Plasma exosomal miR-125b-5p and miR-143-3p have potential to diagnose breast cancer

Plasma exosomal miR-125b-5p and miR-143-3p have potential to diagnose 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 Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Plasma exosomal miR-125b-5p and miR-143-3p have potential to diagnose breast cancer Lei Fan, Yao Li, Jicheng Huang, Bin Hua This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4276021/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 Background Breast cancer (BC) is the most common malignancy in women, posing a serious health risk. Exosomal microRNA (miRNA) has emerged as a promising cancer biomarker. Despite its potential, research on exosomal miRNA in BC remains limited. This study aims to identify plasma exosomal miRNAs for diagnosing BC. Methods Firstly, plasma exosomal miRNA sequencing was conducted on 45 BC patients and 5 healthy controls, followed by Differential Expression Analysis to screen for differentially expressed miRNAs. The diagnostic value of these miRNAs was assessed using receiver operating characteristic (ROC) curves. Subsequent analysis of candidate miRNAs was carried out in the TCGA database. Target genes of differentially expressed miRNAs were predicted using online databases, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed. Finally, a ceRNA network was constructed using Cytoscape. Results Our analysis indicates that, compared to normal controls, plasma exosomal miR-125b-5p and miR-143-3p are significantly downregulated in BC patients. The area under the curve (AUC) for exosomal miR-125b-5p is 0.951 (sensitivity 91.1%, specificity 100%), and the AUC for exosomal miR-143-3p is 0.924 (sensitivity 86.7%, specificity 100%). In the TCGA database, the expression of miR-125b-5p and miR-143-3p is downregulated in BC tissues compared to adjacent normal tissues. Target gene prediction and enrichment analyses reveal pathways closely associated with BC occurrence. Conclusions Our results demonstrate that plasma exosomal miR-125b-5p and miR-143-3p can distinguish between BC patients and normal controls. The downregulation of plasma exosomal miR-125b-5p and miR-143-3p may potentially serve as a marker for the occurrence of BC. exosomes microRNA breast cancer occurrence diagnosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction BC is the most common malignancy in women, the second leading cause of death in women, and the incidence of BC is showing an increasing trend year by year [ 1 ] . Despite continuous advancements in the treatment of BC, a significant proportion of patients still succumb to the disease each year. Successful treatment of BC is highly dependent on early diagnosis. Early-stage BC patients have a 5-year survival rate of over 90%, while patients with distant metastasis have a 5-year survival rate of only 20% [ 2 – 4 ] . Therefore, there is an urgent need for biomarkers with high sensitivity and specificity for early diagnosis of BC [ 5 ] . Traditional biopsies are invasive and susceptible to factors like lesion location and operator experience. Moreover, conventional tumor markers and imaging examinations are limited by their low sensitivity and specificity in disease diagnosis. Liquid biopsy, a novel diagnostic technique emerging in recent years, involves the detection of components like exosomes, and so on. This approach has gained significant attention due to its non-invasiveness and ease of access. Exosomes, present in bodily fluids, serve as mediators for intercellular communication. They carry a diverse range of bioactive molecules, including non-coding RNA, miRNA, DNA, proteins, and others, playing crucial roles in promoting or inhibiting diseases, especially in cancer [ 6 ] . It has been reported that exosomes can be easily obtained through biofluid sampling, allowing for the maximization of their potential value in diagnosing tumors and other diseases [ 7 ] . MiRNA serves as a vital component of exosomes and plays a crucial role in important biological processes such as embryonic development, cell cycle regulation, proliferation differentiation, and apoptosis which may be as diagnosis markers for liquid biopsy [ 8 ] .MiR-125b-5p is significantly downregulated in the exosomes of non-small cell lung cancer patients and serves as a diagnostic and prognostic biomarker for non-small cell lung cancer [ 9 ] . It accurately distinguishes pancreatic cancer patients from healthy controls and acts as an independent biomarker predicting overall survival in pancreatic cancer patients [ 10 ] . Compared to healthy endometrium, miR-125b-5p is downregulated in endometrial cancer, showing promise as a diagnostic biomarker for endometrial cancer [ 11 ] . Meanwhile miR-143-3p is significantly downregulated in urinary exosomes of prostate cancer patients, presenting itself as a promising biomarker [ 12 ] . Serum exosomal miR-143-3p has the potential to serve as a diagnostic biomarker for renal cell carcinoma [ 13 ] . MiR-143-3p is significantly overexpressed in the serum of endometrial cancer patients and can be a potential non-invasive biomarker for endometrial cancer diagnosis [ 14 ] . Despite research on miR-125b-5p and miR-143-3p in various tumor diagnoses, their specific studies in BC diagnosis are currently lacking. In this study, we investigated differentially expressed miRNAs (DEMs) in plasma exosomes from BC patients and normal healthy controls. Our findings revealed significantly downward of miR-125b-5p and miR-143-3p in the plasma exosomes of BC patients. These miRNAs demonstrated potential as effective diagnostic markers for BC. We also predicted the target genes of these miRNAs and conducted pathway enrichment analysis, which provide valuable insights for future research directions. Material and methods Patients and plasma specimens In this study, preoperative peripheral blood samples were collected from 45 BC patients receiving treatment at the Breast Center of Beijing Hospital. The clinical and pathological characteristics were presented in Table 1 . All of these patients had not undergone any relevant treatment before surgery and had their BC diagnosis confirmed through pathological examination. Furthermore, peripheral blood samples were collected from 5 healthy individuals. 5 milliliters of peripheral venous blood were extracted from each patient and subsequently underwent centrifugation. Specifically, the blood samples were centrifuged at 1500 g for 20 minutes, and the supernatant was aspirated into 15-milliliter centrifuge tubes. Next, the supernatant was centrifuged again at 3000 g for 15 minutes, and the upper layer of plasma was aliquoted into 2-milliliter eppendorf tube (EP) and stored at -80°C for further exosomes isolation research. Table 1 The plasma samples and the corresponding clinical data in the present study. clinical data Numbers Breast cancer 45 Age(year) < 50 20 ≥ 50 25 TNM stage Ⅰ 14 Ⅱ 22 Ⅲ 9 T T1 T2-4 22 23 N N0 20 N1-3 25 M M0 45 M1 0 ER (+) 25 (-) 20 PR (+) 23 (-) 22 Her-2 (+) 12 (-) 33 ki-67 < 15 10 ≥ 15 35 Abbreviations: TNM stage: The Tumor, Node, Metastasis staging system; T: Staging of tumor; N: Staging of lymph nodes; M: Staging of metastasis; ER: Estrogen receptor; PR: Progesterone Receptor; Her-2: Human Epidermal Growth Factor Receptor 2; Ki-67: Antigen Identified by Monoclonal Antibody Ki-67. The entire research protocol has been approved by the Ethics Committee of Beijing Hospital in accordance with the principles of the "Helsinki Declaration," and all participants provided written informed consent. Isolation and Identification of Plasma Exosomes Plasma exosomes underwent size exclusion chromatography (SEC) for separation. One milliliter of plasma was initially filtered through a 0.8 µm filter, followed by a 1.5-fold dilution with PBS and additional purification using an Exosupur column (Echobiotech, China). The elution of samples was conducted with PBS, and 2 mL eluates were gathered. Subsequently, the eluates were concentrated to 200 µL using Amicon Ultra centrifugal filters with a molecular weight cutoff of 100 kDa (Millipore, Germany). Exosomes were identified using two techniques: nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). Initially, vesicle suspensions at a concentration of around 1 × 10 8 /mL were analyzed with ZetaView PMX 110, featuring a 405 nm laser, to determine particle size and quantity. Twenty microliters of exosomes underwent NTA analysis (ZetaView 8.02.28) to assess particle motion. Subsequently, exosomes were resuspended in PBS and applied to copper grids for TEM observation. Following a 10-minute incubation at room temperature, negative staining with 1% uranyl acetate was performed for 10 minutes. Observation and imaging were carried out using a TEM (H-7650, Hitachi, Tokyo, Japan). Plasma exosome-derived miRNA sequencing analysis The RNA generated from each plasma exosome sample was extracted using the miRNeasy Kit (Qiagen, Germany). Subsequently, miRNA-seq was performed on the Illumina HiSeq platform. The miRNA-seq data were represented by unique molecular identifiers (UMIs). The screening criteria for DEMs was a median TPM (transcripts per million) ≥ 30, |log 2 fold change (FC)| > 0.5 and FDR (False Discovery Rate) < 0.05. Common target genes of candidate DEMs prediction We employed the online free databases TargetScan ( https://www.targetscan.org/vert_80/ ) and miRTarBase ( https://www.mirnet.ca/ ) for predicting the target genes of each DEM [ 15 , 16 ] . Finally, through the utilization of an online Venn diagram( http://sangerbox.com/login.html)analysi s, we identified the overlapping genes as the common target genes by intersecting the target genes. Pathway enrichment and Gene Ontology analysis of common target genes To gain further insights into the potential molecular mechanisms and interactions of the common target genes, we utilized the STRING database ( https://string-db.org/ ) for protein-protein interaction (PPI) network analysis. In order to determine the specific biological functions of the target genes implicated in BC, we conducted GO and KEGG pathway enrichment analysis using the online platform David ( https://david.ncifcrf.gov/summary.jsp ). Screening for potential target genes Upon importing the PPI network file of the common target genes into Cytoscape software, we applied the CytoHubba plug-in to calculate the Maximum Clique Centrality (MCC) algorithm, which enabled us to identify the top 10 genes as hub genes. Simultaneously, we used the MCODE plugin to identify critical functional modules among the shared target genes. Genes that are present in both hub genes and key functional modules are considered potential target genes of the DEMs [ 17 ] . Statistical analysis SPSS 25.0 software (Chicago, IL, USA) was used for statistical analysis and GraphPad Prism 9.0 was used for plotting and graphing, and Mann-Whitney U-test was used to test the expression levels of exosomal miRNA in the plasma of BC patients and healthy controls. The ROC curve was used to evaluate the diagnostic ability of the candidate exosomal DEMs for BC, and if the AUC value > 0.7, it showed better diagnostic and predictive ability. P -value < 0.05 was considered statistically significant. Results The expression of miR-125b-5p and miR-143-3p is significantly decreased in the plasma exosomes from BC patients. To screen for exosomal miRNAs associated with BC occurrence, we conducted miRNA sequencing on exosomes from the plasma of 45 BC patients and 5 healthy controls. Five miRNAs exhibited differential expression in the plasma exosomes of BC patients compared to healthy controls. The screening criteria was median TPM ≥ 30, |log 2 FC| > 0.5, FDR < 0.05 (Table 2 ). Among them, three were upregulated, including miR-144-3p, miR-144-5p, and miR-15b-5p, while two were downregulated, namely miR-125b-5p and miR-143-3p. DEMs were visualized using volcano map and heatmap (Fig. 1 A-B). Among them, miR-125b-5p and miR-143-3p were most significantly downregulated in plasma exosomes of BC patients (Fig. 1 C). We further compared the expression levels of plasma exosomal miR-125b-5p in breast cancer patients at different TNM stages. The results revealed a significant decrease in the levels of plasma exosomal miR-125b-5p in stage II and stage III BC patients compared to stage I patients (Fig. 1 D). Table 2 Plasma exosomal miRNA differentially expressed between breast cancer patients and healthy controls. ID Log 2 FC FDR miR-144-3p 0.833676769 0.015436401 miR-144-5p 0.733625073 0.040196407 miR-15b-5p 0.607799216 0.042665855 miR-125b-5p -0.726766996 0.002324052 miR-143-3p -0.688851409 0.010703674 Abbreviations: FC: fold change The value of plasma exosomal miR-125b-5p and miR-143-3p in the diagnosis of BC we plotted ROC curves using the expression levels of plasma exosomal miR-125b-5p and miR-143-3p to assess their reliability in diagnosing BC (Fig. 1 E-F). The results indicate that plasma exosomal miR-125b-5p and miR-143-3p exhibit high diagnostic accuracy in distinguishing BC patients from healthy controls (AUC > 0.9). The AUC value for plasma exosomal miR-125b-5p is 0.951 (95% CI: 0.891-1, sensitivity: 91.1%, specificity: 100%), and for plasma exosomal miR-143-3p, the AUC value is 0.924 (95% CI: 0.847-1, sensitivity: 86.7%, specificity: 100%). According to our analysis, plasma exosomal miR-125b-5p and miR-143-3p have the potential to serve as non-invasive biomarkers for diagnosing BC. Analysis of miR-125b-5p and miR-143-3p in the TCGA database In the TCGA database, as shown in the Fig. 2 A, the expression of miR-125b-5p and miR-143-3p in BC tissues is significantly downregulated compared to adjacent normal tissues, consistent with the differential expression in plasma exosomes. Subsequent analysis explored the relationship between the pathological characteristics of BC patients and the expression levels of these two miRNAs. Compared to patients at the T1 stage, the expression of miR-125b-5p and miR-143-3p is significantly decreased in tissues of patients at T2-4 stage (Fig. 2 B). Additionally, the expression of miR-125-5p decreases with the progression of BC (Fig. 2 C). Potential target genes and pathway enrichment analysis of plasma exosomal miR-125b-5p and miR-143-3p Utilizing TargetScan and miRTarBase databases, we predicted a total of 60 common target genes. Specifically, miR-125b-5p targets 30 genes, while miR-143-3p targets 32 genes (Fig. 3 A). Using clusterProfiler, we employed GO functional annotation and KEGG pathway enrichment analysis to investigate the biological roles of the shared target genes of the two miRNAs (Fig. 3 B-C). The GO functional annotation revealed their involvement in processes like " positive regulation of cell proliferation" and " positive regulation of cell growth" in the BP category, " ERBB3:ERBB2 complex" and "caveola" in the CC category, and functions such as " growth factor binding " and " transmembrane receptor protein tyrosine kinase activity " in the MF category. The ERBB3/ERBB2 heterodimer can activate multiple signaling pathways, including MAPK/ERK and PI3K/AKT, thereby promoting the occurrence of BC [ 18 – 20 ] . A series of growth factors and their receptors participate in the occurrence and development of BC. Receptor Tyrosine Kinases are a class of receptors that play a crucial role in the occurrence of BC [ 21 ] . KEGG pathway analysis identified enrichment in eight signaling pathways, including "PI3K-Akt signaling pathway", " Hippo signaling pathway", "HIF − 1 signaling pathway", " Adherens junction", "Focal adhesion", " MAPK signaling pathway", " ErbB signaling pathway" and " NF-kappa B signaling pathway ".The Hippo signaling pathway plays a crucial role in the occurrence of BC, and the overexpression of its core component YAP has been demonstrated to promote the growth of BC cells both in vitro and in vivo [ 22 , 23 ] . The PI3K-Akt signaling pathway has been confirmed to have a major oncogenic role in BC, and its activation can also stimulate the proliferation of BC cells [ 24 ] . In BC, the activation of the NF-κB pathway can drive continuous cell cycle progression, leading to uncontrolled cell proliferation [ 25 ] . Common target gene interaction analysis and potential target gene identification The PPI network is used to elucidate the protein interactions among common target genes. (Fig. 3 D). Importing the PPI network file into Cytoscape software and applying the CytoHubba plug-in with the MCC algorithm, we highlighted the top 10 common target genes (Fig. 3 E). Additionally, utilizing the MCODE plugin with specific parameters (degree cutoff ≥ 2, node score cutoff ≥ 0.2, k-core ≥ 2 and max depth = 100), we pinpointed a significant functional module comprising 9 genes: ERBB2 , PDGFRA , BCL2 , SERPINE1 , CCN2 , PTGS2 , SMAD3 , IGF1R and STAT3 (Fig. 3 F). Importantly, all 9 genes in this functional module overlapped with the top 10 genes in the PPI network, underscoring their crucial role in the network. Therefore, these genes are considered potential target genes of the DEMs. Constructed the DEMs-target genes-pathways network We constructed a network (Fig. 4 ) encompassing DEMs, target genes (including common, potential, and optimal target genes), and KEGG pathways to explore their regulatory connections. Among the 9 optimal target genes, miR-125b-5p regulates ERBB2 and STAT3 , while miR-143-3p regulates CCN2 , SERPINE1 , PDGFRA , PTGS2 , SMAD3 and IGF1R , BCL2 is simultaneously regulated by both miR-125b-5p and miR-143-3p. Specifically, " PI3K-Akt signaling pathway " involves PDGFRA , ERBB2 , BCL2 and IGF1R , "HIF − 1 signaling pathway" involves ERBB2 , STAT3 , SERPINE1 , BCL2 and IGF1R , " Hippo signaling pathway " involves SMAD3 , SERPINE1 and PDGFRA , "Adherens junction" involves SMAD3 , ERBB2 and IGF1R , "MAPK signaling pathway " involves PDGFRA , ERBB2 and IGF1R , " ErbB signaling pathway " involves ERBB2 , " NF-kappa B signaling pathway " involves BCL2 and PTGS2 and "Focal adhesion" involves PDGFRA , ERBB2 , BCL2 and IGF1R . The ceRNA network reveals that the target genes of miR-125b-5p and miR-143-3p, along with the enriched pathways, are closely associated with the occurrence of BC. Discussion BC is the most common type of cancer in women, posing a significant threat to women's health. Its incidence has been steadily increasing, and the current indicators used for diagnosing BC have certain limitations [ 1 ] . There is an urgent need to find effective markers for accurate early diagnosis for BC [ 26 , 27 ] . Exosomes, as emerging stars in liquid biopsy, exhibit unique advantages in the diagnosis of various cancers [ 28 ] . Firstly, exosomes can be produced by various cells and are found in various body fluids, such as blood, urine [ 29 ] , breast milk [ 30 ] , saliva [ 31 ] , cerebrospinal [ 32 ] fluid, and tears. Secondly, exosomes are abundant and easily accessible; there are approximately 10 12 exosomes in 1 ml of blood, and various techniques like ultracentrifugation, ultrafiltration, and exosome isolation kits are widely used for exosome extraction. Thirdly, exosomes contain a rich cargo, including nucleic acids, proteins, metabolites, etc. [ 6 , 33 ] , and these materials can be transferred between cells via exosomes. Lastly, exosomes exhibit strong stability, as their lipid bilayer protects both the exosomes and their cargo from degradation, allowing them to circulate stably in the body [ 34 , 35 ] . These advantages make exosomes promising candidates as biomarkers for cancer diagnosis. An increasing body of research suggests that exosomal miRNAs play a role in tumorigenesis, offering new prospects for identifying potential biomarkers for tumor diagnosis. Li et al.'s study reveals that the expression levels of miR-184 in serum exosomes are significantly elevated in non-small cell lung cancer patients. This miRNA has value in distinguishing the nature of pulmonary diseases (benign or malignant) and is closely associated with patient prognosis [ 36 ] .Yang et al.'s research findings indicate that exosomal miRNAs, including miR-183-5p, miR-19b-3p, and miR-323a-3p, can be employed for diagnosing gliomas. Their impact on the malignant characteristics of glioma cells may be attributed to their negative regulation of MAPK8IP1 / FAM175B , OSMR / CASP3 , FBXO32 , and PTPN2 , respectively [ 37 ] . Ren and their team's study identifies three serum exosomal miRNAs for gastric cancer diagnosis, namely miR-1273g-3p, miR-4793-3p, and miR-619-5p. Through bioinformatic analysis, eight target genes were further determined. Among these, serum exosomal miR-1273g-3p/miR-4793-3p were found to target MELK , and miR-619-5p was found to target NCAPG2 , which are believed to represent novel mechanisms involved in gastric cancer development [ 17 ] . Jin's research results demonstrate that exosomal miRNAs, including miR-181-5p, miR-30a-3p, miR-30e-3p, and miR-361-5p, as well as exosomal miR-10b-5p, miR-15b-5p, and miR-320b, hold promise as prospective candidates for developing highly sensitive and specific non-invasive biomarkers for early Non-Small Cell Lung Carcinoma diagnosis [ 38 ] . Lv et al.'s research results indicate a close correlation between plasma exosomal miR-17-5p and the occurrence of BC. It is suggested that plasma exosomal miR-17-5p may potentially inhibit the occurrence of BC through the MAPK signaling pathway, highlighting it as a novel candidate biomarker for diagnosing BC patients [ 39 ] . Chen and Li established a serum three-miRNA panel (AUC = 0.880) that enhances the diagnostic efficacy of breast invasive ductal carcinoma. Therefore, the serum three-miRNA panel, including miR-9-5p, miR-34b-3p, and miR-146a-5p, holds tremendous potential as non-invasive biomarkers for diagnosing breast invasive ductal carcinoma [ 40 ] . Based on the advantages of exosomes and previous research on exosomal miRNA in tumor diagnosis, we designed this study with the aim of identifying plasma exosomal miRNAs with higher sensitivity and specificity for diagnosing BC. We conducted differential analysis of exosomal miRNA sequencing results from the plasma of 45 BC patients and 5 healthy controls. As a result, we identified 3 upregulated miRNAs (miR-144-3p, miR-144-5p and miR-15b-5p) and 2 downregulated miRNAs (miR-125b-5p and miR-143-3p). Furthermore, when comparing BC patients at different stages, plasma exosomal miR-125b-5p expression is downregulated in stage II and stage III patients compared to stage I patients. The diagnostic capabilities of these 2 DEMs for BC were evaluated using ROC curves. Among them, exosomal miR-125b-5p and exosomal miR-143-3p had AUC values > 0.9, specifically 0.951 and 0.924, respectively. miR-125b-5p exhibited a sensitivity of 91.1% and specificity of 100%, while miR-143-3p showed a sensitivity of 86.1% and specificity of 100%. Subsequently, we predicted the target genes of miR-125b-5p and miR-143-3p using online target gene prediction tools, identifying 60 common target genes. We conducted pathway enrichment analysis for these 60 common target genes. The GO and KEGG analysis results also indicate that miR-125b-5p and miR-143-3p are associated with the occurrence of BC, and 9 potential target genes are also involved in BC occurrence. In the TCGA database, the expression of miR-125b-5p and miR-143-3p is significantly downregulated in BC tissues compared to normal tissues and the expression of miR-125b-5p is significantly decreased in advanced-stage breast cancer. STAT3 is an early tumor diagnostic marker known to promote the onset of BC [ 41 ] . IGF1R , a member of the receptor tyrosine kinase family, exhibits significantly elevated expression in BC cells, making it a promising candidate as an ideal biomarker for diagnosing BC [ 42 ] . There are also reports showing that the oncogenes ERBB2 [ 43 ] undergo focal amplification, which is involved in the occurrence of BC [ 44 ] . These results suggest that plasma exosomal miR-125b-5p and miR-143-3p have the potential to serve as diagnostic biomarkers for BC. While this study has identified plasma exosomal miR-125b-5p and miR-143-3p as having the potential for diagnosing BC, their specific impacts and mechanisms of action in breast cancer are not yet clear. Further experiments are needed to validate their roles. Additionally, it's worth noting that the sample size in this study was limited to 50 cases. To thoroughly assess the diagnostic capabilities of plasma exosomal miR-125b-5p and miR-143-3p for BC, it is essential to expand the study with a larger sample size. Conclusions Through miRNA sequencing analysis, we identified two promising plasma exosomal miRNAs, namely miR-125b-5p and miR-143-3p, that hold potential for diagnosing BC. By utilizing bioinformatics analysis, we successfully determined nine optimal target genes which are closely associated with the occurrence of BC. These differentially expressed miRNAs within plasma exosomes demonstrate significant potential as diagnostic biomarkers for BC. Abbreviations BC: breast cancer; miRNA: microRNA; DEMs: differentially expressed miRNAs; EP: eppendorf tube; SEC: size exclusion chromatography; NTA: nanoparticle tracking analysis; TEM: transmission electron microscopy; FC: fold change; FDR: False Discovery Rate ROC: Receiver Operating Characteristic AUC: Area Under the Curve GO: gene ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; PPI: protein-protein interaction MCC: Maximum Clique Centrality Declarations Ethics approval and consent to participate BC patient peripheral blood samples were obtained from the Breast Center Biobank at Beijing Hospital. The research protocol received approval from the Ethics Committee of Beijing Hospital following the guidelines of the Helsinki Declaration (IRB Ethics Approval Number: 2017BJYYEC-086-05), and written informed consent was obtained from all participating patients. Consent for publication Not applicable. Availability of data and materials Due to privacy/ethical constraints, data can be provided upon request. Competing interests The authors declare that they have no competing interests. Funding This work was supported by the National High Level Hospital Clinical Research Funding, Project No.: BJ-2019-191 and the National High Level Hospital Clinical Research Funding, Project No.: BJ-2023-171. Authors' contributions Hua Bin and conceived and designed the study. Fan Lei and Huang Jicheng analyzed the data. Fan Lei wrote the paper. Hua Bin and Li Yao revised and reviewed the manuscript. All authors have consented to the publication of this article. Acknowledgements Not applicable. 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The Value of Serum Exosomal miR-184 in the Diagnosis of NSCLC [J]. J Healthc Eng, 2022, 2022: 9713218. YANG Q, WEI B, PENG C, et al. Identification of serum exosomal miR-98-5p, miR-183-5p, miR-323-3p and miR-19b-3p as potential biomarkers for glioblastoma patients and investigation of their mechanisms [J]. Curr Res Transl Med, 2022, 70(1): 103315. JIN X, CHEN Y, CHEN H, et al. Evaluation of Tumor-Derived Exosomal miRNA as Potential Diagnostic Biomarkers for Early-Stage Non-Small Cell Lung Cancer Using Next-Generation Sequencing [J]. Clin Cancer Res, 2017, 23(17): 5311-9. LV S, WANG Y, XU W, et al. Serum Exosomal miR-17-5p as a Promising Biomarker Diagnostic Biomarker for Breast Cancer [J]. Clin Lab, 2020, 66(9). CHEN X, LI X, WANG J, et al. Breast invasive ductal carcinoma diagnosis with a three-miRNA panel in serum [J]. Biomark Med, 2021, 15(12): 951-63. MA J H, QIN L, LI X. Role of STAT3 signaling pathway in breast cancer [J]. Cell Commun Signal, 2020, 18(1): 33. XU H, ZHAO Y, GAO X, et al. An innovative fluorescent probe targeting IGF1R for breast cancer diagnosis [J]. Eur J Med Chem, 2021, 219: 113440. VAGHI C, MAURI G, AGOSTARA A G, et al. The predictive role of ERBB2 point mutations in metastatic colorectal cancer: A systematic review [J]. Cancer Treat Rev, 2023, 112: 102488. LEE E Y, MULLER W J. Oncogenes and tumor suppressor genes [J]. Cold Spring Harb Perspect Biol, 2010, 2(10): a003236. Additional Declarations No competing interests reported. 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 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-4276021","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":293019917,"identity":"6082b1ad-ef7a-48db-9014-d87447b5d7ea","order_by":0,"name":"Lei Fan","email":"","orcid":"","institution":"Beijing Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Lei","middleName":"","lastName":"Fan","suffix":""},{"id":293019918,"identity":"dd2feb8c-4404-40f0-9719-696d952e162b","order_by":1,"name":"Yao Li","email":"","orcid":"","institution":"Beijing Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yao","middleName":"","lastName":"Li","suffix":""},{"id":293019921,"identity":"295d3d22-4336-4a17-81ee-689a25c35d0f","order_by":2,"name":"Jicheng Huang","email":"","orcid":"","institution":"Beijing Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jicheng","middleName":"","lastName":"Huang","suffix":""},{"id":293019923,"identity":"8bdf2016-9a6a-46c1-af46-5ce7c1b3a3fc","order_by":3,"name":"Bin Hua","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyUlEQVRIiWNgGAWjYBACAwYGNoYEhgPMBuyNjQ8+kKaF53Cz4QyitTAwHGAwkEhvk+YgRou5RPKzBw933GE3l3zYIM3AYCen20BAi+WMNHODxDPPmC1nJzYYFzAkG5sdIOSwGwlmEolth5kNbic2JM9gOJC4jbCW9G8QLTcPNhzmIU5LDtSWG4yNzcRpOfOm3CCx7RmzwZnEZsYZBsT45Xj6toc/2+4kGxw//vzHhwo7OYJaYCAZagKRykHAjgS1o2AUjIJRMNIAAAhWSf+SISW4AAAAAElFTkSuQmCC","orcid":"","institution":"Beijing Hospital","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Bin","middleName":"","lastName":"Hua","suffix":""}],"badges":[],"createdAt":"2024-04-16 12:27:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4276021/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4276021/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":55509465,"identity":"2d04a65b-57ed-4ab0-9c78-d7f0f2eecb54","added_by":"auto","created_at":"2024-04-29 12:23:59","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":346423,"visible":true,"origin":"","legend":"\u003cp\u003eDiagnostic value of plasma exosomal miR-125b-5p and miR-143-3p (A) Volcano diagrams of the whole differentially expressed exosomal miRNAs. Blue and red dots represent down-regulated and up-regulated DEMs. (B) Heat map of the 5 differentially expressed plasma exosomal miRNAs had |log2FC| \u0026gt; 0.5 in BC. (C) The expression of miR-125b-5p and miR-143-3p in the plasma exosomes of healthy controls (HC）and breast cancer patients (BC）. (D)The expression of miR-125b-5p in the plasma exosomes of breast cancer patients at stage I and stage II-Ⅲ. (E) ROC curve analysis of plasma exosomal miR-125b-5p. (F) ROC curve analysis of plasma exosomal miR-143-3p. (*P \u0026lt;0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001)\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4276021/v1/68b29d44bfe852a30671227a.png"},{"id":55509467,"identity":"545cde6d-4091-4d42-9f4a-f1b12d12918f","added_by":"auto","created_at":"2024-04-29 12:24:00","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":297698,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of miR-125b-5p and miR-143-3p in the TCGA database. (A)The expression of miR-125b-5p and miR-143-3p in adjacent non-cancerous tissues (normal) and tumor tissues (tumor). (B) The expression of miR-125b-5p and miR-143-3p in the BC tissues of BC patients with stage T1 and stage T2-4. (C) The expression of miR-125b-5p in the BC tissues of stage I and stage II-III BC patients. (*P \u0026lt;0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001)\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4276021/v1/8bb345d66c71a16c6a9886cc.png"},{"id":55509466,"identity":"db983b10-95bf-418b-a55c-13dacfe00ac8","added_by":"auto","created_at":"2024-04-29 12:24:00","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":535231,"visible":true,"origin":"","legend":"\u003cp\u003ePrediction the potential target genes of two target DEMs. (A) Common target genes prediction of miR-125b-5p and miR-143-3p. (B) GO analyses of common target genes of miR-125b-5p and miR-143-3p. (C) KEGG analyses of common target genes of miR-125b-5p and miR-143-3p. (D) The PPI network of 60 common target genes. (E) According to the MCC algorithm, the top 10 common target genes were analyzed by CytoHubba plug-in. Different node colors represent the log FC value of the common target genes. (F) the most important functional module of MCODE network.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4276021/v1/8d7b0b9000cd4ad4990d968a.png"},{"id":55509463,"identity":"d66edc22-606d-4834-a43c-aaa3fa29481f","added_by":"auto","created_at":"2024-04-29 12:23:58","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":194356,"visible":true,"origin":"","legend":"\u003cp\u003eThe DEMs-target genes-pathways network represents the relationships among DEMs, common target genes and KEGG pathways. DEMs, common target genes are represented by a red ellipse, blue rectangle, while enrichment pathways are represented by an orange rectangle.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4276021/v1/cbc9b9a5b62a2b1521b72f6c.png"},{"id":55699221,"identity":"16a3674b-a827-4bef-b705-687c9a003ce4","added_by":"auto","created_at":"2024-05-02 02:42:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1105662,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4276021/v1/2e79b4b8-e4c9-4a0e-b6ad-ceaa8bc8e8d3.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Plasma exosomal miR-125b-5p and miR-143-3p have potential to diagnose breast cancer","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBC is the most common malignancy in women, the second leading cause of death in women, and the incidence of BC is showing an increasing trend year by year\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. Despite continuous advancements in the treatment of BC, a significant proportion of patients still succumb to the disease each year. Successful treatment of BC is highly dependent on early diagnosis. Early-stage BC patients have a 5-year survival rate of over 90%, while patients with distant metastasis have a 5-year survival rate of only 20%\u003csup\u003e[\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. Therefore, there is an urgent need for biomarkers with high sensitivity and specificity for early diagnosis of BC \u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTraditional biopsies are invasive and susceptible to factors like lesion location and operator experience. Moreover, conventional tumor markers and imaging examinations are limited by their low sensitivity and specificity in disease diagnosis. Liquid biopsy, a novel diagnostic technique emerging in recent years, involves the detection of components like exosomes, and so on. This approach has gained significant attention due to its non-invasiveness and ease of access.\u003c/p\u003e \u003cp\u003eExosomes, present in bodily fluids, serve as mediators for intercellular communication. They carry a diverse range of bioactive molecules, including non-coding RNA, miRNA, DNA, proteins, and others, playing crucial roles in promoting or inhibiting diseases, especially in cancer \u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. It has been reported that exosomes can be easily obtained through biofluid sampling, allowing for the maximization of their potential value in diagnosing tumors and other diseases\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMiRNA serves as a vital component of exosomes and plays a crucial role in important biological processes such as embryonic development, cell cycle regulation, proliferation differentiation, and apoptosis which may be as diagnosis markers for liquid biopsy \u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e.MiR-125b-5p is significantly downregulated in the exosomes of non-small cell lung cancer patients and serves as a diagnostic and prognostic biomarker for non-small cell lung cancer\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. It accurately distinguishes pancreatic cancer patients from healthy controls and acts as an independent biomarker predicting overall survival in pancreatic cancer patients\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. Compared to healthy endometrium, miR-125b-5p is downregulated in endometrial cancer, showing promise as a diagnostic biomarker for endometrial cancer\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. Meanwhile miR-143-3p is significantly downregulated in urinary exosomes of prostate cancer patients, presenting itself as a promising biomarker\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. Serum exosomal miR-143-3p has the potential to serve as a diagnostic biomarker for renal cell carcinoma\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. MiR-143-3p is significantly overexpressed in the serum of endometrial cancer patients and can be a potential non-invasive biomarker for endometrial cancer diagnosis \u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. Despite research on miR-125b-5p and miR-143-3p in various tumor diagnoses, their specific studies in BC diagnosis are currently lacking.\u003c/p\u003e \u003cp\u003eIn this study, we investigated differentially expressed miRNAs (DEMs) in plasma exosomes from BC patients and normal healthy controls. Our findings revealed significantly downward of miR-125b-5p and miR-143-3p in the plasma exosomes of BC patients. These miRNAs demonstrated potential as effective diagnostic markers for BC. We also predicted the target genes of these miRNAs and conducted pathway enrichment analysis, which provide valuable insights for future research directions.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePatients and plasma specimens\u003c/h2\u003e \u003cp\u003eIn this study, preoperative peripheral blood samples were collected from 45 BC patients receiving treatment at the Breast Center of Beijing Hospital. The clinical and pathological characteristics were presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. All of these patients had not undergone any relevant treatment before surgery and had their BC diagnosis confirmed through pathological examination. Furthermore, peripheral blood samples were collected from 5 healthy individuals. 5 milliliters of peripheral venous blood were extracted from each patient and subsequently underwent centrifugation. Specifically, the blood samples were centrifuged at 1500 g for 20 minutes, and the supernatant was aspirated into 15-milliliter centrifuge tubes. Next, the supernatant was centrifuged again at 3000 g for 15 minutes, and the upper layer of plasma was aliquoted into 2-milliliter eppendorf tube (EP) and stored at -80\u0026deg;C for further exosomes isolation research.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe plasma samples and the corresponding clinical data in the present study.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eclinical data\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNumbers\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eBreast cancer\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e45\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eAge(year)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u0026ge;\u0026thinsp;50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eTNM stage\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eⅠ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eⅡ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eⅢ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eT\u003c/p\u003e \u003cp\u003eT1\u003c/p\u003e \u003cp\u003eT2-4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e22\u003c/p\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eN0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eN1-3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eM0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e45\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eM1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eER\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e(+)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e(-)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003ePR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e(+)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e(-)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eHer-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e(+)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e(-)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e33\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eki-67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u0026ge;\u0026thinsp;15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003eAbbreviations:\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003eTNM stage: The Tumor, Node, Metastasis staging system;\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003eT: Staging of tumor;\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003eN: Staging of lymph nodes;\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003eM: Staging of metastasis;\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003eER: Estrogen receptor;\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003ePR: Progesterone Receptor;\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003eHer-2: Human Epidermal Growth Factor Receptor 2;\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003eKi-67: Antigen Identified by Monoclonal Antibody Ki-67.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e The entire research protocol has been approved by the Ethics Committee of Beijing Hospital in accordance with the principles of the \"Helsinki Declaration,\" and all participants provided written informed consent.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eIsolation and Identification of Plasma Exosomes\u003c/h2\u003e \u003cp\u003ePlasma exosomes underwent size exclusion chromatography (SEC) for separation. One milliliter of plasma was initially filtered through a 0.8 \u0026micro;m filter, followed by a 1.5-fold dilution with PBS and additional purification using an Exosupur column (Echobiotech, China). The elution of samples was conducted with PBS, and 2 mL eluates were gathered. Subsequently, the eluates were concentrated to 200 \u0026micro;L using Amicon Ultra centrifugal filters with a molecular weight cutoff of 100 kDa (Millipore, Germany).\u003c/p\u003e \u003cp\u003eExosomes were identified using two techniques: nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). Initially, vesicle suspensions at a concentration of around 1 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e/mL were analyzed with ZetaView PMX 110, featuring a 405 nm laser, to determine particle size and quantity. Twenty microliters of exosomes underwent NTA analysis (ZetaView 8.02.28) to assess particle motion. Subsequently, exosomes were resuspended in PBS and applied to copper grids for TEM observation. Following a 10-minute incubation at room temperature, negative staining with 1% uranyl acetate was performed for 10 minutes. Observation and imaging were carried out using a TEM (H-7650, Hitachi, Tokyo, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003ePlasma exosome-derived miRNA sequencing analysis\u003c/h2\u003e \u003cp\u003eThe RNA generated from each plasma exosome sample was extracted using the miRNeasy Kit (Qiagen, Germany). Subsequently, miRNA-seq was performed on the Illumina HiSeq platform. The miRNA-seq data were represented by unique molecular identifiers (UMIs). The screening criteria for DEMs was a median TPM (transcripts per million)\u0026thinsp;\u0026ge;\u0026thinsp;30, |log\u003csub\u003e2\u003c/sub\u003e fold change (FC)| \u0026gt; 0.5 and \u003cem\u003eFDR\u003c/em\u003e (False Discovery Rate)\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eCommon target genes of candidate DEMs prediction\u003c/h2\u003e \u003cp\u003eWe employed the online free databases TargetScan (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.targetscan.org/vert_80/\u003c/span\u003e\u003cspan address=\"https://www.targetscan.org/vert_80/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and miRTarBase (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.mirnet.ca/\u003c/span\u003e\u003cspan address=\"https://www.mirnet.ca/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) for predicting the target genes of each DEM\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. Finally, through the utilization of an online Venn diagram(\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://sangerbox.com/login.html)analysi\u003c/span\u003e\u003cspan address=\"http://sangerbox.com/login.html)analysi\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003es, we identified the overlapping genes as the common target genes by intersecting the target genes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003ePathway enrichment and Gene Ontology analysis of common target genes\u003c/h2\u003e \u003cp\u003eTo gain further insights into the potential molecular mechanisms and interactions of the common target genes, we utilized the 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) for protein-protein interaction (PPI) network analysis. In order to determine the specific biological functions of the target genes implicated in BC, we conducted GO and KEGG pathway enrichment analysis using the online platform David (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://david.ncifcrf.gov/summary.jsp\u003c/span\u003e\u003cspan address=\"https://david.ncifcrf.gov/summary.jsp\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eScreening for potential target genes\u003c/h2\u003e \u003cp\u003eUpon importing the PPI network file of the common target genes into Cytoscape software, we applied the CytoHubba plug-in to calculate the Maximum Clique Centrality (MCC) algorithm, which enabled us to identify the top 10 genes as hub genes. Simultaneously, we used the MCODE plugin to identify critical functional modules among the shared target genes. Genes that are present in both hub genes and key functional modules are considered potential target genes of the DEMs \u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eSPSS 25.0 software (Chicago, IL, USA) was used for statistical analysis and GraphPad Prism 9.0 was used for plotting and graphing, and \u003cem\u003eMann-Whitney U-test\u003c/em\u003e was used to test the expression levels of exosomal miRNA in the plasma of BC patients and healthy controls. The ROC curve was used to evaluate the diagnostic ability of the candidate exosomal DEMs for BC, and if the AUC value\u0026thinsp;\u0026gt;\u0026thinsp;0.7, it showed better diagnostic and predictive ability. \u003cem\u003eP\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eThe expression of miR-125b-5p and miR-143-3p is significantly decreased in the plasma exosomes from BC patients.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo screen for exosomal miRNAs associated with BC occurrence, we conducted miRNA sequencing on exosomes from the plasma of 45 BC patients and 5 healthy controls. Five miRNAs exhibited differential expression in the plasma exosomes of BC patients compared to healthy controls. The screening criteria was median TPM\u0026thinsp;\u0026ge;\u0026thinsp;30, |log\u003csub\u003e2\u003c/sub\u003eFC| \u0026gt; 0.5, \u003cem\u003eFDR\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Among them, three were upregulated, including miR-144-3p, miR-144-5p, and miR-15b-5p, while two were downregulated, namely miR-125b-5p and miR-143-3p. DEMs were visualized using volcano map and heatmap (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-B). Among them, miR-125b-5p and miR-143-3p were most significantly downregulated in plasma exosomes of BC patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). We further compared the expression levels of plasma exosomal miR-125b-5p in breast cancer patients at different TNM stages. The results revealed a significant decrease in the levels of plasma exosomal miR-125b-5p in stage II and stage III BC patients compared to stage I patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePlasma exosomal miRNA differentially expressed between breast cancer patients and healthy controls.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLog\u003csub\u003e2\u003c/sub\u003eFC\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eFDR\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003emiR-144-3p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.833676769\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.015436401\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003emiR-144-5p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.733625073\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.040196407\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003emiR-15b-5p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.607799216\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.042665855\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003emiR-125b-5p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-0.726766996\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.002324052\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003emiR-143-3p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-0.688851409\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.010703674\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003eAbbreviations:\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003eFC: fold change\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eThe value of plasma exosomal miR-125b-5p and miR-143-3p in the diagnosis of BC\u003c/h2\u003e \u003cp\u003ewe plotted ROC curves using the expression levels of plasma exosomal miR-125b-5p and miR-143-3p to assess their reliability in diagnosing BC (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE-F). The results indicate that plasma exosomal miR-125b-5p and miR-143-3p exhibit high diagnostic accuracy in distinguishing BC patients from healthy controls (AUC\u0026thinsp;\u0026gt;\u0026thinsp;0.9). The AUC value for plasma exosomal miR-125b-5p is 0.951 (95% CI: 0.891-1, sensitivity: 91.1%, specificity: 100%), and for plasma exosomal miR-143-3p, the AUC value is 0.924 (95% CI: 0.847-1, sensitivity: 86.7%, specificity: 100%). According to our analysis, plasma exosomal miR-125b-5p and miR-143-3p have the potential to serve as non-invasive biomarkers for diagnosing BC.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of miR-125b-5p and miR-143-3p in the TCGA database\u003c/h2\u003e \u003cp\u003eIn the TCGA database, as shown in the Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, the expression of miR-125b-5p and miR-143-3p in BC tissues is significantly downregulated compared to adjacent normal tissues, consistent with the differential expression in plasma exosomes. Subsequent analysis explored the relationship between the pathological characteristics of BC patients and the expression levels of these two miRNAs. Compared to patients at the T1 stage, the expression of miR-125b-5p and miR-143-3p is significantly decreased in tissues of patients at T2-4 stage (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Additionally, the expression of miR-125-5p decreases with the progression of BC (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003ePotential target genes and pathway enrichment analysis of plasma exosomal miR-125b-5p and miR-143-3p\u003c/h2\u003e \u003cp\u003eUtilizing TargetScan and miRTarBase databases, we predicted a total of 60 common target genes. Specifically, miR-125b-5p targets 30 genes, while miR-143-3p targets 32 genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Using clusterProfiler, we employed GO functional annotation and KEGG pathway enrichment analysis to investigate the biological roles of the shared target genes of the two miRNAs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB-C). The GO functional annotation revealed their involvement in processes like \" positive regulation of cell proliferation\" and \" positive regulation of cell growth\" in the BP category, \" ERBB3:ERBB2 complex\" and \"caveola\" in the CC category, and functions such as \" growth factor binding \" and \" transmembrane receptor protein tyrosine kinase activity \" in the MF category. The ERBB3/ERBB2 heterodimer can activate multiple signaling pathways, including MAPK/ERK and PI3K/AKT, thereby promoting the occurrence of BC\u003csup\u003e[\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. A series of growth factors and their receptors participate in the occurrence and development of BC. Receptor Tyrosine Kinases are a class of receptors that play a crucial role in the occurrence of BC\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eKEGG pathway analysis identified enrichment in eight signaling pathways, including \"PI3K-Akt signaling pathway\", \" Hippo signaling pathway\", \"HIF\u0026thinsp;\u0026minus;\u0026thinsp;1 signaling pathway\", \" Adherens junction\", \"Focal adhesion\", \" MAPK signaling pathway\", \" ErbB signaling pathway\" and \" NF-kappa B signaling pathway \".The Hippo signaling pathway plays a crucial role in the occurrence of BC, and the overexpression of its core component YAP has been demonstrated to promote the growth of BC cells both in vitro and in vivo\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. The PI3K-Akt signaling pathway has been confirmed to have a major oncogenic role in BC, and its activation can also stimulate the proliferation of BC cells\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. In BC, the activation of the NF-κB pathway can drive continuous cell cycle progression, leading to uncontrolled cell proliferation\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eCommon target gene interaction analysis and potential target gene identification\u003c/h2\u003e \u003cp\u003eThe PPI network is used to elucidate the protein interactions among common target genes. (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Importing the PPI network file into Cytoscape software and applying the CytoHubba plug-in with the MCC algorithm, we highlighted the top 10 common target genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Additionally, utilizing the MCODE plugin with specific parameters (degree cutoff\u0026thinsp;\u0026ge;\u0026thinsp;2, node score cutoff\u0026thinsp;\u0026ge;\u0026thinsp;0.2, k-core\u0026thinsp;\u0026ge;\u0026thinsp;2 and max depth\u0026thinsp;=\u0026thinsp;100), we pinpointed a significant functional module comprising 9 genes: \u003cem\u003eERBB2\u003c/em\u003e, \u003cem\u003ePDGFRA\u003c/em\u003e, \u003cem\u003eBCL2\u003c/em\u003e, \u003cem\u003eSERPINE1\u003c/em\u003e, \u003cem\u003eCCN2\u003c/em\u003e, \u003cem\u003ePTGS2\u003c/em\u003e, \u003cem\u003eSMAD3\u003c/em\u003e, \u003cem\u003eIGF1R\u003c/em\u003e and \u003cem\u003eSTAT3\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Importantly, all 9 genes in this functional module overlapped with the top 10 genes in the PPI network, underscoring their crucial role in the network. Therefore, these genes are considered potential target genes of the DEMs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eConstructed the DEMs-target genes-pathways network\u003c/h2\u003e \u003cp\u003eWe constructed a network (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) encompassing DEMs, target genes (including common, potential, and optimal target genes), and KEGG pathways to explore their regulatory connections. Among the 9 optimal target genes, miR-125b-5p regulates \u003cem\u003eERBB2\u003c/em\u003e and \u003cem\u003eSTAT3\u003c/em\u003e, while miR-143-3p regulates \u003cem\u003eCCN2\u003c/em\u003e, \u003cem\u003eSERPINE1\u003c/em\u003e, \u003cem\u003ePDGFRA\u003c/em\u003e, \u003cem\u003ePTGS2\u003c/em\u003e, \u003cem\u003eSMAD3\u003c/em\u003e and \u003cem\u003eIGF1R\u003c/em\u003e, \u003cem\u003eBCL2\u003c/em\u003e is simultaneously regulated by both miR-125b-5p and miR-143-3p. Specifically, \" PI3K-Akt signaling pathway \" involves \u003cem\u003ePDGFRA\u003c/em\u003e, \u003cem\u003eERBB2\u003c/em\u003e, \u003cem\u003eBCL2\u003c/em\u003e and \u003cem\u003eIGF1R\u003c/em\u003e, \"HIF\u0026thinsp;\u0026minus;\u0026thinsp;1 signaling pathway\" involves \u003cem\u003eERBB2\u003c/em\u003e, \u003cem\u003eSTAT3\u003c/em\u003e, \u003cem\u003eSERPINE1\u003c/em\u003e, \u003cem\u003eBCL2\u003c/em\u003e and \u003cem\u003eIGF1R\u003c/em\u003e, \" Hippo signaling pathway \" involves \u003cem\u003eSMAD3\u003c/em\u003e, \u003cem\u003eSERPINE1\u003c/em\u003e and \u003cem\u003ePDGFRA\u003c/em\u003e, \"Adherens junction\" involves \u003cem\u003eSMAD3\u003c/em\u003e, \u003cem\u003eERBB2\u003c/em\u003e and \u003cem\u003eIGF1R\u003c/em\u003e, \"MAPK signaling pathway \" involves \u003cem\u003ePDGFRA\u003c/em\u003e, \u003cem\u003eERBB2\u003c/em\u003e and \u003cem\u003eIGF1R\u003c/em\u003e, \" ErbB signaling pathway \" involves \u003cem\u003eERBB2\u003c/em\u003e, \" NF-kappa B signaling pathway \" involves \u003cem\u003eBCL2\u003c/em\u003e and \u003cem\u003ePTGS2\u003c/em\u003e and \"Focal adhesion\" involves \u003cem\u003ePDGFRA\u003c/em\u003e, \u003cem\u003eERBB2\u003c/em\u003e, \u003cem\u003eBCL2\u003c/em\u003e and \u003cem\u003eIGF1R\u003c/em\u003e. The ceRNA network reveals that the target genes of miR-125b-5p and miR-143-3p, along with the enriched pathways, are closely associated with the occurrence of BC.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eBC is the most common type of cancer in women, posing a significant threat to women's health. Its incidence has been steadily increasing, and the current indicators used for diagnosing BC have certain limitations \u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. There is an urgent need to find effective markers for accurate early diagnosis for BC \u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. Exosomes, as emerging stars in liquid biopsy, exhibit unique advantages in the diagnosis of various cancers\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. Firstly, exosomes can be produced by various cells and are found in various body fluids, such as blood, urine\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e, breast milk\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e, saliva\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e, cerebrospinal\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e fluid, and tears. Secondly, exosomes are abundant and easily accessible; there are approximately 10\u003csup\u003e12\u003c/sup\u003e exosomes in 1 ml of blood, and various techniques like ultracentrifugation, ultrafiltration, and exosome isolation kits are widely used for exosome extraction. Thirdly, exosomes contain a rich cargo, including nucleic acids, proteins, metabolites, etc.\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e, and these materials can be transferred between cells via exosomes. Lastly, exosomes exhibit strong stability, as their lipid bilayer protects both the exosomes and their cargo from degradation, allowing them to circulate stably in the body\u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e. These advantages make exosomes promising candidates as biomarkers for cancer diagnosis.\u003c/p\u003e \u003cp\u003eAn increasing body of research suggests that exosomal miRNAs play a role in tumorigenesis, offering new prospects for identifying potential biomarkers for tumor diagnosis. Li et al.'s study reveals that the expression levels of miR-184 in serum exosomes are significantly elevated in non-small cell lung cancer patients. This miRNA has value in distinguishing the nature of pulmonary diseases (benign or malignant) and is closely associated with patient prognosis\u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e.Yang et al.'s research findings indicate that exosomal miRNAs, including miR-183-5p, miR-19b-3p, and miR-323a-3p, can be employed for diagnosing gliomas. Their impact on the malignant characteristics of glioma cells may be attributed to their negative regulation of \u003cem\u003eMAPK8IP1\u003c/em\u003e/\u003cem\u003eFAM175B\u003c/em\u003e, \u003cem\u003eOSMR\u003c/em\u003e/\u003cem\u003eCASP3\u003c/em\u003e, \u003cem\u003eFBXO32\u003c/em\u003e, and \u003cem\u003ePTPN2\u003c/em\u003e, respectively\u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e. Ren and their team's study identifies three serum exosomal miRNAs for gastric cancer diagnosis, namely miR-1273g-3p, miR-4793-3p, and miR-619-5p. Through bioinformatic analysis, eight target genes were further determined. Among these, serum exosomal miR-1273g-3p/miR-4793-3p were found to target \u003cem\u003eMELK\u003c/em\u003e, and miR-619-5p was found to target \u003cem\u003eNCAPG2\u003c/em\u003e, which are believed to represent novel mechanisms involved in gastric cancer development\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. Jin's research results demonstrate that exosomal miRNAs, including miR-181-5p, miR-30a-3p, miR-30e-3p, and miR-361-5p, as well as exosomal miR-10b-5p, miR-15b-5p, and miR-320b, hold promise as prospective candidates for developing highly sensitive and specific non-invasive biomarkers for early Non-Small Cell Lung Carcinoma diagnosis\u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e. Lv et al.'s research results indicate a close correlation between plasma exosomal miR-17-5p and the occurrence of BC. It is suggested that plasma exosomal miR-17-5p may potentially inhibit the occurrence of BC through the MAPK signaling pathway, highlighting it as a novel candidate biomarker for diagnosing BC patients\u003csup\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e. Chen and Li established a serum three-miRNA panel (AUC\u0026thinsp;=\u0026thinsp;0.880) that enhances the diagnostic efficacy of breast invasive ductal carcinoma. Therefore, the serum three-miRNA panel, including miR-9-5p, miR-34b-3p, and miR-146a-5p, holds tremendous potential as non-invasive biomarkers for diagnosing breast invasive ductal carcinoma\u003csup\u003e[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eBased on the advantages of exosomes and previous research on exosomal miRNA in tumor diagnosis, we designed this study with the aim of identifying plasma exosomal miRNAs with higher sensitivity and specificity for diagnosing BC. We conducted differential analysis of exosomal miRNA sequencing results from the plasma of 45 BC patients and 5 healthy controls. As a result, we identified 3 upregulated miRNAs (miR-144-3p, miR-144-5p and miR-15b-5p) and 2 downregulated miRNAs (miR-125b-5p and miR-143-3p). Furthermore, when comparing BC patients at different stages, plasma exosomal miR-125b-5p expression is downregulated in stage II and stage III patients compared to stage I patients. The diagnostic capabilities of these 2 DEMs for BC were evaluated using ROC curves. Among them, exosomal miR-125b-5p and exosomal miR-143-3p had AUC values\u0026thinsp;\u0026gt;\u0026thinsp;0.9, specifically 0.951 and 0.924, respectively. miR-125b-5p exhibited a sensitivity of 91.1% and specificity of 100%, while miR-143-3p showed a sensitivity of 86.1% and specificity of 100%. Subsequently, we predicted the target genes of miR-125b-5p and miR-143-3p using online target gene prediction tools, identifying 60 common target genes. We conducted pathway enrichment analysis for these 60 common target genes. The GO and KEGG analysis results also indicate that miR-125b-5p and miR-143-3p are associated with the occurrence of BC, and 9 potential target genes are also involved in BC occurrence. In the TCGA database, the expression of miR-125b-5p and miR-143-3p is significantly downregulated in BC tissues compared to normal tissues and the expression of miR-125b-5p is significantly decreased in advanced-stage breast cancer.\u003c/p\u003e \u003cp\u003e \u003cem\u003eSTAT3\u003c/em\u003e is an early tumor diagnostic marker known to promote the onset of BC\u003csup\u003e[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e. \u003cem\u003eIGF1R\u003c/em\u003e, a member of the receptor tyrosine kinase family, exhibits significantly elevated expression in BC cells, making it a promising candidate as an ideal biomarker for diagnosing BC\u003csup\u003e[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/sup\u003e. There are also reports showing that the oncogenes \u003cem\u003eERBB2\u003c/em\u003e\u003csup\u003e[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/sup\u003e undergo focal amplification, which is involved in the occurrence of BC\u003csup\u003e[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]\u003c/sup\u003e. These results suggest that plasma exosomal miR-125b-5p and miR-143-3p have the potential to serve as diagnostic biomarkers for BC.\u003c/p\u003e \u003cp\u003eWhile this study has identified plasma exosomal miR-125b-5p and miR-143-3p as having the potential for diagnosing BC, their specific impacts and mechanisms of action in breast cancer are not yet clear. Further experiments are needed to validate their roles. Additionally, it's worth noting that the sample size in this study was limited to 50 cases. To thoroughly assess the diagnostic capabilities of plasma exosomal miR-125b-5p and miR-143-3p for BC, it is essential to expand the study with a larger sample size.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThrough miRNA sequencing analysis, we identified two promising plasma exosomal miRNAs, namely miR-125b-5p and miR-143-3p, that hold potential for diagnosing BC. By utilizing bioinformatics analysis, we successfully determined nine optimal target genes which are closely associated with the occurrence of BC. These differentially expressed miRNAs within plasma exosomes demonstrate significant potential as diagnostic biomarkers for BC.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eBC: breast cancer;\u003c/p\u003e\n\u003cp\u003emiRNA: microRNA;\u003c/p\u003e\n\u003cp\u003eDEMs: differentially expressed miRNAs;\u003c/p\u003e\n\u003cp\u003eEP: eppendorf tube;\u003c/p\u003e\n\u003cp\u003eSEC: size exclusion chromatography;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNTA: nanoparticle tracking analysis;\u003c/p\u003e\n\u003cp\u003eTEM: transmission electron microscopy;\u003c/p\u003e\n\u003cp\u003eFC: fold change;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFDR: False Discovery Rate\u003c/p\u003e\n\u003cp\u003eROC: Receiver Operating Characteristic\u003c/p\u003e\n\u003cp\u003eAUC: Area Under the Curve\u003c/p\u003e\n\u003cp\u003eGO: gene ontology;\u003c/p\u003e\n\u003cp\u003eKEGG: Kyoto Encyclopedia of Genes and Genomes;\u003c/p\u003e\n\u003cp\u003ePPI: protein-protein interaction\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMCC: Maximum Clique Centrality\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBC patient peripheral blood samples were obtained from the Breast Center Biobank at Beijing Hospital. The research protocol received approval from the Ethics Committee of Beijing Hospital following the guidelines of the Helsinki Declaration (IRB Ethics Approval Number: 2017BJYYEC-086-05), and written informed consent was obtained from all participating patients.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDue to privacy/ethical constraints, data can be provided upon request.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National High Level Hospital Clinical Research Funding, Project No.: BJ-2019-191\u0026nbsp;and the National High Level Hospital Clinical Research Funding, Project No.: BJ-2023-171.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHua Bin and conceived and designed the study. Fan Lei and Huang Jicheng analyzed the data. Fan Lei wrote the paper. Hua Bin and Li Yao revised and reviewed the manuscript. All authors have consented to the publication of this article.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGIAQUINTO A N, SUNG H, MILLER K D, et al. Breast Cancer Statistics, 2022 [J]. CA Cancer J Clin, 2022, 72(6): 524-41.\u003c/li\u003e\n\u003cli\u003eYIN W B, YAN M G, FANG X, et al. Circulating circular RNA hsa_circ_0001785 acts as a diagnostic biomarker for breast cancer detection [J]. Clin Chim Acta, 2018, 487: 363-8.\u003c/li\u003e\n\u003cli\u003eASADIRAD A, KHODADADI A, TALAIEZADEH A, et al. Evaluation of miRNA-21-5p and miRNA-10b-5p levels in serum-derived exosomes of breast cancer patients in different grades [J]. 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Cold Spring Harb Perspect Biol, 2010, 2(10): a003236.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"exosomes, microRNA, breast cancer, occurrence, diagnosis","lastPublishedDoi":"10.21203/rs.3.rs-4276021/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4276021/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eBreast cancer (BC) is the most common malignancy in women, posing a serious health risk. Exosomal microRNA (miRNA) has emerged as a promising cancer biomarker. Despite its potential, research on exosomal miRNA in BC remains limited. This study aims to identify plasma exosomal miRNAs for diagnosing BC.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eFirstly, plasma exosomal miRNA sequencing was conducted on 45 BC patients and 5 healthy controls, followed by Differential Expression Analysis to screen for differentially expressed miRNAs. The diagnostic value of these miRNAs was assessed using receiver operating characteristic (ROC) curves. Subsequent analysis of candidate miRNAs was carried out in the TCGA database. Target genes of differentially expressed miRNAs were predicted using online databases, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed. Finally, a ceRNA network was constructed using Cytoscape.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eOur analysis indicates that, compared to normal controls, plasma exosomal miR-125b-5p and miR-143-3p are significantly downregulated in BC patients. The area under the curve (AUC) for exosomal miR-125b-5p is 0.951 (sensitivity 91.1%, specificity 100%), and the AUC for exosomal miR-143-3p is 0.924 (sensitivity 86.7%, specificity 100%). In the TCGA database, the expression of miR-125b-5p and miR-143-3p is downregulated in BC tissues compared to adjacent normal tissues. Target gene prediction and enrichment analyses reveal pathways closely associated with BC occurrence.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eOur results demonstrate that plasma exosomal miR-125b-5p and miR-143-3p can distinguish between BC patients and normal controls. The downregulation of plasma exosomal miR-125b-5p and miR-143-3p may potentially serve as a marker for the occurrence of BC.\u003c/p\u003e","manuscriptTitle":"Plasma exosomal miR-125b-5p and miR-143-3p have potential to diagnose breast cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-29 12:22:48","doi":"10.21203/rs.3.rs-4276021/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":"446c818d-eeed-4195-827c-45a37c09ab9d","owner":[],"postedDate":"April 29th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-05-02T02:41:37+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-29 12:22:48","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4276021","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4276021","identity":"rs-4276021","version":["v1"]},"buildId":"zQwnuV7TCBrMSSSToR1PI","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}