that plant-derived miRs can influence mammalian gene expression in a cross-kingdom manner (23). We detected 78 miRNAs belonging to 25 families several of which belonged to highly conserved families across various species; overall, most of the identified miRs originated from \u003cem\u003eO. europaea\u003c/em\u003e and, among these, \u003cem\u003eoeu\u003c/em\u003e-miR156, \u003cem\u003eoeu\u003c/em\u003e-miR159, \u003cem\u003eoeu\u003c/em\u003e-miR166, and \u003cem\u003eoeu\u003c/em\u003e-miR169 were the most represented. Plant miR159 has been previously identified in human sera and its level has been inversely associated with breast cancer progression and incidence (59).\u003c/p\u003e \u003cp\u003eAmong the EMT-related genes most frequently recognised by the TOFS miRs were CDH2, SMAD2 and ZEB1, which are typically upregulated during the EMT process. These genes appear to be potential targets of TOFS miRs, which could then compensate for the reduced levels of endogenous human miRs (60) that usually regulate their expression.\u003c/p\u003e \u003cp\u003eWe evaluated the ability of PC3 cells transfected with miRs to migrate through a wound healing assay. Cell migration, indeed, is strictly correlated to activation of the EMT process (41). We observed that miRs treatment induces a decrease in the cells' migratory capacity, allowing us to hypothesize that miRs treatment could counteract EMT.\u003c/p\u003e \u003cp\u003eOur EMT analysis focused on evaluating the level of expression of TGF-β1. The significant reduction in TGF-β1 observed in PC3 cells induced by TOFS miRs, has a powerful impact on EMT dynamics since it significantly alter TGF-β1 pathway, leading a slight decrease in the expression of transcription factor Snail protein and β-catenin, which results in an increase of E-cadherin expression, resulting in restored cell-to-cell adhesion and reduced migration.\u003c/p\u003e \u003cp\u003eThe subcellular distribution of β-catenin was next evaluated in PC3 cells forced to undergo EMT \u003cem\u003evia\u003c/em\u003e TGF-β1 treatment. As expected, β-catenin localized mainly in the cytoplasm and nucleus with TGF-β1 treatment but miRs treatment promoted a re-localization to the membrane post treatment. These data confirm the ability of TOFS miRs to restore an epithelial phenotype. Overall, the results confirm the ability of TOFS miRs to down regulated cell migration in PC3 cells by targeting the expression of key proteins involved in EMT.\u003c/p\u003e \u003cp\u003eThe use of a supplement made of biofortified tomato obtained with a prolonged patented heating process together with the implementation of an extraction protocol to recover microRNAs from TOFS, outlines a scenario of dual economic and strategic advantage. On one hand, the utilization of edible plants for the production of therapeutic small RNAs represents a ground-breaking and economical alternative to the currently expensive chemical synthesis required for artificial miRNAs(23). On the other hand, this practice is deeply rooted in the circular economy and waste-to-resource models, converting abundant agro-industrial residues, such as olive mill wastewater and pomace, from environmental liabilities requiring costly disposal into valuable raw materials for the nutraceutical industry (61).\u003c/p\u003e \u003cp\u003eConsequently, this approach not only drastically reduces the production costs of molecules capable of regulating human gene expression, but also generates new revenue streams for producers, effectively closing the loop of two of the most globally widespread agricultural chains (61).\u003c/p\u003e \u003cp\u003eFrom a translational perspective, the ability of TOFS-derived miRs to restore epithelial markers and suppress EMT-associated signalling suggests a potential role in limiting prostate cancer cell invasiveness and metastatic potential. While not intended as a standalone therapeutic approach, such diet-derived miRs may represent a promising adjunct strategy to complement existing treatments aimed at controlling disease progression.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThrough a multiparametric analysis, the present study demonstrates that a newly available food supplement of well-defined formulation based on \u003cem\u003eSolanum lycopersicum\u003c/em\u003e and \u003cem\u003eOlea europaea\u003c/em\u003e contains plant-derived miRs capable of modulating PC3 cells behaviour. Specifically, TOFS-derived miRs interfere with the expression of key EMT-related proteins, re-establishing a non-cancerogenic epithelial phenotype and suppressing features associated with metastatic potential including cell migratory activity. Beyond the observed effects on EMT regulation, the miRs identified in TOFS may act in concert with other bioactive micronutrients naturally present in the supplement, including highly bioavailable lycopene, quercetin, narigenin, and verbascoside, thereby contributing to a broader protective molecular environment that supports prostate health.\u003c/p\u003e \u003cp\u003eImportantly, these findings support the concept that biofortified functional foods can act not only as sources of antioxidant compounds but also as carriers of biologically active plant-derived miRs with cross-kingdom regulatory potential. While their bioavailability and long-term effects in humans require further investigation, our data provide mechanistic \u003cem\u003ein vitro\u003c/em\u003e evidence that dietary miRs can modulate cancer-relevant pathways.\u003c/p\u003e \u003cp\u003eFrom a translational and preventive perspective, TOFS-derived miRs are not intended to replace established prostate cancer therapies, but may represent a complementary approach to limit disease progression or tumour aggressiveness, particularly in early-stage disease. Future studies on TOFS will be needed to validate these effects \u003cem\u003ein vivo\u003c/em\u003e and to assess their clinical relevance. Indeed, previous findings from epidemiological trials already raised awareness that processed tomato products were associated with a 30\u0026ndash;40% reduction of PC risk (49).\u003c/p\u003e \u003cp\u003eFinally, the production of TOFS within a circular economy framework, leveraging agro-industrial by-products such as olive mill wastewater, highlights a sustainable and economically viable strategy for developing nutraceuticals with added biological value.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e3'UTR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e3' untranslated regions\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBkg\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eBackground\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBSA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eBovine Serum Albumin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCDH2\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eN-cadherin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eEMT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eEpithelial-Mesenchymal Transition\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFBS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFetal Bovine Serum\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGene Ontology\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLipofectamine\u0026trade; 2000\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMiRs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMicroRNAs\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003eOeu\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e \u003cem\u003eOlea europaea\u003c/em\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003eOeu\u003c/em\u003e miRs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e \u003cem\u003eO. europaea\u003c/em\u003e miRs\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eProstate Cancer\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRPMI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRoswell Park Memorial Institute\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRoom Temperature\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eStandard Deviation\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003eSly\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e \u003cem\u003eSolanum lycopersicum\u003c/em\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003eSly\u003c/em\u003e miRs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e \u003cem\u003eS. lycopersicum\u003c/em\u003e miRs\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSMAD2\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSMAD family member 2\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTGF-β1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTransforming Growth Factor-β1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTOFS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTomato and Olive Food Supplement\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTRITC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTetramethyl Rhodamine Isothiocyanate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eUMI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eUnique Molecular Identifiers\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":" \u003cp\u003e \u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e \u003cp\u003eNot applicable\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eNot applicable\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003ePGN has a professional affiliation with Janus Pharma S.r.l. (Rome, Italy). The other authors declare no conflicts of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by the CANVAS project under Grant Agreement No. 101079510.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization and study design: CM, MP, PGN, VC; Wet-lab experiments and sample preparation: AM, FC, CT. NGS Sequencing: SF. Bioinformatic and biostatistical analyses: FI, AM; Validation: MP, CM, PGN, VC. Writing\u0026mdash;original draft preparation, AM. Writing\u0026mdash;review and editing: all authors have read, contributed equally to the revision, and approved the final version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003ePGN acknowledges partial support from the Federico Calabresi Foundation (Rome, Italy).\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets analysed during the current study are included in this published article and its additional file, and are also available in the Zenodo repository [ [10.5281/zenodo.18387556](https:/doi.org/10.5281/zenodo.18387556) ].\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSchafer EJ, Laversanne M, Sung H, Soerjomataram I, Briganti A, Dahut W, et al. Recent Patterns and Trends in Global Prostate Cancer Incidence and Mortality: An Update. Eur Urol. 2025 Mar;87(3):302–13.\u003c/li\u003e\n\u003cli\u003eWang L, Lu B, He M, Wang Y, Wang Z, Du L. 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Environmental Science and Pollution Research. 2024 Feb 26;31(14):20853–80.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"microRNA1, Solanum lycopersicum 2, Olea europaea 3, EMT4, prostate cancer5, Cross-kingdom interaction6","lastPublishedDoi":"10.21203/rs.3.rs-8743228/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8743228/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eProstate cancer (PC) is the second most common malignancy among men worldwide. Treatments for metastatic PC, especially in the castration-resistant stage, often yield suboptimal outcomes and rarely achieve a cure. This underscores the urgent need for improved preventive and therapeutic strategies. In recent years, plant-derived microRNAs (miRs) have emerged as potential epigenetic regulators across kingdoms, potentially influencing pathways involved in tumour progression. Within a translational medicine framework, we evaluated a newly developed tomato-based food supplement composed of on 98% heated, spray dried whole tomato (\u003cem\u003eSolanum lycopersicum\u003c/em\u003e) and 2% olive wastewater (\u003cem\u003eOlea europaea\u003c/em\u003e), both key nutrients of the Mediterranean diet. Here, we investigate the anti-tumoral potential of miRs extracted from a resuspension of the supplement (called TOFS), with particular emphasis on their modulation of epithelial\u0026ndash;mesenchymal transition (EMT), using the prostate cancer PC3 cell line as an in vitro model. TOFS miRs were quantified and analysed by NGS-based analysis, identifying miRNAs from 25 families with known human targets involved in tumorigenesis. The regulation by miRs of EMT-related factors, including TGF-β1, Snail, β-catenin, and E-cadherin, was assessed as indicators of cell\u0026ndash;cell adhesion, cytoskeletal stability, and intercellular communication using ELISA and Western blot analyses.\u003c/p\u003e \u003cp\u003eThe results indicate that TOFS miRs modulate key cellular processes in PC3 cells, particularly their migratory activity. This finding further prove the capability of TOFS to modulate experimental prostate carcinogenesis and the potential to provide clinical benefits in benign prostatic hyperplasia in humans.\u003c/p\u003e","manuscriptTitle":"Solanum lycopersicum and Olea europaea microRNAs as Epithelial-Mesenchymal-Transition regulators in prostate cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-05 10:15:56","doi":"10.21203/rs.3.rs-8743228/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"aae9ec63-7a91-4ef5-b35b-a82dbe4a1f36","owner":[],"postedDate":"February 5th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-02-14T07:09:57+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-05 10:15:56","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8743228","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8743228","identity":"rs-8743228","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}