Identification of novel exosomal miRNAs and their role in diagnosis and prognosis of Triple Negative Breast Cancer

Identification of novel exosomal miRNAs and their role in diagnosis and prognosis of Triple Negative Breast 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 Identification of novel exosomal miRNAs and their role in diagnosis and prognosis of Triple Negative Breast Cancer Ananya Choudhary, Satish S. Poojary, Priyanka Jain, Harit Chaturvedi, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5910171/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 Exosomal miRNAs have been identified as key secretory biomarkers facilitating intercellular communication in the tumor microenvironment, with substantial potential as diagnostic, prognostic, and therapeutic targets. Our research focuses on the role of exosomal miRNAs in triple-negative breast cancer (TNBC) and cancer stem cells, contributing to tumor progression and relapse. Through meta-analysis and data mining, we identified five differentially expressed miRNAs- hsa-miR-6803, hsa-miR-1180, hsa-miR-4728, hsa-miR-1915 and hsa-miR-940 in breast cancer. Target predictions, GO and KEGG enrichment analyses have indicated the potential role of these oncomiRs in Wnt, Notch and EGFR signalling pathways involved in tumor progression. While the panel of five miRNAs was found to be over-expressed in breast cancers, they have not been reported in TNBC and TNBCSCs, making them ideal biomarkers for TNBC. These oncomiRs were consistently detected across TNBC cell lines, TNBCSCs and further validated in TNBC tumor tissues (n = 15). Interestingly, a highly significant overexpression of two of the five specific miRNAs- hsa-miR-1180 and hsa-miR-4728 and their high tumorigenic validation in an invasion, migration and siRNA analysis indicates their potential as prognostic and therapeutic target(s). The presence of these miRNAs in TNBC cell lines, TNBCSCs and circulatory exosomes and their elevated expression in tumor tissue highlights their significance as potential biomarkers for TNBC. We suggest that these five specific secretory miRNAs could function as a liquid biopsy tool, not only for the diagnosis of tumor progression but may also provide a reference value for early detection as well as for monitoring the prognosis of TNBCs TNBC miRNA extracellular vesicles exosomes CSC prognosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Triple negative breast cancer (TNBC) is an aggressive subtype of breast cancer characterized by the absence of estrogen, progesterone, and human epidermal growth factor receptor 2 (HER2) expression. This subtype is frequently associated with a poor prognosis and a higher likelihood of recurrence. Increasing evidence indicates the presence of a small population of cancer stem cells (CSCs) that possess self-renewal abilities and the capacity to regenerate a heterogeneous tumor cell population, which may play a role in disease recurrence. 4 , 5 . CSCs are also characterized by an increased proliferation potential, cell lineage independent migration, metastasis and high telomerase expression. These cells can survive radiation therapy and chemotherapy treatment owing to efficient drug efflux mechanisms thereby making the cancer very difficult to treat. To directly target these CSCs, it is critical to identify novel pathways and develop new therapeutic strategies. Currently, TNBC poses significant treatment challenges, characterized by a high recurrence rate and a limited availability of effective targeted therapies. Treatment options for TNBC are constrained by the lack of hormone receptor expression and HER2 amplification. It is crucial to elucidate the molecular mechanisms underlying TNBC and to identify specific therapeutic targets to improve treatment outcomes for this aggressive breast cancer subtype. Recent research has underscored the potential role of microRNAs (miRNAs) in TNBC development and progression. miRNAs are small non-coding RNAs that modulate gene expression by binding to the 3' untranslated regions of target mRNAs, resulting in either their degradation or inhibition of translation. Numerous studies have indicated that the dysregulated expression of specific miRNAs is linked to the progression of TNBC and associated with a poor prognosis 8 – 10 . For example, miR-21 and miR-155 have been found to be often overexpressed in TNBC and are linked to cell proliferation, invasion, and resistance to chemotherapy 11 , 12 . Furthermore, studies have also found specific miRNAs to be under-expressed in TNBC such as miR-31 13,14 and miR-203 15–17 which have been shown to play a vital role in controlling cell migration and cell death. Novel miRNA-based therapies 18 have previously been proposed as a potential strategies for the treatment of TNBC wherein they can either restore the expression of tumor-suppressor miRNAs or inhibit the expression of oncogenic miRNAs 19 , 20 , specifically those secreted by exosomes. Therefore it is crucial to investigate the mechanisms of action of miRNAs associated with TNBC and to develop novel miRNA-based therapies for the treatment of this aggressive cancer subtype Exosomes are nanosized extracellular vesicles (EVs) secreted by cells and found in various biological fluids 21 , 22 .They contribute to intracellular communication within the tumor microenvironment. Tumor derived EVs contain a variety of biomolecules, including microRNAs (miRNAs), which can be used as biomarkers for the progression, prognosis and hence can serve as therapeutic targets of breast cancer 23 , 24 . Recent research has demonstrated that miRNAs within exosomes exhibit differential expression in breast cancer tissues compared to normal tissues 25 – 29 . Elevated levels of miR-141 30,31 and miR-375 32–34 were found in the exosomes of breast cancer patients, linked with poor prognosis. Conversely, miR-29b expression was reduced in the exosomes of breast cancer patients leading to good prognosis 35 , 36 . Thus, exosomal miRNAs may offer insights in effective treatment of resistant TNBCs and hold potential as promising targets for effective therapies. After a comprehensive literature survey of global databases (Web of Science, Cochrane Library, PubMed) and subsequent meta-analysis on GEO datasets, we shortlisted a set of five miRNAs -hsa-miR-4728, hsa-miR-6803, hsa-miR-1180, hsa-miR-940 and hsa-miR-1915 which are expressed in TNBCs. Overall survival analysis indicated that these miRNAs are associated with a negative disease prognosis. This study was the first to detect their presence in TNBC and TNBC stem cells and their respective exosomes. Thus, the secretory nature of these miRNAs was confirmed. All five miRNAs were consistently expressed in TNBC, TNBCSCs and their exosomes. Subsequently, upon clinical correlation in patient samples (n = 15), we detected all five miRNAs in tissue biopsies where hsa-miR-1180 and hsa-miR-4728 were found to be highly upregulated. To our knowledge, these exosomal miRNAs have not previously been reported in TNBC and TNBCSCs. The potential molecular mechanisms by which these miRNAs contribute to TNBC carcinogenesis were also explored through in vitro functional assays. Materials and Methods Identification and analysis of miRNA profiling datasets specific to TNBC : A search was conducted on PubMed and Web of Science electronic databases to find all the relevant literature studies on miRNA expression in TNBC. Referencing a method described formerly by Chen et.al. 81 , The search algorithms applied included“((microRNA OR micro RNA OR micro ribonucleic acid OR miRNA) AND ((breast carcinoma OR breast carcinomas OR breast cancer OR breast cancers OR triple negative breast cancer OR Triple Negative Breast cancers OR breast tumors) OR (TNBC carcinoma OR TNBC carcinomas OR TNBC OR adenocarcinoma of breast OR TNBC cancers OR TNBC)) AND (Humans [Mesh] AND English[lang]))”. Additionally, a total of 120 miRNA datasets were searched within the Gene Expression Omnibus (GEO) database ( https://www.ncbi.nlm.nih.gov/geo/ ), with each dataset's title, abstract, and full text thoroughly reviewed. The selection criteria included: (a) Original experimental studies that compare miRNA expression among different groups (TNBC vs. normal, TNBC vs. non-TNBC, and breast cancer vs. normal) using human samples, and (b) studies that report both upregulated and downregulated miRNAs, including specific cutoff parameters such as fold change and p-values. These specific inclusion criteria enabled the identification of all qualifying miRNA expression datasets. Comparably, we excluded datasets according to the criteria: (1) Any duplicated publications; (2) in-vitro or pre-clinical studies; (3) reviews, reports, editorials and (4) studies not characterised by miRNA expression analysis. A total of 53 studies on breast cancer were mined from the public domain and literature survey was conducted. The expression value of miRNA was calculated from each study. After selecting the datasets, data matrices were downloaded, and differential analyses were conducted using tools available on GEO. Various miRNA microarray platforms were employed, and uniquely expressed miRNAs from each dataset were annotated using miRbase. The fold change (FC) in miRNA expression was normalized by expressing it as log2FC to standardize the obtained miRNA expression values. Five miRNA showing high differential expression in breast cancer were selected for downstream experiments. miRNA target prediction Potential miRNA–target interactions were predicted using established software tools, including TargetScan, miRTarbase, miRDB, and miRmap. The selection criteria included a prediction score of less than 0 for TargetScan, a cumulative score exceeding 95 for miRmap, and a prediction score range of 75 to 100 for miRDB, while all targets from miRTarbase were included for consideration. These miRNA sequences were utilized as input in conjunction with reference cDNA sequences in the miRanda tool. Gene ontology and enrichment analysis Gene Ontology (GO) analysis is commonly utilized to assess the enrichment of differentially expressed genes (DEGs) in relation to biological processes, cellular components, and molecular functions. The candidate miRNA target genes were subjected to Gene Ontology (GO) and pathway enrichment analyses to elucidate their roles in critical biological pathways. Functional enrichment of predicted target genes for the selected five miRNAs was carried out using KEGG, Biocarta, Panther, and Reactome databases. Exosome isolation and analysis Cells were grown in exosome-depleted, serum free media. Exosomes were isolated from cell free conditioned media collected at 48 hrs. by use of Exosome isolation kit (ExoCan Life Sciences, India). The exosome-containing supernatant was filtered through 0.2 µm membrane filters to remove particles exceeding 200 nm in size. Following filtration, the supernatant underwent centrifugation at 20,000×g for 40 minutes at 4°C to isolate the exosomes. The resulting pellet was then resuspended in 1× PBS for further processing to identify miRNA. Exosomal characterization (Physical properties) EV characterization was conducted according to the International Society of Extracellular Vesicles guidelines. The physical properties of exosomes from cancer cell lines and stem cells were analysed using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). A 100 µL aliquot of exosomes was placed on a formvar carbon-coated nickel grid for 1 hour. The grid was then cleaned over drops of 0.1 M sodium cacodylate (pH 7.6) and treated with a solution of 2% paraformaldehyde and 2.5% glutaraldehyde in the same buffer for 10 minutes. The grids were rinsed with 0.1 M sodium cacodylate (pH 7.6), stained with 2% uranyl acetate for 15 minutes to enhance contrast, washed, treated with 0.4% uranyl acetate for 10 minutes, air-dried for 5 minutes, and then examined at 100 kV using a transmission electron microscope. Exosomal characterization (Western blotting) : The BCA Protein Assay Kit (Thermo Fisher Scientific) was employed to quantify exosomal proteins. Following electrophoresis on 4–15% gradient SDS-PAGE gels, 30 micrograms of protein were transferred to PVDF membranes, which were blocked using 5% bovine serum albumin (BSA) and subsequently incubated with specific primary antibodies, including CD63, CD81, and Calnexin (Biolegend, USA) at a dilution of 1:1000 for 24 hours at 4°C. Protein levels were assessed by probing with secondary antibodies against rabbit and mouse conjugated with horseradish peroxidase (HRP) at a dilution of 1:10,000, following three to five washing steps (10 minutes each). The bound complexes were detected using chemiluminescence methods (ECL; BioRad, USA), and images were acquired using the Amersham ChemiDoc Imaging System Exosomal miRNA isolation Total RNA, including miRNA, was isolated from breast cancer cell lines in vitro utilizing the Trizol precipitation method. RNA quantity and quality were assessed using the NanoDrop 1000 spectrophotometer. Cell Culture In this study, the human mammary epithelial cell line MCF-10A and TNBC cell lines MDA-MB-231 and MDA-MB-468, sourced from the American Type Culture Collection (ATCC, USA) were employed. Flow cytometric analysis revealed an enrichment of TNBC stem cells within the TNBC cell lines. The cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% exosome-depleted fetal bovine serum (FBS), 100 IU/mL penicillin, and 100 µg/mL streptomycin. Cultures were routinely maintained at 37°C in a 5% CO₂ environment Immunocytochemical staining and immunofluorescence analysis : Cells were cultured on 0.13 mm thick coverslips (Corning® Cover glass, Corning Life Sciences) for 2 days, followed by washing with PBS and fixation in 1 mL of 100% methanol for 10 minutes. Permeabilization was then performed using 100% acetone for 30 seconds, followed by blocking with 1% BSA for 2 hours at RT. Primary antibodies (1–2 µg) were incubated with the samples overnight at a dilution of 1:1000, after which the samples were incubated with corresponding secondary antibodies. Finally, cells were mounted using VECTASHIELD mounting medium containing 0.5 µg/mL DAPI (VECTASHIELD; VectorLabs, CA) Western Blotting : For all western blot analyses of cell lines, proteins were extracted from cells that had reached approximately 80% confluence. The protein concentration in the cell lysates was determined using the BCA Protein Assay Kit (Thermo Fisher Scientific), and 30 µg of protein was loaded into each lane. Protein samples were subjected to SDS–PAGE (12%) and transferred onto PVDF membranes. The immunodetection protocol included transferring the proteins (15 V for 15 minutes per membrane) and blocking the membranes with a western blot blocking solution overnight. Following two washes with 1X TBST, membranes were incubated overnight at 4°C with primary antibodies against Sox2 and OCT4 (1:5000) and with GAPDH (1:5000) for 1 hour at room temperature. After five additional TBST washes, HRP-conjugated secondary antibodies (1:10000) were applied for 1 hour at room temperature, and detection was performed using ECL chemiluminescence. Culture of Tumorspheres We assessed the capability of cell lines to generate spheres in an anchorage-independent suspension culture. Human breast adenocarcinoma cell lines MDA-MB-231 and MDA-MB-468 cell lines were cultured in DMEM (Gibco, ThermoFisher) containing 10% FBS (Gibco, ThermoFisher) and incubated at 37°C with 5% CO2 for 48 h. Following collection and washing, cells were resuspended in serum-free DMEM/F12 (Gibco, ThermoFisher) supplemented with 10 ng/ml fibroblast growth factor (FGF), 1% B27 and 20 ng/ml epidermal growth factor (EGF). At a density of up to 5000 cells/m, the cells were seeded in ultra-low attachment 6-well plates (Corning, USA) and incubated in a humidified environment with 5% CO2, set at 37°C for 4–6 days. Afterwards, the plates were examined for the growth of tumorspheres and measured with an inverted microscope. The tumorspheres were collected through mild centrifugation, followed by dissociation using Accutase (Sigma, USA) to produce individual cells, which were subsequently suspended in a serum-free medium to reform tumorspheres.. These tumorspheres were passaged every 5 days. Once the primary tumorspheres grew to a diameter of approximately 100µm, the samples were collected for downstream analysis. Flow cytometric analysis and CSC characterization The expression of the molecular markers CD133⁺, CD44⁺, and CD24⁻ was evaluated by flow cytometry. Trypsin was used to carefully disperse tumorspheres into a single-cell solution after they were collected. Cells were labelled with anti-CD44-FITC, anti-CD133-PE, and anti-CD24 AlexaFluor antibodies (BD Biosciences, USA), incubated for 30 minutes in the dark at 4°C.The cells were analysed using a flow cytometer (BD FACS ARIA III). The acquisition was set for 10,000 events per sample. Data analysis was performed using the BD FACSDiva™ software (FACSDiva™, BD, USA). Cells were sorted based on the surface antigen expression. Side Population analysis To isolate and identify SP and non-SP fractions, TNBC cells were treated with trypsin, then resuspended in pre-warmed DMEM containing 1% FBS.Dye Cycle Violet reagent (DCV) at a concentration of 5 µg/mL was added both in the presence and absence of verapamil (Sigma) and incubated at 37°C for 90 minutes with periodic shaking. Following incubation, the cells were washed with PBS containing 1% FBS, cold centrifuged, and resuspended in ice-cold sheath fluid (BD).Cells were preincubated with the ABCG2 inhibitor fumitremorgin-C(FTC) at a concentration of 10 µg/ml at 37°C for 30 minutes before DCV addition. Propidium iodide was added to the cells at a concentration of 1 µg/mL to differentiate viable cells. The Hoechst 33342 dye was excited at 357 nm, and its fluorescence was subsequently analysed using FACS AriaIII (BD Biosciences, San Diego, CA). The gating for forward and side scatter was stringent, ensuring that debris and non-viable cells were excluded from the analysis. Software used for analysis was BD FACSDiva™ (FACSDiva™, BD, USA). Immunofluorescence analysis : For tumorsphere immunostaining, cells were plated on glass coverslips (0.11 mm, Corning, USA) in DMEM with 10% FBS for 4 hours. Cells were then fixed with 4% paraformaldehyde and incubated with primary antibodies against SOX2 (mouse monoclonal IgG, Santa Cruz; 1:1000), OCT4 (mouse monoclonal IgG, BioLegend; 1:500), and ALDH1 (mouse monoclonal IgG, BioLegend; 1:500). Corresponding goat anti-mouse secondary antibodies conjugated with FITC, PE, and Cy3 were applied. Tumorspheres were incubated at 37°C for 60 minutes, followed by DAPI staining (Sigma) to visualize nuclei. Images were captured with a Zeiss fluorescence microscope, and processed using ZEN Blue and ZEN Black microscopy software (Zeiss) Transfection TNBC cells were transfected with 50 nmol/L miRNA mimics of hsa-miR-1180 and hsa-miR-4728 using Lipofectamine 3000 reagent (Invitrogen). The cells were treated with the miRNA mimics and corresponding scramble controls (IDT Technologies) in Opti-MEM medium for 4 hours, then switched to standard growth medium as per the manufacturer’s instructions. Analysis was conducted 48 hours after transfection. Migration and Invasion assays Matrigel (Corning, USA) was coated on top of the transwell chamber and the (serum starved) transfected cells were added in the upper chamber, seeded at 1 × 10⁴ /well. Subsequently, 500 µl of cell growth medium was added to the bottom chamber. Growth media serves as a chemoattractant. For 24 to 36 hours, the cells were incubated at 37°C. After the cells migrated to the lower chamber, they were fixed with 70% ethanol and stained using crystal violet. The quantity of migrated (stained) cells was measured by tallying the number of stained cells, and the mean cell count per field was computed for each well. Three replicate wells were utilized for every experiment, with representative images captured from randomly chosen fields in each well. Wound healing assay TNBC cells (5 × 10⁵ cells/well) were seeded into 6-well plates and grown as a monolayer for 24–48 hours. Once cells reach a 80% confluence, a horizontal scratch is created in each well using a 200uL pipette tip. Detached cells were removed by rinsing with 500 µL PBS, followed by the addition of 500 µL of fresh medium. The plates were incubated for 12, 18, and 24 hours, and images were captured at each interval with an inverted microscope to observe scratch closure progress. Real-time PCR for miRNA expression analysis Total RNA was isolated using TRIzol reagent (Invitrogen), with concentration measured at A260 and purity assessed via the A260/A280 ratio. RNA quality was evaluated using 2% agarose gels A total of 1 µg of RNA was reverse-transcribed into cDNA using random primers (Thermo), resulting in a final volume of 20 µL of cDNA. Subsequent amplification was performed via PCR for the miRNAs miR-940, 155, 6803, and 4728, with primer sets listed in Supplementary Table S1 . RT-qPCR was conducted using SYBR Green chemistry, normalizing to GAPDH expression levels, and the ∆∆Ct method was used to calculate normalized fold expression for the target miRNAs. Statistical analysis All experimental data were quantified from three independent sets of experiments and expressed as mean ± standard deviation (SD). Statistical analyses were conducted using GraphPad Prism software. Differences between groups were assessed using Student's t-test, one-way ANOVA, and two-way ANOVA, with significance levels determined as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. Additional study workflows are illustrated and included in supplementary file S1(Fig. 1 , 2 ). Results Identification of differentially expressed miRNAs by meta-analysis in breast cancer For the meta-analysis, a total of 120 miRNA datasets related to breast cancer were obtained from the Gene Expression Omnibus (GEO) databases. Of these, a sum of 53 datasets on breast cancer were shortlisted and mined from the public domain based on various exclusion and inclusion criteria. Following the dataset selection, we downloaded data matrices and differential analyses were conducted using the GEO2R tool provided on the GEO platform. Across these datasets that reported multivariate analyses, expression values of miRNAs were calculated from each study. A set of five miRNAs showing high differential expression in invasive breast cancers was selected for downstream experiments. The predictive significance of the chosen miRNAs in breast cancer patients was validated by performing Kaplan-Meier analysis which demonstrated that all five miRNAs : hsa-miR-6803, hsa-miR-1180, hsa-miR-4728, hsa-miR-1915 and hsa-miR-940 were significantly associated with poor overall survival (OS). Survival analysis indicates increased expression of these miRNAs is correlated with poor prognosis for patients with breast cancer as presented in Fig. 1 (a-e). While these miRNAs have shown poor prognosis across breast cancers, there is limited data to support the same in case of TNBCs as these miRNAs have been previously unreported in TNBCs and TNBCSCs. Given the prognostic promise and limited availability of data, we explored the prognostic potential of these miRNAs in TNBC. Target prediction and Functional annotation for target genes To further identify their target genes of these five selected miRNAs, target prediction was performed using four electronic databases-TargetScan, miRTarBase and miRDB and miRmap. We created a Venn diagram to illustrate the intersections between five miRNA targets, summarizing common targets across breast cancers in the initial cohort (Fig. 2 a). The number of predicted targets for miR-4728-3p, miR-940, miR-1180, miR-6803-5p and miR-1915-3p was 3749, 6171, 2948, 4709 and 7071, respectively. (Fig. 2 b) Gene set enrichment analysis is widely used to evaluate the enrichment of differentially expressed genes (DEGs) in a range of biological processes, cellular components, and molecular functions. Analysis of DEGs using Gene Ontology revealed significant enrichment in processes such as endothelial cell migration, signalling pathways involving Wnt, NOTCH, EGFR, the JNK cascade, along with cell division and DNA replication. (Fig. 2 c-d). These results indicate the potential involvement of key signaling pathways and biological processes with our selected miRNAs, leading to tumor progression. Isolation and characterization of exosomes derived from TNBC cells Exosomes were collected from the conditioned media of TNBC cell lines MDA-MD 231 and MDA-MB 468 after 48h. We performed scanning electron microscopy (SEM) and transmission electron microscopy (TEM) to examine cell culture-derived EVs. Further, Confocal microscopy was employed to qualitatively analyse localization of exosomes within TNBC cells. Exosomes were successfully identified as double-membrane vesicles through TEM Fig. 3 (d,h)) and their size range was estimated to lie between 50–200 nm as indicated by SEM (Fig. 3 (c,g)). The exosome marker CD81 was successfully found localized in TNBC cells as indicated by Immunofluorescence (Fig. 4 (a-c) and western blot Fig. 4 (d) Presence of cancer stem-like cells in TNBC Using flow cytometric techniques, we identified the presence of stem-like cells in the TNBC population. These cells have the potential to generate 3D spheroids in culture, exhibiting stem-like properties. Our results exhibit increased expression of stemness markers SOX2, ALDH1 and ABCG2 in tumor-derived spheroids. This was confirmed through RTqPCR analysis (Fig. 6 G) and Immunoblotting (Fig. 6 H). Furthermore, expression of stemness marker genes in TNBCSCs was found to be two-fold higher compared to TNBC cells (Fig. 6 H). Flow cytometric analysis was carried out in TNBC cell lines MDA-MB 231 and MDA-MB 468 to evaluate for expression of stemness marker CD133 and CD44 and identification of cancer stem cells. FACS analysis revealed a presence of a small population of SP cells (0.9–1.1%) that exhibit dye exclusion properties of cancer stem-like cells (Fig. 5 (e-h)) The SP cells, which disappear in the presence of verapamil (f,h), are outlined and shown as a percentage of the total Cancer stem-like cell population. SP cells were subsequently grown in cell culture and developed tumorspheres as seen in representative phase contrast images (b,d). Further, marker-based selection of TNBC stem cells by FACS also revealed presence of small population (1–2%) of stem-like cells expressing markers CD133⁺, CD44⁺, and CD24⁻ (Fig. 5 (i-l)). TNBC stem-like cells were characterized by immunostaining of tumorspheres with SOX2, ALDH1 and ABCG2 markers (as seen in Fig. 6 (A-F) and immunoblotting to determine the expression level of ALDH1 (Fig. 6 G). Targeting hsa-miR 1180 and hsa-miR 4728 inhibits invasion and migration in vitro To explore the role of miR1180 and miR-4728 on cell growth and proliferation in TNBC, we performed loss of function experiments by transfecting TNBC cells with anti-miR-1180 and anti-miR-4728 (24 h) in TNBC cells. As shown in Fig. 7 (A) in the wound healing assay, knockdown of miR-1180 and miR-4728 inhibited cell migration and proliferation when compared with WT conditions. Figure 7 (B) shows a drastic reduction in TNBC cell invasion abilities when miRNAs-1180, 4728 were silenced compared with the WT control. These results indicated the role of miR-1180 and miR-4728 in the cell proliferation, migration and invasion in TNBC cells. Exo-miRNAs consistently expressed in CSCs and breast cancer tissues RT-qPCR was employed to assess the levels of oncogenic miRNAs in TNBC cells and stem cells in vitro (Fig. 8 a, b, d, e). We found a significant upregulation of our target miRNAs miR 4728, miR 6803, miR-940, miR − 1915 and miR-1180 in TNBC cell lines MDA-MB 231, MDA-MB 468 in comparison to their corresponding CSCs. A similar expression pattern was observed in TNBC and TNBCSCs derived exosomes (Fig. 8 c and f). Our panel of five miRNAs- miR 6803, miR 1180, miR 4728, miR 1915 and miR 940 were found to be highly expressed in TNBC cells, TNBCSCs and enriched in circulating exosomes. Given their circulatory nature and consistent expression across TNBC cells and stem cells, these results indicate miRNAs miR 4728, miR 6803, miR-940, miR − 1915 and miR-1180 may have a significant role to play in TNBC extracellular communications leading to disease progression or better prognosis if these miRs are targeted for therapy. Clinical Correlation To clinically validate our findings, the presence of these miRNAs was evaluated in breast cancer tissues. Tissue biopsies were obtained from 15 TNBC patients, along with corresponding adjacent normal tissue samples for comparison. RT-qPCR analysis of resected tumor tissue RNA revealed consistent expression of these miRNAs in patients with TNBC (Fig. 8 g). Amongst the five miRNAs, hsa-miR 1180 and hsa-miR 4728 were found to be significantly upregulated in TNBC biopsies when compared with their adjacent (normal) tissues. Thus, our results demonstrated a positive correlation between the elevated expression of these miRNAs and the progression of TNBC. Second, the presence of hsa-miR 1180 and hsa-miR 4728 in circulatory tumor-derived vesicles and the detection of the same miRNAs in clinical tissue samples highlights their significance as potential biomarkers in TNBC. Discussion Triple negative breast cancer (TNBC), characterized by the absence of the estrogen receptor (ER), progesterone receptor (PR), and HER2/neu receptor expression 2 is known to be a highly aggressive cancer that shows the worst prognosis among all breast cancer types, coupled with rapid relapse. Currently there are hardly any targeted therapies for combat this aggressive and debilitating disease. Through intercellular communication with the tumor microenvironment and potentiated by CSC population, TNBC cells often acquire treatment resistance leading to metastasis and chemoresistance 26 , 44 – 47 . Additionally, the molecular heterogeneity of TNBC as revealed by the presence of many molecular markers poses challenges to effective treatment. 48 Several studies have outlined promising predictive and prognostic markers in breast cancer 49 – 51 . However, very few studies have been able to identify TNBC specific therapeutic biomarkers 52 , 53 . At present, there is no universal biomarker available for diagnosing and targeting TNBC, unlike other breast cancer subtypes, which have biomarkers such as HER2 and specific hormone receptors 49 , 50 There is a critical need to identify reliable, non-invasive markers specific to TNBC for rapid screening, early diagnosis, risk assessment, and ultimately for effective treatment and management of breast cancer. This study highlights the clinical utility of specific exosomal miRNAs as non-invasive diagnostic and prognostic markers, as well as potential therapeutic targets. After comprehensive literature review and subsequent meta-analysis of 120 publicly available breast cancer datasets, 53 relevant datasets were identified based on various exclusion and inclusion criteria. Upon analysis of these datasets, a set of five, highly oncogenic miRNAs were found to be expressed in TNBCs : hsa-miR 6803, hsa-miR 1180, hsa-miR 4728, hsa-miR 1915 and hsa-miR 940. Though these miRNAs were often found in breast cancers, they have never been shown to be exclusively reported in TNBC, making them ideal candidates for therapeutic interventions. Additionally, since there was so far no evidence linking these oncomiRs with disease relapse, we were eager to explore their gene expression profile in TNBCSCs. Of the five miRNAs, two were found to show exclusively high expression in TNBC clinical tissue. RT-qPCR analysis revealed a notable increase in the expression of hsa-miR-1180 and hsa-miR-4728 in TNBC tissues when compared to non-tumor breast tissues. Our study has confirmed that hsa-miR-1180 and hsa-miR-4728 act as tumor promoters, suggesting that their upregulation in tumor tissues may contribute to the progression and metastasis of TNBC. Decreased expression of these miRNAs attenuated tumor progression, as demonstrated by wound healing and invasion assays (Fig. 7 A, B) Additionally, miRNA target prediction was carried out using four electronic databases :TargetScan, miRTarBase and miRDB and miRmap, which revealed 14 common targets between the two selected miRNAs - hsa-miR-1180 and hsa-miR-4728. PPI and RNA-protein association analysis of targets using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database ( https://string-db.org ) revealed amongst others, their role in epigenetic regulation of HATs, TP53 regulation of metabolic pathways and PPARɑ mediated gene expression in breast cancer (Fig. 9 ) Using gene enrichment analysis and target prediction models, these dysregulated miRNAs were also found to be involved in a wide range of crucial biological processes and pathways including regulation of RNA pol II, Wnt signaling, Notch signaling, MAPK signaling pathway and the regulation of JNK signaling cascade. Previous studies have highlighted the correlation of Wnt signaling pathways with maintenance of stem cell niche and expression in cancer stem cells 35 – 41 . Notch receptor and ligand overexpression is linked to TNBC progression 38 , 42 . Notch receptors are also involved in modulating the behaviour of tumor-initiating cells and in the initiation of TNBC 38 , 42 , 43 . These pathways are pertinent to the therapeutic targeting of CSCs or other cells responsible for diverse prolapse as they play key roles in genesis and regulation of cell survival and their fate 44 . Given that many of these pathways are known to support tumor cell proliferation, cancer stem cell (CSC) survival, epithelial-to-mesenchymal transition (EMT), and invasion, it is likely that these miRNAs contribute to the progression and expansion of TNBC and TNBC stem cells. Exosome-encapsulated miRNAs have a great potential as prognostic biomarkers. Several studies have demonstrated that exosomes are robust and may be stored for extended periods without compromising the integrity of encased miRNAs. These features greatly increase their potential applicability in a diagnostic or clinical setting. Secretory miRNAs are reflective of their parent cell status and thus may reveal a more specific tumor profile than conventional miRNA profile derived from whole blood or serum. From our identified set of five oncogenic miRNAs, miR 6803 has been previously identified as a diagnostic marker in colorectal cancer 54 , 55 . miR 1180 has been found to play a role in tumor progression in Lung cancer, melanoma, ovarian cancer, and hepatocellular carcinoma. 56 – 61 . miR 1915 has been reported in gastric cancer 62 , 63 , colorectal carcinoma (CRC) 64 – 66 and most recently in Breast cancer 67 . Interestingly, miR 4728 is found to have dual functions. It acts as a tumor suppressor in CRC 68 while being tumorigenic in breast cancer 69 – 71 . miR 4728 has been identified as a marker of HER2 status in BrCa patients. miR 940 has been widely reported as an oncogenic marker for CRC 72 , gastric cancer 73 , cervical cancer 74 and breast cancer 75 , 76 . It is involved in four critical pathways: the Wnt/β-catenin pathway, the MAPK pathway, the PD-1 pathway, and the phosphatidylinositol 3-kinase (PI3K)-Akt pathway, all of which are significantly implicated in breast carcinogenesis 77 , 78 . While many of these selected miRNAs have been identified in various types of cancer, their secretory roles in TNBC and TNBC stem cells (TNBCSCs) were previously unrecognized. Our study reveals the presence of secretory miRNAs (miRNAs miR 6803, miR 1180, miR 4728, miR 1915 and miR 940) in TNBC and TNBC stem cells highlighting their diagnostic value and clinical utility for better management of TNBC. Furthermore, these secretory oncomiRs were found consistently overexpressed in TNBC and TNBCSCs. We subsequently correlated our findings with TNBC tumor tissue samples (n = 15) and found a consistent high expression of all five oncomiRs across tumor biopsies. From this panel of five miRNAs, two oncomiRs -hsa-miR-1180 and hsa-miR-4728 were found to be significantly upregulated across all tumor tissue samples in TNBC patients. The presence of these two secretory miRNAs in TNBC and TNBCSC along with their overexpression in clinical tissue samples indicate their possible role in TNBC progression and metastasis and may serve as reliable prognostic as well as therapeutic marker(s). We recognise that our integrated analysis has some limitations. During the systematic literature review, the initial screening was conducted solely on three databases (PubMed, Web of Science and Cochrane Library), which may have resulted in the omission of some relevant studies. Ideally, we could have searched for additional data sources such as pre-print server archives, relevant books, Google Scholar and other similar resources. Additionally, the limited availability of publicly listed TNBC clinical data sets in GEO and TCGA may have narrowed the scope of our estimates. We have analyzed potential circulatory prognostic markers for TNBC, validated our findings in TNBC tumor tissues and further validated them in vitro using TNBC cell lines. Since TNBC is a highly metastatic disease, it is very challenging to obtain fresh surgical tissue samples. We were able to include a small number of tumor tissue samples which may limit the clinical relevance and translational impact of our study. We recognize that analysis of larger datasets and clinical correlation using higher numbers of TNBC tissues could provide greater insights on prognostic significance of these TNBC derived exosomal miRNAs. Further, addition of population-based studies and data stratification could provide better understanding and help in effective therapy and/or prognostic monitoring of TNBC. Nonetheless, the identification of novel secretory miRNAs in TNBC and TNBCSCs is promising with potential application in the diagnosis and treatment of hormone refractory, metastatic breast cancers. Our study identifies novel oncogenic exomiRs in TNBC and TNBCSCs and highlights their utility as therapeutic targets for TNBC. The mechanisms through which these exomiRs influence the development and progression of TNBC are not yet fully elucidated. Nonetheless, it is believed that they may modulate the expression of genes associated with cell signalling pathways, including the Wnt, Notch, and MAPK pathways, which are known to play a role in the progression and relapse of TNBC. Previous studies have demonstrated similar prognostic potential of several other miRNAs in breast cancer such as miR-9 79 , miR-21 9 , miR-29b 9 , 34 and miR 331 80 . Our study is the first to assess prognostic behaviour of these novel TNBC and TNBCSC derived exosomal miRNAs. Elucidating the role of secretory miRNAs in the molecular mechanisms driving TNBC initiation and progression is crucial for advancing miRNA-based therapeutics, which hold promise as effective treatment options for TNBC patients. Conclusion A set of five novel secretory oncomiRs (hsa-miR 6803, hsa-miR 1180, hsa-miR 4728, hsa-miR 1915 and hsa-miR 940) were exclusively found to be expressed in TNBC and TNBCSCs, demonstrating great prognostic potential. These miRNAs were also found consistently expressed in TNBC tissue samples. Two of the oncomiRs : hsa-miR-1180 and hsa-miR-4728 were found to be significantly upregulated in TNBC tumor tissue. In this study, these miRNAs were found to function as oncomiRs. Given the secretory nature of these oncomiRs and their consistent expression across clinical cases, they can be explored as potential therapeutic as well as prognostic markers for TNBC. Abbreviations TNBC: Triple Negative Breast Cancer; miRNA: micro-RNA; TEM: transmission electron microscope; SEM: Scanning electron microscope; FBS: fetal bovine serum; DMEM, Dulbecco's Modified Eagle Medium; GAPDH: glyceraldehyde 3-phosphate 26 dehydrogenase; PVDF: polyvinyl difluoride; RTqPCR: Reverse transcription-quantitative PCR; RIPA: Radio-Immunoprecipitation Assay; SDS: sodium dodecyl sulfate, PPI: Protein-Protein interactions, GO: Gene ontology, GEO : Gene expression omnibus ; DEG: Differentially expressed genes Declarations Acknowledgements This study was supported in part by the Department of Science and Technology (DST), Ministry of Science and Technology, Government of India by awarding the INSPIRE fellowship to A.Choudhary for her Ph.D. study. Confocal microscopy was performed at the Translational Health Science and Technology Institute (THSTI), in Faridabad, India. We thank Mr. Suraj Tewari (THSTI) for his technical expertise in Super-resolution microscopy and confocal imaging; Max Institute of Cancer Care, Max super specialty hospital, Saket, New Delhi for the provision of TNBC tissue samples for clinical validation; Mr. Akshey Kaushal, application engineer at the Indian Institute of Technology (IIT) Delhi, for his technical help with HR-TEM and Cryo-TEM employed for exosome visualization; Dr Prasanna Venkatraman, Deputy Director at the Cancer Research Institute, ACTREC Mumbai, India for sharing with us MCF10A cell lines; Mr. Manoj Gupta and Dr Pradeep K Rai for assistance with FACS analysis. Author Contributions : B. C. Das: Conceptualization, Supervision, Reviewing and Editing; Ananya Choudhary: Data curation, Methodology, Investigation, Writing, Original draft preparation, Reviewing and Editing; Funding acquisition; Satish S. Poojary : Visualization, Methodology, Supervision; Priyanka Jain : Software, Validation, Formal Analysis ; Harit Chaturvedi : Resources Funding : This study was supported in part by the Department of Science and Technology (DST), Ministry of Science and Technology, Government of India by awarding the INSPIRE fellowship to A.Choudhary for her Ph.D. study Availability of supporting data: All data relevant to the study are included in the article or uploaded as supplementary information. Ethics approval and consent to participate : Informed consent was obtained from all participants prior to their involvement in the study, and all procedures were conducted in accordance with the ethical guidelines of Indian Council of Medical Research (ICMR). Participants were fully informed about the study objectives, potential risks and benefits, and their right to withdraw at any time. Written consent forms were obtained from all participants, ensuring their privacy and confidentiality of data collected. All personal data and biopsy samples were handled with strict confidentiality. Identifiable information has been removed or anonymized in all published findings. All clinical samples were collected with signed informed consent and ethics approval also obtained from Max Institute of Cancer Care, Max Hospital Saket, New Delhi Consent for publication: The content of this paper has not been submitted to any other scientific publications. All the authors have declared that no financial conflict of interest exists. All authors have approved the submission of this work for publication in Breast Cancer Research. Competing interests : The authors declare no competing interests. References Dent, R. et al. Triple-negative breast cancer: Clinical features and patterns of recurrence. Clinical Cancer Research 13 , 4429–4434 (2007). Irvin, W. J. & Carey, L. A. 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Chen Z, Zhan Y, Chi J, Guo S, Zhong X, He A, Zheng J, Gong Y, Li X, Zhou L. Using microRNAs as Novel Predictors of Urologic Cancer Survival: An Integrated Analysis. EBioMedicine. 2018 Aug;34:94-107. doi: 10.1016/j.ebiom.2018.07.014. Epub 2018 Jul 21. PMID: 30037718; PMCID: PMC6116416. Additional Declarations No competing interests reported. Supplementary Files SupplementaryInformationBCRJan30.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. 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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-5910171","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":428314345,"identity":"fd623564-b5f1-4b76-8bec-bacbf57a4431","order_by":0,"name":"Ananya Choudhary","email":"","orcid":"","institution":"Amity University Uttar Pradesh","correspondingAuthor":false,"prefix":"","firstName":"Ananya","middleName":"","lastName":"Choudhary","suffix":""},{"id":428314349,"identity":"86d964b4-053d-4d7a-acfc-a6f396359bcd","order_by":1,"name":"Satish S. 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Increased miRNA expression is linked to poorer prognosis in breast cancer patients.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5910171/v1/71798f3c5778224b7d7fab3f.png"},{"id":84536240,"identity":"0516b03f-52c2-4f74-b636-80e8560a711e","added_by":"auto","created_at":"2025-06-13 07:13:15","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1173835,"visible":true,"origin":"","legend":"\u003cp\u003e(a-d): Intersection of predicted target genes from selected miRNA pool of miR-4728-3p, miR-940, miR-1180, miR-6803-5p and miR-1915-3p. (a-b): Indicates predicted target genes for differentially expressed miRNAs 4728-3p, 940, 1180, 6803-3p, and 1915-3p. Functional enrichment analysis for predicted target genes of the selected five miRNA. (c-d) Pathway enrichment analysis was performed using the KEGG, Biocarta, Panther, and Reactome databases, in addition to Gene Ontology enrichment analysis.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5910171/v1/836478a1769178572d19d062.png"},{"id":84536244,"identity":"779ce811-5725-4807-beb5-168badb6e4af","added_by":"auto","created_at":"2025-06-13 07:13:15","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1878527,"visible":true,"origin":"","legend":"\u003cp\u003e(a-h) Characterization of TNBC exosomes using advanced microscopy techniques. Phase contrast images of TNBC cells MDA-MB 231 and MDA-MB-468 (a,e), Scanning electron microscopy images of TNBC cells (b,f) scale 3um and of TNBC derived exosomes (c,g) scale 200um. High resolution transmission electron microscopy (HRTEM) imaging of TNBC derived exosomes (d, h), scale 50 nm\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5910171/v1/ece997977eb25ccf04e953e7.png"},{"id":84536574,"identity":"b47b5016-bdd0-4524-aa3d-99ed6c5c22b1","added_by":"auto","created_at":"2025-06-13 07:21:15","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":640854,"visible":true,"origin":"","legend":"\u003cp\u003e(a-c): Representative Immunofluorescence images showing CD81 staining in TNBC cells (arrows indicate presence of exosomes). Scale bar 20um. Fig 4(d): Immunoblot showing expression of CD81 exosomal marker in TNBC cell line MDA-MB 231 derived exosome samples. Calnexin is used as a negative control while β-Actin is used as a positive control.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5910171/v1/a149388f780252789a49427a.png"},{"id":84535428,"identity":"654e4de0-fe82-4902-95ba-3faefadca48c","added_by":"auto","created_at":"2025-06-13 07:05:15","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1423788,"visible":true,"origin":"","legend":"\u003cp\u003e(a-l): Isolation and characterisation of TNBC stem-like cells by FACS. Phase contrast imaging of TNBC cells and TNBC Stem-like cells (a-d). CSCs are observed as spheroids in culture (b,d). Flow cytometric analysis of TNBC cells using DCV based side-population assay (e-h). FACS analysis using marker-based selection methods expressing markers CD133⁺, CD44⁺, and CD24⁻ (i-l)\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5910171/v1/678bcc166c399146b89cf00e.png"},{"id":84536243,"identity":"e59387d9-44b1-4042-a9dc-7bf64ed631cd","added_by":"auto","created_at":"2025-06-13 07:13:15","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1124925,"visible":true,"origin":"","legend":"\u003cp\u003e(A-H) Characterization of TNBC derived stem-like cells. Immunofluorescence imaging of TNBC stem-like cells (A-F) Tumorspheres indicate presence of stemness markers SOX2, OCT4 and EpCAM. DAPI is used as a nuclear stain. SOX2 is tagged with FITC dye and thus emits green fluorescence, OCT4 is indicated in red (AlexaFluor 647) and EpCAM is marked by yellow dye (PE). Scale bar is 10um. Expression levels of stemness marker genes SOX2, ALDH1 and ABCG2 in TNBC and Cancer stem-like cells (CLSC) by qRT-PCR (G) Representative immunoblot showing expression levels of SOX2 in TNBC tumorspheres and cancer cells. GAPDH was used as positive control (H)\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-5910171/v1/f43e6c8a67583e40515555c2.png"},{"id":84535436,"identity":"11ccd3d0-1f20-4de9-8ac9-2d9d56732ca3","added_by":"auto","created_at":"2025-06-13 07:05:15","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2757613,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Wound healing assay of TNBC cells MDA-MB 231 (WT) and KD miR-1180 and miR-4728. Photographs were taken at intervals of 0h and 18h respectively. (B) Cell invasion assay using WT TNBC and KD of miR-1180, miR-4728. After 48h, cells were fixed with methanol and stained with crystal violet. Subsequently photographs were taken in phase contrast microscope. Transiently transfected cells with inhibition of miR-1180, miR-4728 show significant reduction in cell invasion in TNBC. These results reveal that miR-1180 and miR-4728 play a significant role in TNBC proliferation, migration and invasion.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-5910171/v1/113acf30b92d3a92b976273f.png"},{"id":84535429,"identity":"82c8ed7e-ec4c-44d9-9afd-ccc024d8b0fa","added_by":"auto","created_at":"2025-06-13 07:05:15","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":843007,"visible":true,"origin":"","legend":"\u003cp\u003e(a-g): RT-PCR was employed to assess the levels of oncogenic miRNAs in TNBC cells, stem cells and tumor derived exosomes in-vitro. Expression analysis of miRNAs in TNBC cells (a,e). miRNAs were detected in TNBC exosomes (c, f). Selected miRNAs expressed in breast cancer tissues (n=15) as seen in 8(g). hsa-miR 1180 and hsa-miR 4728 are found to be highly upregulated in TNBC tumor tissue when compared with adjacent (normal) tissue\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-5910171/v1/08b60366207bd50ee6af18b2.png"},{"id":84536246,"identity":"cdcd9d88-b728-4ed6-9356-135a355901bf","added_by":"auto","created_at":"2025-06-13 07:13:15","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":813681,"visible":true,"origin":"","legend":"\u003cp\u003ePPI and RNA-protein association analysis using STRING. (A) Venn diagram shows intersection of 14 miRNA targets between hsa-miR-1180 and hsa-miR-4728. (B, C) RNA–protein Association and Interaction Networks (RAIN) were identified. (D) Interaction of miRNA targets. STRING diagram of target PPI of hsa-miR-1180 and hsa-miR-4728 target proteins indicate potential role of epigenetic modifiers of HATs (red nodes), TP53 regulation of metabolic pathways (Blue nodes) and PPARɑ regulated gene expression in breast cancer (Green nodes). Lines indicate interaction evidence.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-5910171/v1/d6d8d7689ae63c377a907138.png"},{"id":84537622,"identity":"17dc0f04-78ed-4add-96ef-9c9f54f4aaac","added_by":"auto","created_at":"2025-06-13 07:37:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":16285759,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5910171/v1/83c6bd78-8fbd-4b92-ad7a-3aa859251296.pdf"},{"id":84535423,"identity":"49c18012-b1c2-43f0-8d7a-98f457dbfaaf","added_by":"auto","created_at":"2025-06-13 07:05:15","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1648240,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformationBCRJan30.docx","url":"https://assets-eu.researchsquare.com/files/rs-5910171/v1/d8cd495a8d832220c3c1468e.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Identification of novel exosomal miRNAs and their role in diagnosis and prognosis of Triple Negative Breast Cancer","fulltext":[{"header":"Introduction","content":" \u003cp\u003eTriple negative breast cancer (TNBC) is an aggressive subtype of breast cancer characterized by the absence of estrogen, progesterone, and human epidermal growth factor receptor 2 (HER2) expression. This subtype is frequently associated with a poor prognosis and a higher likelihood of recurrence. Increasing evidence indicates the presence of a small population of cancer stem cells (CSCs) that possess self-renewal abilities and the capacity to regenerate a heterogeneous tumor cell population, which may play a role in disease recurrence.\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. CSCs are also characterized by an increased proliferation potential, cell lineage independent migration, metastasis and high telomerase expression. These cells can survive radiation therapy and chemotherapy treatment owing to efficient drug efflux mechanisms thereby making the cancer very difficult to treat. To directly target these CSCs, it is critical to identify novel pathways and develop new therapeutic strategies. Currently, TNBC poses significant treatment challenges, characterized by a high recurrence rate and a limited availability of effective targeted therapies. Treatment options for TNBC are constrained by the lack of hormone receptor expression and HER2 amplification. It is crucial to elucidate the molecular mechanisms underlying TNBC and to identify specific therapeutic targets to improve treatment outcomes for this aggressive breast cancer subtype. Recent research has underscored the potential role of microRNAs (miRNAs) in TNBC development and progression.\u003c/p\u003e \u003cp\u003emiRNAs are small non-coding RNAs that modulate gene expression by binding to the 3' untranslated regions of target mRNAs, resulting in either their degradation or inhibition of translation. Numerous studies have indicated that the dysregulated expression of specific miRNAs is linked to the progression of TNBC and associated with a poor prognosis\u003csup\u003e\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. For example, miR-21 and miR-155 have been found to be often overexpressed in TNBC and are linked to cell proliferation, invasion, and resistance to chemotherapy\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Furthermore, studies have also found specific miRNAs to be under-expressed in TNBC such as miR-31\u003csup\u003e13,14\u003c/sup\u003e and miR-203\u003csup\u003e15\u0026ndash;17\u003c/sup\u003e which have been shown to play a vital role in controlling cell migration and cell death. Novel miRNA-based therapies\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e have previously been proposed as a potential strategies for the treatment of TNBC wherein they can either restore the expression of tumor-suppressor miRNAs or inhibit the expression of oncogenic miRNAs\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, specifically those secreted by exosomes. Therefore it is crucial to investigate the mechanisms of action of miRNAs associated with TNBC and to develop novel miRNA-based therapies for the treatment of this aggressive cancer subtype\u003c/p\u003e \u003cp\u003eExosomes are nanosized extracellular vesicles (EVs) secreted by cells and found in various biological fluids\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e.They contribute to intracellular communication within the tumor microenvironment. Tumor derived EVs contain a variety of biomolecules, including microRNAs (miRNAs), which can be used as biomarkers for the progression, prognosis and hence can serve as therapeutic targets of breast cancer\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Recent research has demonstrated that miRNAs within exosomes exhibit differential expression in breast cancer tissues compared to normal tissues\u003csup\u003e\u003cspan additionalcitationids=\"CR26 CR27 CR28\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Elevated levels of miR-141\u003csup\u003e30,31\u003c/sup\u003e and miR-375\u003csup\u003e32\u0026ndash;34\u003c/sup\u003e were found in the exosomes of breast cancer patients, linked with poor prognosis. Conversely, miR-29b expression was reduced in the exosomes of breast cancer patients leading to good prognosis\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Thus, exosomal miRNAs may offer insights in effective treatment of resistant TNBCs and hold potential as promising targets for effective therapies.\u003c/p\u003e \u003cp\u003eAfter a comprehensive literature survey of global databases (Web of Science, Cochrane Library, PubMed) and subsequent meta-analysis on GEO datasets, we shortlisted a set of five miRNAs -hsa-miR-4728, hsa-miR-6803, hsa-miR-1180, hsa-miR-940 and hsa-miR-1915 which are expressed in TNBCs. Overall survival analysis indicated that these miRNAs are associated with a negative disease prognosis. This study was the first to detect their presence in TNBC and TNBC stem cells and their respective exosomes. Thus, the secretory nature of these miRNAs was confirmed. All five miRNAs were consistently expressed in TNBC, TNBCSCs and their exosomes. Subsequently, upon clinical correlation in patient samples (n\u0026thinsp;=\u0026thinsp;15), we detected all five miRNAs in tissue biopsies where hsa-miR-1180 and hsa-miR-4728 were found to be highly upregulated. To our knowledge, these exosomal miRNAs have not previously been reported in TNBC and TNBCSCs. The potential molecular mechanisms by which these miRNAs contribute to TNBC carcinogenesis were also explored through in vitro functional assays.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e \u003cb\u003eIdentification and analysis of miRNA profiling datasets specific to TNBC\u003c/b\u003e: A search was conducted on PubMed and Web of Science electronic databases to find all the relevant literature studies on miRNA expression in TNBC. Referencing a method described formerly by Chen et.al.\u003csup\u003e\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e\u003c/sup\u003e, The search algorithms applied included\u0026ldquo;((microRNA OR micro RNA OR micro ribonucleic acid OR miRNA) AND ((breast carcinoma OR breast carcinomas OR breast cancer OR breast cancers OR triple negative breast cancer OR Triple Negative Breast cancers OR breast tumors) OR (TNBC carcinoma OR TNBC carcinomas OR TNBC OR adenocarcinoma of breast OR TNBC cancers OR TNBC)) AND (Humans [Mesh] AND English[lang]))\u0026rdquo;. Additionally, a total of 120 miRNA datasets were searched within the Gene Expression Omnibus (GEO) database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/geo/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/geo/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), with each dataset's title, abstract, and full text thoroughly reviewed. The selection criteria included: (a) Original experimental studies that compare miRNA expression among different groups (TNBC vs. normal, TNBC vs. non-TNBC, and breast cancer vs. normal) using human samples, and (b) studies that report both upregulated and downregulated miRNAs, including specific cutoff parameters such as fold change and p-values. These specific inclusion criteria enabled the identification of all qualifying miRNA expression datasets. Comparably, we excluded datasets according to the criteria: (1) Any duplicated publications; (2) in-vitro or pre-clinical studies; (3) reviews, reports, editorials and (4) studies not characterised by miRNA expression analysis. A total of 53 studies on breast cancer were mined from the public domain and literature survey was conducted. The expression value of miRNA was calculated from each study. After selecting the datasets, data matrices were downloaded, and differential analyses were conducted using tools available on GEO. Various miRNA microarray platforms were employed, and uniquely expressed miRNAs from each dataset were annotated using miRbase. The fold change (FC) in miRNA expression was normalized by expressing it as log2FC to standardize the obtained miRNA expression values. Five miRNA showing high differential expression in breast cancer were selected for downstream experiments.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003emiRNA target prediction\u003c/strong\u003e \u003cp\u003ePotential miRNA\u0026ndash;target interactions were predicted using established software tools, including TargetScan, miRTarbase, miRDB, and miRmap. The selection criteria included a prediction score of less than 0 for TargetScan, a cumulative score exceeding 95 for miRmap, and a prediction score range of 75 to 100 for miRDB, while all targets from miRTarbase were included for consideration. These miRNA sequences were utilized as input in conjunction with reference cDNA sequences in the miRanda tool.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eGene ontology and enrichment analysis\u003c/strong\u003e \u003cp\u003eGene Ontology (GO) analysis is commonly utilized to assess the enrichment of differentially expressed genes (DEGs) in relation to biological processes, cellular components, and molecular functions. The candidate miRNA target genes were subjected to Gene Ontology (GO) and pathway enrichment analyses to elucidate their roles in critical biological pathways. Functional enrichment of predicted target genes for the selected five miRNAs was carried out using KEGG, Biocarta, Panther, and Reactome databases.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eExosome isolation and analysis\u003c/strong\u003e \u003cp\u003eCells were grown in exosome-depleted, serum free media. Exosomes were isolated from cell free conditioned media collected at 48 hrs. by use of Exosome isolation kit (ExoCan Life Sciences, India). The exosome-containing supernatant was filtered through 0.2 \u0026micro;m membrane filters to remove particles exceeding 200 nm in size. Following filtration, the supernatant underwent centrifugation at 20,000\u0026times;g for 40 minutes at 4\u0026deg;C to isolate the exosomes. The resulting pellet was then resuspended in 1\u0026times; PBS for further processing to identify miRNA.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e\u003cb\u003eExosomal characterization (Physical properties)\u003c/b\u003e EV characterization was conducted according to the International Society of Extracellular Vesicles guidelines. The physical properties of exosomes from cancer cell lines and stem cells were analysed using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). A 100 \u0026micro;L aliquot of exosomes was placed on a formvar carbon-coated nickel grid for 1 hour. The grid was then cleaned over drops of 0.1 M sodium cacodylate (pH 7.6) and treated with a solution of 2% paraformaldehyde and 2.5% glutaraldehyde in the same buffer for 10 minutes. The grids were rinsed with 0.1 M sodium cacodylate (pH 7.6), stained with 2% uranyl acetate for 15 minutes to enhance contrast, washed, treated with 0.4% uranyl acetate for 10 minutes, air-dried for 5 minutes, and then examined at 100 kV using a transmission electron microscope.\u003c/p\u003e \u003cp\u003e \u003cb\u003eExosomal characterization (Western blotting)\u003c/b\u003e: The BCA Protein Assay Kit (Thermo Fisher Scientific) was employed to quantify exosomal proteins. Following electrophoresis on 4\u0026ndash;15% gradient SDS-PAGE gels, 30 micrograms of protein were transferred to PVDF membranes, which were blocked using 5% bovine serum albumin (BSA) and subsequently incubated with specific primary antibodies, including CD63, CD81, and Calnexin (Biolegend, USA) at a dilution of 1:1000 for 24 hours at 4\u0026deg;C. Protein levels were assessed by probing with secondary antibodies against rabbit and mouse conjugated with horseradish peroxidase (HRP) at a dilution of 1:10,000, following three to five washing steps (10 minutes each). The bound complexes were detected using chemiluminescence methods (ECL; BioRad, USA), and images were acquired using the Amersham ChemiDoc Imaging System\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eExosomal miRNA isolation\u003c/strong\u003e \u003cp\u003eTotal RNA, including miRNA, was isolated from breast cancer cell lines in vitro utilizing the Trizol precipitation method. RNA quantity and quality were assessed using the NanoDrop 1000 spectrophotometer.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCell Culture\u003c/strong\u003e \u003cp\u003eIn this study, the human mammary epithelial cell line MCF-10A and TNBC cell lines MDA-MB-231 and MDA-MB-468, sourced from the American Type Culture Collection (ATCC, USA) were employed. Flow cytometric analysis revealed an enrichment of TNBC stem cells within the TNBC cell lines. The cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% exosome-depleted fetal bovine serum (FBS), 100 IU/mL penicillin, and 100 \u0026micro;g/mL streptomycin. Cultures were routinely maintained at 37\u0026deg;C in a 5% CO₂ environment\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eImmunocytochemical staining and immunofluorescence analysis\u003c/b\u003e: Cells were cultured on 0.13 mm thick coverslips (Corning\u0026reg; Cover glass, Corning Life Sciences) for 2 days, followed by washing with PBS and fixation in 1 mL of 100% methanol for 10 minutes. Permeabilization was then performed using 100% acetone for 30 seconds, followed by blocking with 1% BSA for 2 hours at RT. Primary antibodies (1\u0026ndash;2 \u0026micro;g) were incubated with the samples overnight at a dilution of 1:1000, after which the samples were incubated with corresponding secondary antibodies. Finally, cells were mounted using VECTASHIELD mounting medium containing 0.5 \u0026micro;g/mL DAPI (VECTASHIELD; VectorLabs, CA)\u003c/p\u003e \u003cp\u003e \u003cb\u003eWestern Blotting\u003c/b\u003e: For all western blot analyses of cell lines, proteins were extracted from cells that had reached approximately 80% confluence. The protein concentration in the cell lysates was determined using the BCA Protein Assay Kit (Thermo Fisher Scientific), and 30 \u0026micro;g of protein was loaded into each lane. Protein samples were subjected to SDS\u0026ndash;PAGE (12%) and transferred onto PVDF membranes. The immunodetection protocol included transferring the proteins (15 V for 15 minutes per membrane) and blocking the membranes with a western blot blocking solution overnight. Following two washes with 1X TBST, membranes were incubated overnight at 4\u0026deg;C with primary antibodies against Sox2 and OCT4 (1:5000) and with GAPDH (1:5000) for 1 hour at room temperature. After five additional TBST washes, HRP-conjugated secondary antibodies (1:10000) were applied for 1 hour at room temperature, and detection was performed using ECL chemiluminescence.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCulture of Tumorspheres\u003c/strong\u003e \u003cp\u003eWe assessed the capability of cell lines to generate spheres in an anchorage-independent suspension culture. Human breast adenocarcinoma cell lines MDA-MB-231 and MDA-MB-468 cell lines were cultured in DMEM (Gibco, ThermoFisher) containing 10% FBS (Gibco, ThermoFisher) and incubated at 37\u0026deg;C with 5% CO2 for 48 h. Following collection and washing, cells were resuspended in serum-free DMEM/F12 (Gibco, ThermoFisher) supplemented with 10 ng/ml fibroblast growth factor (FGF), 1% B27 and 20 ng/ml epidermal growth factor (EGF). At a density of up to 5000 cells/m, the cells were seeded in ultra-low attachment 6-well plates (Corning, USA) and incubated in a humidified environment with 5% CO2, set at 37\u0026deg;C for 4\u0026ndash;6 days. Afterwards, the plates were examined for the growth of tumorspheres and measured with an inverted microscope. The tumorspheres were collected through mild centrifugation, followed by dissociation using Accutase (Sigma, USA) to produce individual cells, which were subsequently suspended in a serum-free medium to reform tumorspheres.. These tumorspheres were passaged every 5 days. Once the primary tumorspheres grew to a diameter of approximately 100\u0026micro;m, the samples were collected for downstream analysis.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eFlow cytometric analysis and CSC characterization\u003c/strong\u003e \u003cp\u003eThe expression of the molecular markers CD133⁺, CD44⁺, and CD24⁻ was evaluated by flow cytometry. Trypsin was used to carefully disperse tumorspheres into a single-cell solution after they were collected. Cells were labelled with anti-CD44-FITC, anti-CD133-PE, and anti-CD24 AlexaFluor antibodies (BD Biosciences, USA), incubated for 30 minutes in the dark at 4\u0026deg;C.The cells were analysed using a flow cytometer (BD FACS ARIA III). The acquisition was set for 10,000 events per sample. Data analysis was performed using the BD FACSDiva\u0026trade; software (FACSDiva\u0026trade;, BD, USA). Cells were sorted based on the surface antigen expression.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eSide Population analysis\u003c/strong\u003e \u003cp\u003eTo isolate and identify SP and non-SP fractions, TNBC cells were treated with trypsin, then resuspended in pre-warmed DMEM containing 1% FBS.Dye Cycle Violet reagent (DCV) at a concentration of 5 \u0026micro;g/mL was added both in the presence and absence of verapamil (Sigma) and incubated at 37\u0026deg;C for 90 minutes with periodic shaking. Following incubation, the cells were washed with PBS containing 1% FBS, cold centrifuged, and resuspended in ice-cold sheath fluid (BD).Cells were preincubated with the ABCG2 inhibitor fumitremorgin-C(FTC) at a concentration of 10 \u0026micro;g/ml at 37\u0026deg;C for 30 minutes before DCV addition. Propidium iodide was added to the cells at a concentration of 1 \u0026micro;g/mL to differentiate viable cells. The Hoechst 33342 dye was excited at 357 nm, and its fluorescence was subsequently analysed using FACS AriaIII (BD Biosciences, San Diego, CA). The gating for forward and side scatter was stringent, ensuring that debris and non-viable cells were excluded from the analysis. Software used for analysis was BD FACSDiva\u0026trade; (FACSDiva\u0026trade;, BD, USA).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eImmunofluorescence analysis\u003c/b\u003e: For tumorsphere immunostaining, cells were plated on glass coverslips (0.11 mm, Corning, USA) in DMEM with 10% FBS for 4 hours. Cells were then fixed with 4% paraformaldehyde and incubated with primary antibodies against SOX2 (mouse monoclonal IgG, Santa Cruz; 1:1000), OCT4 (mouse monoclonal IgG, BioLegend; 1:500), and ALDH1 (mouse monoclonal IgG, BioLegend; 1:500). Corresponding goat anti-mouse secondary antibodies conjugated with FITC, PE, and Cy3 were applied. Tumorspheres were incubated at 37\u0026deg;C for 60 minutes, followed by DAPI staining (Sigma) to visualize nuclei. Images were captured with a Zeiss fluorescence microscope, and processed using ZEN Blue and ZEN Black microscopy software (Zeiss)\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eTransfection\u003c/strong\u003e \u003cp\u003eTNBC cells were transfected with 50 nmol/L miRNA mimics of hsa-miR-1180 and hsa-miR-4728 using Lipofectamine 3000 reagent (Invitrogen). The cells were treated with the miRNA mimics and corresponding scramble controls (IDT Technologies) in Opti-MEM medium for 4 hours, then switched to standard growth medium as per the manufacturer\u0026rsquo;s instructions. Analysis was conducted 48 hours after transfection.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eMigration and Invasion assays\u003c/strong\u003e \u003cp\u003eMatrigel (Corning, USA) was coated on top of the transwell chamber and the (serum starved) transfected cells were added in the upper chamber, seeded at 1 \u0026times; 10⁴ /well. Subsequently, 500 \u0026micro;l of cell growth medium was added to the bottom chamber. Growth media serves as a chemoattractant. For 24 to 36 hours, the cells were incubated at 37\u0026deg;C. After the cells migrated to the lower chamber, they were fixed with 70% ethanol and stained using crystal violet. The quantity of migrated (stained) cells was measured by tallying the number of stained cells, and the mean cell count per field was computed for each well.\u003c/p\u003e \u003c/p\u003e \u003cp\u003eThree replicate wells were utilized for every experiment, with representative images captured from randomly chosen fields in each well.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eWound healing assay\u003c/strong\u003e \u003cp\u003eTNBC cells (5 \u0026times; 10⁵ cells/well) were seeded into 6-well plates and grown as a monolayer for 24\u0026ndash;48 hours. Once cells reach a 80% confluence, a horizontal scratch is created in each well using a 200uL pipette tip. Detached cells were removed by rinsing with 500 \u0026micro;L PBS, followed by the addition of 500 \u0026micro;L of fresh medium. The plates were incubated for 12, 18, and 24 hours, and images were captured at each interval with an inverted microscope to observe scratch closure progress.\u003c/p\u003e \u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eReal-time PCR for miRNA expression analysis\u003c/h2\u003e \u003cp\u003eTotal RNA was isolated using TRIzol reagent (Invitrogen), with concentration measured at A260 and purity assessed via the A260/A280 ratio. RNA quality was evaluated using 2% agarose gels A total of 1 \u0026micro;g of RNA was reverse-transcribed into cDNA using random primers (Thermo), resulting in a final volume of 20 \u0026micro;L of cDNA. Subsequent amplification was performed via PCR for the miRNAs miR-940, 155, 6803, and 4728, with primer sets listed in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. RT-qPCR was conducted using SYBR Green chemistry, normalizing to GAPDH expression levels, and the ∆∆Ct method was used to calculate normalized fold expression for the target miRNAs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll experimental data were quantified from three independent sets of experiments and expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Statistical analyses were conducted using GraphPad Prism software. Differences between groups were assessed using Student's t-test, one-way ANOVA, and two-way ANOVA, with significance levels determined as *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\u003eAdditional study workflows are illustrated and included in supplementary file S1(Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eIdentification of differentially expressed miRNAs by meta-analysis in breast cancer\u003c/h2\u003e \u003cp\u003eFor the meta-analysis, a total of 120 miRNA datasets related to breast cancer were obtained from the Gene Expression Omnibus (GEO) databases. Of these, a sum of 53 datasets on breast cancer were shortlisted and mined from the public domain based on various exclusion and inclusion criteria. Following the dataset selection, we downloaded data matrices and differential analyses were conducted using the GEO2R tool provided on the GEO platform. Across these datasets that reported multivariate analyses, expression values of miRNAs were calculated from each study. A set of five miRNAs showing high differential expression in invasive breast cancers was selected for downstream experiments. The predictive significance of the chosen miRNAs in breast cancer patients was validated by performing Kaplan-Meier analysis which demonstrated that all five miRNAs : hsa-miR-6803, hsa-miR-1180, hsa-miR-4728, hsa-miR-1915 and hsa-miR-940 were significantly associated with poor overall survival (OS). Survival analysis indicates increased expression of these miRNAs is correlated with poor prognosis for patients with breast cancer as presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e (a-e). While these miRNAs have shown poor prognosis across breast cancers, there is limited data to support the same in case of TNBCs as these miRNAs have been previously unreported in TNBCs and TNBCSCs. Given the prognostic promise and limited availability of data, we explored the prognostic potential of these miRNAs in TNBC.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eTarget prediction and Functional annotation for target genes\u003c/h3\u003e\n\u003cp\u003eTo further identify their target genes of these five selected miRNAs, target prediction was performed using four electronic databases-TargetScan, miRTarBase and miRDB and miRmap. We created a Venn diagram to illustrate the intersections between five miRNA targets, summarizing common targets across breast cancers in the initial cohort (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). The number of predicted targets for miR-4728-3p, miR-940, miR-1180, miR-6803-5p and miR-1915-3p was 3749, 6171, 2948, 4709 and 7071, respectively. (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb) Gene set enrichment analysis is widely used to evaluate the enrichment of differentially expressed genes (DEGs) in a range of biological processes, cellular components, and molecular functions. Analysis of DEGs using Gene Ontology revealed significant enrichment in processes such as endothelial cell migration, signalling pathways involving Wnt, NOTCH, EGFR, the JNK cascade, along with cell division and DNA replication. (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec-d). These results indicate the potential involvement of key signaling pathways and biological processes with our selected miRNAs, leading to tumor progression.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eIsolation and characterization of exosomes derived from TNBC cells\u003c/h2\u003e \u003cp\u003eExosomes were collected from the conditioned media of TNBC cell lines MDA-MD 231 and MDA-MB 468 after 48h. We performed scanning electron microscopy (SEM) and transmission electron microscopy (TEM) to examine cell culture-derived EVs. Further, Confocal microscopy was employed to qualitatively analyse localization of exosomes within TNBC cells. Exosomes were successfully identified as double-membrane vesicles through TEM Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e (d,h)) and their size range was estimated to lie between 50\u0026ndash;200 nm as indicated by SEM (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e(c,g)). The exosome marker CD81 was successfully found localized in TNBC cells as indicated by Immunofluorescence (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e (a-c) and western blot Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e(d)\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePresence of cancer stem-like cells in TNBC\u003c/h3\u003e\n\u003cp\u003eUsing flow cytometric techniques, we identified the presence of stem-like cells in the TNBC population. These cells have the potential to generate 3D spheroids in culture, exhibiting stem-like properties. Our results exhibit increased expression of stemness markers SOX2, ALDH1 and ABCG2 in tumor-derived spheroids. This was confirmed through RTqPCR analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG) and Immunoblotting (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH). Furthermore, expression of stemness marker genes in TNBCSCs was found to be two-fold higher compared to TNBC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH).\u003c/p\u003e \u003cp\u003eFlow cytometric analysis was carried out in TNBC cell lines MDA-MB 231 and MDA-MB 468 to evaluate for expression of stemness marker CD133 and CD44 and identification of cancer stem cells. FACS analysis revealed a presence of a small population of SP cells (0.9\u0026ndash;1.1%) that exhibit dye exclusion properties of cancer stem-like cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e (e-h)) The SP cells, which disappear in the presence of verapamil (f,h), are outlined and shown as a percentage of the total Cancer stem-like cell population. SP cells were subsequently grown in cell culture and developed tumorspheres as seen in representative phase contrast images (b,d). Further, marker-based selection of TNBC stem cells by FACS also revealed presence of small population (1\u0026ndash;2%) of stem-like cells expressing markers CD133⁺, CD44⁺, and CD24⁻ (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e (i-l)). TNBC stem-like cells were characterized by immunostaining of tumorspheres with SOX2, ALDH1 and ABCG2 markers (as seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e (A-F) and immunoblotting to determine the expression level of ALDH1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eTargeting hsa-miR 1180 and hsa-miR 4728 inhibits invasion and migration in vitro\u003c/h3\u003e\n\u003cp\u003eTo explore the role of miR1180 and miR-4728 on cell growth and proliferation in TNBC, we performed loss of function experiments by transfecting TNBC cells with anti-miR-1180 and anti-miR-4728 (24 h) in TNBC cells. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e(A) in the wound healing assay, knockdown of miR-1180 and miR-4728 inhibited cell migration and proliferation when compared with WT conditions. Figure\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e(B) shows a drastic reduction in TNBC cell invasion abilities when miRNAs-1180, 4728 were silenced compared with the WT control. These results indicated the role of miR-1180 and miR-4728 in the cell proliferation, migration and invasion in TNBC cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eExo-miRNAs consistently expressed in CSCs and breast cancer tissues\u003c/h2\u003e \u003cp\u003eRT-qPCR was employed to assess the levels of oncogenic miRNAs in TNBC cells and stem cells in vitro (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea, b, d, e). We found a significant upregulation of our target miRNAs miR 4728, miR 6803, miR-940, miR \u0026minus;\u0026thinsp;1915 and miR-1180 in TNBC cell lines MDA-MB 231, MDA-MB 468 in comparison to their corresponding CSCs. A similar expression pattern was observed in TNBC and TNBCSCs derived exosomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ec and f). Our panel of five miRNAs- miR 6803, miR 1180, miR 4728, miR 1915 and miR 940 were found to be highly expressed in TNBC cells, TNBCSCs and enriched in circulating exosomes. Given their circulatory nature and consistent expression across TNBC cells and stem cells, these results indicate miRNAs miR 4728, miR 6803, miR-940, miR \u0026minus;\u0026thinsp;1915 and miR-1180 may have a significant role to play in TNBC extracellular communications leading to disease progression or better prognosis if these miRs are targeted for therapy.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eClinical Correlation\u003c/h2\u003e \u003cp\u003eTo clinically validate our findings, the presence of these miRNAs was evaluated in breast cancer tissues. Tissue biopsies were obtained from 15 TNBC patients, along with corresponding adjacent normal tissue samples for comparison. RT-qPCR analysis of resected tumor tissue RNA revealed consistent expression of these miRNAs in patients with TNBC (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eg). Amongst the five miRNAs, hsa-miR 1180 and hsa-miR 4728 were found to be significantly upregulated in TNBC biopsies when compared with their adjacent (normal) tissues. Thus, our results demonstrated a positive correlation between the elevated expression of these miRNAs and the progression of TNBC. Second, the presence of hsa-miR 1180 and hsa-miR 4728 in circulatory tumor-derived vesicles and the detection of the same miRNAs in clinical tissue samples highlights their significance as potential biomarkers in TNBC.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eTriple negative breast cancer (TNBC), characterized by the absence of the estrogen receptor (ER), progesterone receptor (PR), and HER2/neu receptor expression\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e is known to be a highly aggressive cancer that shows the worst prognosis among all breast cancer types, coupled with rapid relapse. Currently there are hardly any targeted therapies for combat this aggressive and debilitating disease. Through intercellular communication with the tumor microenvironment and potentiated by CSC population, TNBC cells often acquire treatment resistance leading to metastasis and chemoresistance\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan additionalcitationids=\"CR45 CR46\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Additionally, the molecular heterogeneity of TNBC as revealed by the presence of many molecular markers poses challenges to effective treatment.\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e Several studies have outlined promising predictive and prognostic markers in breast cancer\u003csup\u003e\u003cspan additionalcitationids=\"CR50\" citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. However, very few studies have been able to identify TNBC specific therapeutic biomarkers\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e,\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. At present, there is no universal biomarker available for diagnosing and targeting TNBC, unlike other breast cancer subtypes, which have biomarkers such as HER2 and specific hormone receptors\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e,\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e There is a critical need to identify reliable, non-invasive markers specific to TNBC for rapid screening, early diagnosis, risk assessment, and ultimately for effective treatment and management of breast cancer. This study highlights the clinical utility of specific exosomal miRNAs as non-invasive diagnostic and prognostic markers, as well as potential therapeutic targets.\u003c/p\u003e \u003cp\u003eAfter comprehensive literature review and subsequent meta-analysis of 120 publicly available breast cancer datasets, 53 relevant datasets were identified based on various exclusion and inclusion criteria. Upon analysis of these datasets, a set of five, highly oncogenic miRNAs were found to be expressed in TNBCs : hsa-miR 6803, hsa-miR 1180, hsa-miR 4728, hsa-miR 1915 and hsa-miR 940. Though these miRNAs were often found in breast cancers, they have never been shown to be exclusively reported in TNBC, making them ideal candidates for therapeutic interventions. Additionally, since there was so far no evidence linking these oncomiRs with disease relapse, we were eager to explore their gene expression profile in TNBCSCs. Of the five miRNAs, two were found to show exclusively high expression in TNBC clinical tissue. RT-qPCR analysis revealed a notable increase in the expression of hsa-miR-1180 and hsa-miR-4728 in TNBC tissues when compared to non-tumor breast tissues. Our study has confirmed that hsa-miR-1180 and hsa-miR-4728 act as tumor promoters, suggesting that their upregulation in tumor tissues may contribute to the progression and metastasis of TNBC. Decreased expression of these miRNAs attenuated tumor progression, as demonstrated by wound healing and invasion assays (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, B)\u003c/p\u003e \u003cp\u003eAdditionally, miRNA target prediction was carried out using four electronic databases :TargetScan, miRTarBase and miRDB and miRmap, which revealed 14 common targets between the two selected miRNAs - hsa-miR-1180 and hsa-miR-4728. PPI and RNA-protein association analysis of targets using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://string-db.org\u003c/span\u003e\u003cspan address=\"https://string-db.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e)\u003c/span\u003e revealed amongst others, their role in epigenetic regulation of HATs, TP53 regulation of metabolic pathways and PPARɑ mediated gene expression in breast cancer (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e)\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eUsing gene enrichment analysis and target prediction models, these dysregulated miRNAs were also found to be involved in a wide range of crucial biological processes and pathways including regulation of RNA pol II, Wnt signaling, Notch signaling, MAPK signaling pathway and the regulation of JNK signaling cascade. Previous studies have highlighted the correlation of Wnt signaling pathways with maintenance of stem cell niche and expression in cancer stem cells\u003csup\u003e\u003cspan additionalcitationids=\"CR36 CR37 CR38 CR39 CR40\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Notch receptor and ligand overexpression is linked to TNBC progression\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Notch receptors are also involved in modulating the behaviour of tumor-initiating cells and in the initiation of TNBC\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. These pathways are pertinent to the therapeutic targeting of CSCs or other cells responsible for diverse prolapse as they play key roles in genesis and regulation of cell survival and their fate\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Given that many of these pathways are known to support tumor cell proliferation, cancer stem cell (CSC) survival, epithelial-to-mesenchymal transition (EMT), and invasion, it is likely that these miRNAs contribute to the progression and expansion of TNBC and TNBC stem cells.\u003c/p\u003e \u003cp\u003eExosome-encapsulated miRNAs have a great potential as prognostic biomarkers. Several studies have demonstrated that exosomes are robust and may be stored for extended periods without compromising the integrity of encased miRNAs. These features greatly increase their potential applicability in a diagnostic or clinical setting. Secretory miRNAs are reflective of their parent cell status and thus may reveal a more specific tumor profile than conventional miRNA profile derived from whole blood or serum. From our identified set of five oncogenic miRNAs, miR 6803 has been previously identified as a diagnostic marker in colorectal cancer\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e,\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. miR 1180 has been found to play a role in tumor progression in Lung cancer, melanoma, ovarian cancer, and hepatocellular carcinoma.\u003csup\u003e\u003cspan additionalcitationids=\"CR57 CR58 CR59 CR60\" citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. miR 1915 has been reported in gastric cancer\u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e,\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e, colorectal carcinoma (CRC)\u003csup\u003e\u003cspan additionalcitationids=\"CR65\" citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e and most recently in Breast cancer\u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e. Interestingly, miR 4728 is found to have dual functions. It acts as a tumor suppressor in CRC\u003csup\u003e\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e while being tumorigenic in breast cancer\u003csup\u003e\u003cspan additionalcitationids=\"CR70\" citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/sup\u003e. miR 4728 has been identified as a marker of HER2 status in BrCa patients. miR 940 has been widely reported as an oncogenic marker for CRC\u003csup\u003e\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u003c/sup\u003e, gastric cancer\u003csup\u003e\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e\u003c/sup\u003e, cervical cancer\u003csup\u003e\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e\u003c/sup\u003e and breast cancer\u003csup\u003e\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e,\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e\u003c/sup\u003e. It is involved in four critical pathways: the Wnt/β-catenin pathway, the MAPK pathway, the PD-1 pathway, and the phosphatidylinositol 3-kinase (PI3K)-Akt pathway, all of which are significantly implicated in breast carcinogenesis\u003csup\u003e\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e,\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e\u003c/sup\u003e. While many of these selected miRNAs have been identified in various types of cancer, their secretory roles in TNBC and TNBC stem cells (TNBCSCs) were previously unrecognized. Our study reveals the presence of secretory miRNAs (miRNAs miR 6803, miR 1180, miR 4728, miR 1915 and miR 940) in TNBC and TNBC stem cells highlighting their diagnostic value and clinical utility for better management of TNBC. Furthermore, these secretory oncomiRs were found consistently overexpressed in TNBC and TNBCSCs. We subsequently correlated our findings with TNBC tumor tissue samples (n\u0026thinsp;=\u0026thinsp;15) and found a consistent high expression of all five oncomiRs across tumor biopsies. From this panel of five miRNAs, two oncomiRs -hsa-miR-1180 and hsa-miR-4728 were found to be significantly upregulated across all tumor tissue samples in TNBC patients. The presence of these two secretory miRNAs in TNBC and TNBCSC along with their overexpression in clinical tissue samples indicate their possible role in TNBC progression and metastasis and may serve as reliable prognostic as well as therapeutic marker(s).\u003c/p\u003e \u003cp\u003eWe recognise that our integrated analysis has some limitations. During the systematic literature review, the initial screening was conducted solely on three databases (PubMed, Web of Science and Cochrane Library), which may have resulted in the omission of some relevant studies. Ideally, we could have searched for additional data sources such as pre-print server archives, relevant books, Google Scholar and other similar resources. Additionally, the limited availability of publicly listed TNBC clinical data sets in GEO and TCGA may have narrowed the scope of our estimates. We have analyzed potential circulatory prognostic markers for TNBC, validated our findings in TNBC tumor tissues and further validated them in vitro using TNBC cell lines. Since TNBC is a highly metastatic disease, it is very challenging to obtain fresh surgical tissue samples. We were able to include a small number of tumor tissue samples which may limit the clinical relevance and translational impact of our study. We recognize that analysis of larger datasets and clinical correlation using higher numbers of TNBC tissues could provide greater insights on prognostic significance of these TNBC derived exosomal miRNAs. Further, addition of population-based studies and data stratification could provide better understanding and help in effective therapy and/or prognostic monitoring of TNBC.\u003c/p\u003e \u003cp\u003eNonetheless, the identification of novel secretory miRNAs in TNBC and TNBCSCs is promising with potential application in the diagnosis and treatment of hormone refractory, metastatic breast cancers. Our study identifies novel oncogenic exomiRs in TNBC and TNBCSCs and highlights their utility as therapeutic targets for TNBC. The mechanisms through which these exomiRs influence the development and progression of TNBC are not yet fully elucidated. Nonetheless, it is believed that they may modulate the expression of genes associated with cell signalling pathways, including the Wnt, Notch, and MAPK pathways, which are known to play a role in the progression and relapse of TNBC. Previous studies have demonstrated similar prognostic potential of several other miRNAs in breast cancer such as miR-9\u003csup\u003e79\u003c/sup\u003e, miR-21\u003csup\u003e9\u003c/sup\u003e, miR-29b\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e and miR 331\u003csup\u003e80\u003c/sup\u003e. Our study is the first to assess prognostic behaviour of these novel TNBC and TNBCSC derived exosomal miRNAs. Elucidating the role of secretory miRNAs in the molecular mechanisms driving TNBC initiation and progression is crucial for advancing miRNA-based therapeutics, which hold promise as effective treatment options for TNBC patients.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eA set of five novel secretory oncomiRs (hsa-miR 6803, hsa-miR 1180, hsa-miR 4728, hsa-miR 1915 and hsa-miR 940) were exclusively found to be expressed in TNBC and TNBCSCs, demonstrating great prognostic potential. These miRNAs were also found consistently expressed in TNBC tissue samples. Two of the oncomiRs : hsa-miR-1180 and hsa-miR-4728 were found to be significantly upregulated in TNBC tumor tissue. In this study, these miRNAs were found to function as oncomiRs. Given the secretory nature of these oncomiRs and their consistent expression across clinical cases, they can be explored as potential therapeutic as well as prognostic markers for TNBC.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eTNBC: Triple Negative Breast Cancer; miRNA: micro-RNA; TEM: transmission electron microscope; SEM: Scanning electron microscope; FBS: fetal bovine serum; DMEM, Dulbecco\u0026apos;s Modified Eagle Medium; GAPDH: glyceraldehyde 3-phosphate 26 dehydrogenase; PVDF: polyvinyl difluoride; RTqPCR: Reverse transcription-quantitative PCR; RIPA: Radio-Immunoprecipitation Assay; SDS: \u0026nbsp;sodium dodecyl sulfate, PPI: Protein-Protein interactions, GO: Gene ontology, GEO : Gene expression omnibus ; DEG: Differentially expressed genes\u003c/p\u003e\n"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported in part by the Department of Science and Technology (DST), Ministry of Science and Technology, Government of India by awarding the INSPIRE fellowship to A.Choudhary for her Ph.D. study. Confocal microscopy was performed at the Translational Health Science and Technology Institute (THSTI), in Faridabad, India. \u0026nbsp;We thank Mr. Suraj Tewari (THSTI) for his technical expertise in Super-resolution microscopy and confocal imaging; Max Institute of Cancer Care, Max super specialty hospital, Saket, New Delhi for the provision of TNBC tissue samples for clinical validation; Mr. Akshey Kaushal, application engineer at the Indian Institute of Technology (IIT) Delhi, for his technical help with HR-TEM and Cryo-TEM employed for exosome visualization; \u0026nbsp;Dr Prasanna Venkatraman, Deputy Director at the Cancer Research Institute, ACTREC Mumbai, India for sharing with us MCF10A cell lines; Mr. Manoj Gupta and Dr Pradeep K Rai for assistance with FACS analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e: B. C. Das:\u003c/strong\u003e Conceptualization, Supervision, Reviewing and Editing; \u003cstrong\u003eAnanya Choudhary:\u0026nbsp;\u003c/strong\u003eData curation, Methodology, Investigation, Writing, Original draft preparation, Reviewing and Editing; Funding acquisition; \u0026nbsp;\u003cstrong\u003eSatish S. Poojary\u003c/strong\u003e: Visualization, Methodology, Supervision; \u003cstrong\u003ePriyanka Jain\u003c/strong\u003e:\u0026nbsp;Software, Validation, Formal Analysis\u003cstrong\u003e; Harit Chaturvedi :\u003c/strong\u003e Resources\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding :\u003c/strong\u003e This study was supported in part by the Department of Science and Technology (DST), Ministry of Science and Technology, Government of India by awarding the INSPIRE fellowship to A.Choudhary for her Ph.D. study\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of supporting data:\u003c/strong\u003e All data relevant to the study are included in the article or uploaded as supplementary information.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate :\u003c/strong\u003e Informed consent was obtained from all participants prior to their involvement in the study, and all procedures were conducted in accordance with the ethical guidelines of Indian Council of Medical Research (ICMR). Participants were fully informed about the study objectives, potential risks and benefits, and their right to withdraw at any time. Written consent forms were obtained from all participants, ensuring their privacy and confidentiality of data collected. All personal data and biopsy samples were handled with strict confidentiality. Identifiable information has been removed or anonymized in all published findings. All clinical samples were collected with signed informed consent and ethics approval also obtained from Max Institute of Cancer Care, Max Hospital Saket, New Delhi\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u003c/strong\u003e The content of this paper has not been submitted to any other scientific publications. All the authors have declared that no financial conflict of interest exists. All authors have approved the submission of this work for publication in Breast Cancer Research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e: The authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eDent, R. \u003cem\u003eet al.\u003c/em\u003e Triple-negative breast cancer: Clinical features and patterns of recurrence. \u003cem\u003eClinical Cancer Research\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 4429\u0026ndash;4434 (2007).\u003c/li\u003e\n\u003cli\u003eIrvin, W. J. \u0026amp; Carey, L. A. 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PMID: 30037718; PMCID: PMC6116416.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"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":"TNBC, miRNA, extracellular vesicles, exosomes, CSC, prognosis","lastPublishedDoi":"10.21203/rs.3.rs-5910171/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5910171/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eExosomal miRNAs have been identified as key secretory biomarkers facilitating intercellular communication in the tumor microenvironment, with substantial potential as diagnostic, prognostic, and therapeutic targets. Our research focuses on the role of exosomal miRNAs in triple-negative breast cancer (TNBC) and cancer stem cells, contributing to tumor progression and relapse. Through meta-analysis and data mining, we identified five differentially expressed miRNAs- hsa-miR-6803, hsa-miR-1180, hsa-miR-4728, hsa-miR-1915 and hsa-miR-940 in breast cancer. Target predictions, GO and KEGG enrichment analyses have indicated the potential role of these oncomiRs in Wnt, Notch and EGFR signalling pathways involved in tumor progression. While the panel of five miRNAs was found to be over-expressed in breast cancers, they have not been reported in TNBC and TNBCSCs, making them ideal biomarkers for TNBC. These oncomiRs were consistently detected across TNBC cell lines, TNBCSCs and further validated in TNBC tumor tissues (n\u0026thinsp;=\u0026thinsp;15). Interestingly, a highly significant overexpression of two of the five specific miRNAs- hsa-miR-1180 and hsa-miR-4728 and their high tumorigenic validation in an invasion, migration and siRNA analysis indicates their potential as prognostic and therapeutic target(s). The presence of these miRNAs in TNBC cell lines, TNBCSCs and circulatory exosomes and their elevated expression in tumor tissue highlights their significance as potential biomarkers for TNBC. We suggest that these five specific secretory miRNAs could function as a liquid biopsy tool, not only for the diagnosis of tumor progression but may also provide a reference value for early detection as well as for monitoring the prognosis of TNBCs\u003c/p\u003e","manuscriptTitle":"Identification of novel exosomal miRNAs and their role in diagnosis and prognosis of Triple Negative Breast Cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-13 07:05:10","doi":"10.21203/rs.3.rs-5910171/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":"ff25465e-bc02-4f92-bbde-4b91967f03e4","owner":[],"postedDate":"June 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-04T18:38:24+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-13 07:05:10","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5910171","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5910171","identity":"rs-5910171","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}