The Role of the circDUSP1-Mediated miR-429/DLC1 Signaling Axis in the Proliferation, Migration, and Invasion of TNBC Cells

The Role of the circDUSP1-Mediated miR-429/DLC1 Signaling Axis in the Proliferation, Migration, and Invasion of TNBC Cells | 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 Article The Role of the circDUSP1-Mediated miR-429/DLC1 Signaling Axis in the Proliferation, Migration, and Invasion of TNBC Cells Canhui Jian, Xiaoxue Tian, Shuai Luo, Jiafei Zeng, Jin Li, Ting Xu, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6147334/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 20 Jul, 2025 Read the published version in Scientific Reports → Version 1 posted 12 You are reading this latest preprint version Abstract TNBC is a highly invasive and heterogeneous breast cancer subtype, lacking specific targeted therapies and linked to high morbidity and mortality rates. In recent years, circular RNA (circRNA), a significant non-coding RNA, has garnered substantial attention in cancer research. Through the competing endogenous RNA (ceRNA) mechanism, circRNAs can modulate miRNA activity, thereby influencing downstream gene expressions. This study investigated the expression profiles and biological roles of circDUSP1 in TNBC, demonstrating that circDUSP1 functions as a molecular sponge, adsorbing miR-429 and subsequently modulating the its target gene expression, DLC1, which suppresses the malignant progression of TNBC. These findings indicate that circDUSP1 holds promise as both a biomarker and a therapeutic target in TNBC. Biological sciences/Cancer Biological sciences/Cell biology TNBC triple-negative breast cancer circDUSP1 miR‐429 DLC1 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Breast cancer ranks among the most prevalent malignancies affecting women globally, with its incidence steadily rising, posing a significant threat to women's health. TNBC is a distinct and heterogeneous subtype lacking the three primary targets: estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) [ 1 ] . Consequently, patients do not benefit from existing endocrine and targeted therapies. Chemotherapy remains the primary treatment for TNBC [ 2 ] , yet its effectiveness is limited, and prognosis remains poor. Recently, the combination of cytotoxic drugs with immune checkpoint inhibitors and antibody-drug conjugates has emerged as a promising treatment avenue. [ 2 ] However, no targeted drugs for TNBC have received approval from the U.S. Food and Drug Administration (FDA). Thus, investigating the molecular mechanisms of TNBC and identifying new biomarkers and targets are crucial for enhancing patient survival and prognosis, offering innovative treatment strategies. Circular RNA (circRNA), a novel class of non-coding RNA, plays a crucial regulatory role in cancer development. Formed from pre-mRNA via back splicing, circRNAs possess a closed-loop structure and are abundant in miRNA binding sites [ 3 ] . Dysregulation of circRNA can promote tumorigenesis and metastasis, making it a promising target for cancer therapy [ 4 ] . Numerous studies have linked circRNA to tumor cell proliferation, apoptosis, and metastasis [ 5 ] . Notably, circRNAs exhibit high stability and specificity and can modulate miRNA activity through the competing endogenous RNA (ceRNA) mechanism [ 4 ] , influencing downstream gene expression [ 6 ] . circDUSP1 (hsa_circ_0075043), originating from exon 4 of the dual-specificity phosphatase 1 (DUSP1) gene, is a circular transcript of 1043 nucleotides formed by back splicing. Research indicates that circDUSP1 expression is reduced in TNBC (TNBC) and inversely correlates with TNBC tumorigenesis and progression [ 7 ] . MicroRNAs (miRNAs) are a class of conserved endogenous non-coding single-stranded RNAs, approximately 18–25 nucleotides in length, prevalent in eukaryotes and highly conserved across species. miRNAs regulate post-transcriptional gene expression by binding to the 3' untranslated region (3'UTR) of target mRNAs through base pairing. They function as oncogenes or tumor suppressors [ 8 ] , influencing tumor cell survival, proliferation, invasion, metastasis, apoptosis, and drug response [ 9 ] . Consequently, miRNAs hold significant potential for early tumor diagnosis, prognosis, and therapeutic target development. Our previous research indicates that miR-429 is markedly overexpressed in TNBC, enhancing TNBC cell proliferation, migration, and invasion by targeting DLC1. DLC 1 (Deleted in Liver Cancer 1) is a crucial tumor suppressor gene [ 10 ] within the RhoGTP enzyme-activating protein (RhoGAP) family, known for inhibiting RhoGTP enzyme activity [ 11 ] . As a GTP enzyme-activating protein, DLC 1 is pivotal in processes like cell migration and proliferation by terminating RhoGTP enzyme signaling and regulating cytoskeletal reorganization and RhoGTPase pathways [ 12 ] . Beyond its role as a tumor biomarker, DLC 1 is a promising therapeutic target with significant clinical implications. Our previous research demonstrated that DLC 1 is underexpressed in TNBC and is regulated by miR-429. Based on the ceRNA mechanism hypothesis, circDUSP1 might upregulate DLC 1 by sponging miR-429. Currently, the specific interactions among circDUSP1, miR-429, and DLC 1 remain unexplored. This study aims to elucidate the mechanisms involving circDUSP1, miR-429, and DLC 1 in TNBC using molecular techniques, offering new molecular targets for TNBC prediction and treatment. The study demonstrates that circDUSP1 expression is reduced in TNBC, facilitating the proliferation, migration, and invasion of TNBC cells in vitro and enhancing tumor growth in vivo. Mechanistic investigations reveal that circDUSP1 acts as a sponge for miR-429, thereby upregulating the tumor suppressor gene DLC1. Consequently, circDUSP1 may serve as both a prognostic biomarker and a potential therapeutic target for TNBC. Methods Tissue samples Freshly resected tissue specimens were obtained from three patients diagnosed with TNBC, with pathologic confirmation, at the Affiliated Hospital of Zunyi Medical University. The samples included three pairs of cancerous and adjacent normal breast tissues. All patients were newly diagnosed with breast cancer and had not undergone prior radiotherapy or chemotherapy. Informed consent was obtained from each participant before surgery, and the study received approval from the hospital’s Ethics Committee. Post-surgical samples were immediately stored in liquid nitrogen. Cell culture Human mammary epithelial cells (MCF-10A), TNBC cells (MDA-MB-231 and MDA-MB-468), and 293T cells were sourced from Procell. MCF-10A cells were cultured in a specialized MCF-10A medium (Procell, China), while MDA-MB-231 and MDA-MB-468 cells were maintained in DMEM containing 10% FBS (Procell, China). Cell lines were cultured in an incubator with 5% CO2 at 37°C.s were incubated at 37°C with 5% CO 2 . Cell transfection Lentiviral transfection was performed using knockdown lentiviruses (Lv-NC-sh, Lv-hsa-circ-0075043-sh) and overexpression lentiviruses (Lv-NC-oe, Lv-hsa-circ-0075043-oe), synthesized by Fenghui Biotechnology Co., Ltd. Twenty-four hours before transfection, adherent cells were plated in 6-well plates at 1×105 cells per well and incubated overnight under standard conditions (37°C, 5% CO2). After reaching 50% confluence, the medium was replaced with 2 mL fresh medium (0.5 µg/mL polybrene), and lentiviral solution was added. The cells were then incubated under the same conditions for 48 hours. Afterward, the viral-containing medium was replaced with fresh culture medium, and successful transfection was confirmed by the appearance of green fluorescence in over 80% of the cells under a fluorescence microscope. At this point, the medium was replaced with culture medium supplemented with 2 µg/mL puromycin, with daily observation and medium replacement. Once all cells in the blank control group had died, the infected cells were expanded to establish a stable transfected cell line. The RNA of hsa-miR-429 mimics, hsa-miR-429 inhibitors, and their respective negative controls (NC) were synthesized by Sangon Biotech Co., Ltd. (Shanghai). TNBC cell lines with stable knockdown or overexpression of hsa-circ-0075043 (circDUSP1) were seeded into 6-well plates and incubated for 24 hours. Subsequently, two 1.5 ml sterile EP tubes were prepared: 100 µl of serum-free medium was added to each, with 100 nm RNA placed in one tube and 12 µl of enhanced transfection reagent (Biodragon, China) in the other. After 5 minutes of incubation, the contents of both tubes were combined and allowed to stand for 20 minutes. The resulting mixture was then added to the appropriate (0.2 ml) wells of the 6-well plate for continued incubation. Cells were subsequently harvested for expression analysis and functional assays. Quantitative real-time PCR (qRT-PCR) Total RNA was extracted using the Beyotime kit (China), then reverse-transcribed into cDNA with Takara's PrimeScript Reverse Transcription Kit (China). cDNA amplification was carried out using either the TB Green PCR Kit (Takara, China) or the TB Green Master Mix Kit (Takara, China). GAPDH (circDUSP1 and DLC1) and U6 (miR-429) served as internal controls to assess the expressions of circDUSP1, miR-429 and DLC1 mRNA. The 2-ΔΔCt method was employed to obtain relative expression. Table 1 showed primer sequences. RNase R digestion Total RNA was extracted using the RNA extraction kit, and the RNA was divided into two equal aliquots (1 µg/aliquot). One aliquot was left untreated, while RNase R (Epicentre, USA) was added to the other, with incubation at 37°C for 20 minutes for digestion. Both undigested and digested RNA samples were analyzed via qRT-PCR to quantify the expression levels of circDUSP1 and DUSP1. Genes from the control group were used as internal references, and relative gene expression was assessed by the 2-ΔCT method. Nucleocytoplasmic separation assay Cell pellets were harvested, and nuclear and cytoplasmic fractions were isolated using a nuclear protein extraction kit (Solarbio, China). Total RNA was subsequently extracted, and the expression of circDUSP1 in both the nucleus and cytoplasm was quantified via qRT-PCR. KRT19P3 and GAPDH were employed as internal controls for the nucleus and cytoplasm, respectively. The target gene's relative expression was obtained in each compartment (2-ΔCT method). Extraction of cell genomic DNA (gDNA) g-DNA was extracted by an animal tissue/cell g-DNA extraction kit (Solarbio, China). CircDUSP1, DUSP1, and GAPDH expression were analyzed via qRT-PCR. Divergent primers for circDUSP1 were designed and synthesized by Sangon Biotech Co., Ltd. (Shanghai), with primer sequences provided in Table 1. Agarose gel electrophoresis Agarose gel electrophoresis The cDNA and gDNA amplification products from the preceding experiment were analyzed via agarose gel electrophoresis. A 40 mL solution of 3% agarose gel was prepared and allowed to cool naturally to approximately 60°C. Nucleic acid dye was then added at a 1:5000 ratio, mixed thoroughly, and the solution poured into a gel casting tray with a comb, where it was left to solidify at room temperature. After the gel solidified, the comb was removed, and the gel was transferred to an electrophoresis chamber filled with 1×TBE buffer. The 6× loading buffer was diluted to 1×, and the PCR products were further diluted 1:1 with the buffer. Both the marker and the diluted samples were loaded into the gel wells. Electrophoresis was initiated immediately after loading. Upon completion, the gel was visualized and photographed using a UV gel imaging system. FISH CircDUSP1 and miR-429 FISH probes (Shanghai) were designed and synthesized by GenePharma Pharmaceutical Co., Ltd. (Shanghai), with the probe sequences detailed in Table 2. Localization of circDUSP1 in TNBC cells was assessed using the RNA FISH kit (GenePharma). Briefly, fixed and permeabilized cell slides were prehybridized at 55°C for 2 hours. The probe and hybridization solution were denatured in a PCR instrument, and the denatured mixture was applied to the cell slides for 24 hours of hybridization. Following this, cell nuclei were stained with DAPI and observed under a fluorescence microscope. CCK-8 assay Stable transfected TNBC cell lines (2 × 103 cells/well) were seeded in 96-well plates, following by assessment of cell proliferation through CCK-8 reagent (GLPBIO, China) at 0, 24, 48, 72, and 96 hours. The fresh medium containing 10% CCK-8 solution was utilized. Following a 90-minute incubation at 37°C in the dark, absorbance at 450 nm was measured with an ELISA reader. Colony Formation Assay Stably transfected TNBC cells lines were seeded into 6-well plates at a density of 600 cells per well, followed by the addition of 2 mL complete culture medium with thorough mixing. The plates were then incubated in a humidified cell culture incubator. The culture medium was replaced with fresh complete medium every 3 days. Visible colonies emerged 13 days post-seeding. The colonies were gently washed with PBS and fixed with 4% paraformaldehyde for 15 minutes. After fixation, the colonies were stained with crystal violet solution for 10 minutes, followed by PBS washes to remove residual dye. Microscopic images were captured for documentation, and the number of colonies was quantitatively analyzed using Image J software. EdU proliferation assay Stably transfected TNBC cells were seeded into 24-well plates at 1 × 105 cells/well. After 24 hours of standard culture, prewarmed EdU working solution (BeyoClick™ EdU-594 Cell Proliferation Detection Kit, Beyotime, China) was added, and cells were incubated for 12 hours. Following EdU labeling, cells were fixed, permeabilized, and treated with an appropriate volume of Click reaction solution. Cells were incubated in the dark for 30 minutes, after which the nuclei were counterstained with DAPI. Fluorescence microscopy was used for observation. Wound healing assay Stably transfected TNBC cells were cultured in 6-well plates for 24 hours. Using a sterile 10-µL pipette tip to create a scratch on the cell monolayer. PBS washing was performed, supplied with serum-free medium, and incubated for another 24 hours. Scratch area was monitored at 0 and 24 hours under a microscope. Transwell assay Stably transfected TNBC cells were digested, centrifuged, and resuspended in serum-free DMEM. The cell suspension was added to the upper chamber of an 8 µm Transwell, with DMEM containing 12% FBS in the lower chamber. After 24 hours of incubation, the upper chamber was removed, and non-migrated cells were gently wiped off with a cotton swab. Migrated cells on the membrane underside were fixed with 4% paraformaldehyde, stained with 0.1% crystal violet for 5 minutes, imaged under a microscope, and quantified using ImageJ software. For the invasion assay, a 0.5 mg/ml matrix gel (pore size 8 µm, Corning, USA) was pre-coated on the upper chamber of the Transwell. Other steps followed the same procedure as in the migration assay after the gel had solidified. Dual-luciferase reporter gene assay The psi-CHECK2-circDUSP1-WT and psi-CHECK2-circDUSP1-Mut vectors were synthesized by Sangon Biotech Co., Ltd. (Shanghai). Approximately 6 × 10 4 293T cells were plated in a 24-well plate and cultured for 24 hours. Subsequently, 50 µL of DMEM was mixed with 0.4 µg of WT or MUT plasmid and 1 µL of 20 pmol/µL hsa-miR-429 mimics or NC, incubating for 30 minutes (solution A). Separately, 48 µL of DMEM was combined with 2 µL of transfection reagent (solution B). Mix both solutions, incubate for 30 minutes, and add to cells. After 48 hours of incubation, luciferase activity was measured using the Dual-Luciferase Reporter Gene Detection Kit (Sangon Biotech, Shanghai, E608001). RNA-pull down assay Biotin-labeled circDUSP1 and control probes were synthesized by GenePharma Pharmaceutical Co., Ltd. (Shanghai). Following cell lysis with lysis buffer, the RNA-protein binding reaction mixture was prepared, and the RNA complex was incubated with the biotin-labeled circDUSP1 probe at room temperature for 1 hour. The biotin-labeled RNA complex was then incubated with Streptavidin Magnetic Beads (BEAVER, Suzhou, China) for an additional hour. The magnetic beads bound to the biotin-labeled RNA complex were isolated using a magnetic stand, followed by elution with elution buffer. The RNA complex was collected, and total RNA was extracted for subsequent detection of circDUSP1 and miR-429 enrichment via qRT-PCR. Western blot Total protein from TNBC cells was extracted by RIPA lysis buffer (EpiZyme, PC101, Shanghai, China). Protein concentration was tested with the BCA kit (EpiZyme, PC101, Shanghai, China). Proteins were resolved by 7.5% SDS-PAGE (EpiZyme, PG114, Shanghai, China) and transferred to PVDF membranes (0.45 µm, Merck, GER). The membranes were blocked for 30 minutes at room temperature using a rapid blocking solution. Overnight incubation with rabbit primary antibodies targeting DLC1 and β-actin was performed at 4°C, followed by incubation with secondary HRP-linked antibodies (1:50000, HUABIO, China) by DLC1 antibody (1: 1000, Abcam, USA) and β-actin (HUABIO, 1: 40000, China) for 1 hour. Afterward, the membrane was developed using ECL solution and imaged with a gel imager. Data analysis was conducted with ImageJ. In vivo tumor growth assay Three-week-old female BALB/C nude mice were purchased from Chongqing Tengxin Huafu Laboratory Animal Sales Co., Ltd.. MDA-MB-231 cells with stable overexpression or knockdown of circDUSP1 were injected subcutaneously into the axillary region of each mouse (5 mice per group), with 100 µL of cell suspension (containing 3 × 10 6 cells) administered per mouse. Tumor volume was measured weekly. After 30 days, all mice were euthanized via tail vein injection of 3% pentobarbital sodium solution. Upon confirmation of unconsciousness and absence of pain response, complete euthanasia was ensured by cervical dislocation. And the tumors were excised for measurement of tumor weight. HE and IHC immunohistochemical staining HE staining: Paraffin-embedded sections were dewaxed, rehydrated, and stained with hematoxylin to visualize nuclei and eosin for cytoplasmic staining. Sections were then dehydrated and mounted. Immunohistochemical staining involved rehydrating dewaxed paraffin sections and performing antigen retrieval with sodium citrate buffer. Endogenous peroxidase activity was inhibited by a 15-minute incubation with 3% hydrogen peroxide. Then, block sections by 10% goat serum for 30 minutes. Primary and secondary antibodies were applied in sequence. DBA chromogen was added for 2 minutes, followed by a 3-minute rinse under running water. The sections were counterstained by hematoxylin, dehydrated, and mounted using graded ethanol. DLC1 cytoplasmic staining (1:100, Abcam, USA) was considered positive. The average optical density of DLC1 was quantified using ImageJ 6.4 software. Statistic analysis Statistical analysis utilized GraphPad Prism 9.5, presenting data as mean ± standard deviation. Group comparisons employed t-tests or one-way ANOVA, with statistical significance set at p < 0.05. Significance levels were indicated as follows: * for p < 0.05, ** for p < 0.01, *** for p < 0.001, **** for p < 0.0001, and ns for p ≥ 0.05. Results circDUSP1 level was decreased in TNBC tissues and cells Initial studies revealed elevated expression of miR-429 in TNBC tissues and cells, with evidence supporting a targeted regulatory relationship between miR-429 and DLC1 that influenced TNBC cell processes. To investigate the upstream regulatory mechanism of miR-429 in TNBC through DLC1 targeting, online databases such as circRNADisease.V2.0 ( http://cgga.org.cn:9091/circRNADisease/ ), starBase, and circBank were utilized to identify circRNAs that were underexpressed in breast cancer and could potentially interact with miR-429. A Venn diagram intersection analysis revealed four candidate circRNAs: circCDYL, circDUSP1, circPTK2, and circSMAD2 (Fig. 1 A). Notably, circDUSP1 (circBase ID: hsa_circ_0075043) had been documented to be significantly downregulated in TNBC and linked to patient prognosis. StarBase predictions confirmed a binding site between circDUSP1 and miR-429 (Fig. 1 B), leading to the selection of circDUSP1 as the focal point for subsequent investigations. This study analyzed tissue samples from three pairs of TNBC patients using qRT-PCR. The results demonstrated significantly lower levels of circDUSP1 in tumor tissues (Fig. 1 C). Furthermore, circDUSP1 expression was downregulated in TNBC cell lines (Fig. 1 D), consistent with previous reports. According to the circBase database, the full-length sequence of circDUSP1 spans 1043 bp and is derived from the 5' and 3' ends of exon 4 of the DUSP1 gene on chromosome 5 through reverse splicing. Specific primers targeting the reverse splicing junction of circDUSP1 were designed, and the qRT-PCR products underwent Sanger sequencing, confirming the reverse splicing formation of circDUSP1 (Fig. 1 E). cDNA and gDNA were extracted from MDA-MB-231 and MDA-MB-468 cells, respectively, and amplified by qRT-PCR, followed by agarose gel electrophoresis. The results revealed that circDUSP1 was only detected in the cDNA samples (Fig. 1 F), confirming that the reverse-spliced product of circDUSP1 originated from pre-RNA and was absent from the gDNA. RNase R digestion experiments further confirmed that circDUSP1 exhibited higher resistance to RNase R and greater stability than linear DUSP1 mRNA (Fig. 1 G). Nuclear-cytoplasmic separation and FISH assays revealed that circDUSP1 was primarily localized in the cytoplasm (Fig. 1 H-I). Results demonstrate that circDUSP1 is downregulated and exhibits exceptional stability in TNBC. circDUSP1 suppressed proliferation and metastasis of TNBC cells in vitro To assess the impact of circDUSP1 on the biological behavior of TNBC cells, in vitro functional assays were conducted. MDA-MB-231 and MDA-MB-468 cells were transfected with stable circDUSP1 overexpression lentivirus (circDUSP1) and stable circDUSP1 knockdown lentivirus (si-circDUSP1), respectively. The efficiency of circDUSP1 overexpression and knockdown was confirmed by qRT-PCR (Fig. 2 A). CCK8, colony formation, and EdU were employed to evaluate the effect of circDUSP1 on cell proliferation. Results showed that circDUSP1 knockdown promoted cell proliferation, while its overexpression suppressed proliferation (Fig. 2 B- 2 F). Furthermore, Transwell and scratch assays revealed that circDUSP1 overexpression reduced cell migration and invasion, whereas circDUSP1 knockdown exerted the opposite effect (Fig. 2 G- 2 L). Data indicate that circDUSP1 inhibits the proliferation, migration, and invasion of TNBC cells in vitro. circDUSP1 suppressed tumor growth in TNBC in vivo To assess the impact of circDUSP1 on tumor growth in vivo, MDA-MB-231 cells stably overexpressing or knocking down circDUSP1 were subcutaneously injected into female nude mice. Tumor volume and weight were significantly reduced in the circDUSP1 overexpression group, while they were markedly increased in the circDUSP1 knockdown group (Fig. 3 A- 3 C). Immunohistochemical analysis of the Ki-67 proliferation index revealed low Ki-67 expression in tumors from the circDUSP1 overexpression group, whereas tumors from the circDUSP1 knockdown group exhibited significantly higher Ki-67 positivity (Fig. 3 D, 3 E). Examination of DLC1 expression via immunohistochemistry showed a notable increase in DLC1 levels in tumors from the circDUSP1 overexpression group, while DLC1 expression was significantly reduced in the knockdown group (Fig. 3 D, 3 F). Additionally, qRT-PCR analysis of circDUSP1, miR-429, and DLC1 expression in the tumor tissues confirmed that in the overexpression group, circDUSP1 expression was elevated, miR-429 was downregulated, and DLC1 mRNA levels were upregulated, whereas the knockdown group showed the opposite trend (Fig. 3 G- 3 I). These results support the conclusion that circDUSP1 suppresses tumor growth in vivo. In summary, both in vitro and in vivo data confirm the tumor suppressor role of circDUSP1 in TNBC cells. In TNBC, circDUSP1 functioned as a molecular sponge by binding miR-429 The binding site of circDUSP1 and miR-429 was first predicted using the online tool Circular RNA Interactome, and a schematic diagram of this interaction was constructed (Fig. 4 A). Subcellular localization of circDUSP1 and miR-429 in TNBC cells was then confirmed through FISH experiments. The circDUSP1 probe was labeled with Cy3 (red), while the miR-429 probe was labeled with FAM (green). Results showed that in MDA-MB-231 and MDA-MB-468 cells, circDUSP1 and miR-429 co-localized in the cytoplasm (Fig. 4 B), supporting the ceRNA mechanism. To further confirm circDUSP1’s role as a sponge for miR-429, dual-luciferase reporter plasmids with wild-type and mutant circDUSP1 were constructed. The binding effect was assessed by measuring luciferase activity. miR-429 showed no significant effect on the luciferase activity of the mutant circDUSP1-3'UTR, indicating that miR-429 inhibited luciferase activity by binding to circDUSP1-3'UTR (Fig. 4 C). To validate this interaction, RNA pull-down assays using biotin-labeled circDUSP1 were performed. The results demonstrated that circDUSP1-specific probes significantly enriched circDUSP1 and miR-429 in both cells (Fig. 4 D- 4 E). Additionally, qRT-PCR results showed that stable overexpression of circDUSP1 led to a downregulation of miR-429, while circDUSP1 knockdown resulted in miR-429 upregulation (Fig. 4 F). Collectively, these data confirm that circDUSP1 regulates miR-429 through the ceRNA mechanism. circDUSP1 regulated proliferation, migration, and invasion of TNBC cells via the miR-429/DLC1 axis Prior work established that miR-429 was overexpressed in TNBC tissues and cells, promoting these cell processes, while inhibiting the tumor-suppressive function of DLC1. Preliminary data also indicated an interaction between circDUSP1 and miR-429. To investigate the regulatory relationship among circDUSP1, miR-429, and DLC1, the impact of circDUSP1 on DLC1 expression was assessed via qRT-PCR and Western blot. Overexpression of circDUSP1 caused a significant increase in mRNA and protein levels of DLC1, while silencing circDUSP1 resulted in a marked reduction in DLC1 expression at both the mRNA and protein levels (Figs. 5 A- 5 C). In vivo experiments also corroborated these observations, revealing corresponding increases or decreases in DLC1 mRNA levels in tumor tissues with stable overexpression or knockdown of circDUSP1 (Fig. 3 F). These consistent results from both in vitro and in vivo experiments further supported that circDUSP1 regulated DLC1 expression. To examine the functional role of circDUSP1 in TNBC cells, rescue assays were conducted by co-transfecting miR-429 mimics and inhibitors alongside circDUSP1 overexpression and knockdown vectors into MDA-MB-231 and MDA-MB-468 cells. Expression levels of circDUSP1, miR-429, and DLC1 mRNA were then assessed by qRT-PCR (Figs. 5 D- 5 F). The results indicated that miR-429 mimics suppressed the upregulation of circDUSP1 and DLC1 expression induced by circDUSP1 overexpression, while simultaneously increasing miR-429 expression. Conversely, miR-429 inhibitors restored the expression of circDUSP1 and DLC1 following circDUSP1 knockdown, accompanied by a reduction in miR-429 levels. Western blot analysis further confirmed that miR-429 mimics reduced DLC1 protein levels, whereas miR-429 inhibitors reversed this effect (Figs. 5 G- 5 H). A series of rescue assays (CCK-8, colony formation, etc.) demonstrated that miR-429 mimics significantly reduced the inhibitory effects of circDUSP1 overexpression on these TNBC cell processes. In contrast, miR-429 inhibitors counteracted the promoting effects of circDUSP1 knockdown on these processes (Figs. 6 A- 6 K). These data provide additional evidence that circDUSP1 functions as a miR-429 sponge. In summary, circDUSP1 binds miR-429 via sponging, inhibiting TNBC cell processes, with miR-429 partially reversing the tumor-suppressive effects of circDUSP1. Rescue assays demonstrated that co-transfection of circDUSP1 and miR-429 counteracted the inhibitory effects of circDUSP1 overexpression on TNBC cell processes, while miR-429 inhibition reversed the promoting effect of circDUSP1 knockdown on TNBC cell growth, migration, and invasion. Discussion TNBC is an aggressive and recurrent subtype of breast cancer [ 13 ] , with treatment primarily involving a combination of chemotherapy, surgery, and radiotherapyy [ 14 ] . However, due to its high heterogeneity and the absence of effective targeted therapies, treatment efficacy remains limited, and drug resistance frequently develops [ 1 ] . These challenges continue to complicate TNBC management. Therefore, a thorough investigation of the molecular mechanisms driving TNBC onset and progression, along with the identification of potential therapeutic targets, is essential for improving treatment outcomes and prognosis. Recent studies have highlighted the significant roles of circRNA and miRNA, emerging non-coding RNAs [ 15 ] . In various cancers. Our previous research demonstrated that miR-429 promoted TNBC cell proliferation and invasion by targeting DLC1. Expanding on the ceRNA hypothesis, this study further examined the upstream regulatory mechanisms of miR-429 in modulating DLC1 in TNBC, identified circDUSP1 as a key regulator, and explored its impact on TNBC cell proliferation, migration, and invasion via the miR-429/DLC1 signaling axis, providing insights into its potential role in TNBC biology. .. .. . CircRNAs are a class of non-coding RNA molecules that have emerged as key regulators in the onset, progression, and metastasis of various cancers [ 16 ] . Among them, circDUSP1, a novel circRNA, has been identified as a tumor suppressor in TNBC [ 7 ] . DUSP1, the host gene of circDUSP1, functions as a critical regulatory factor in the MAPK signaling pathway, modulating the activities of ERK1/2, JNK1/2, and p38 through dephosphorylation8 [ 17 ] . This regulation is integral to processes such as cell proliferation, differentiation, and apoptosis [ 18 ] . DUSP1 participates in the pathogenesis of multiple cancers; for instance, it suppresses the progression of ESCC by inhibiting the ERK signaling pathway, while its downregulation in prostate cancer leads to MAPK pathway activation, promoting metastatic spread [ 19 ] . Although a functional relationship between circDUSP1 and DUSP1 exists, the precise mechanisms by which circDUSP1 modulates TNBC cell behavior through alternative pathways remain incompletely understood. The functions of circRNAs are primarily mediated through interactions with miRNAs [ 20 ] , a class of non-coding RNAs that inhibit target mRNA expression by binding to them [ 21 ] . In the context of cancer initiation and progression, miRNAs have been classified as either oncogenes or tumor suppressors and are increasingly utilized as biomarkers for cancer diagnosis and prognosis. miR-429, a member of a specific miRNA family, is critical in the pathogenesis of various cancers. It is widely involved in regulating critical processes such as cell growth, migration, invasion, and EMT [ 22 – 25 ] . Studies indicate that the role of miR-429 in different cancers can be either pro-oncogenic or anti-oncogenic, influenced by the tumor’s genetic background and microenvironment. In TNBC, miR-429 exerts a dual function: while its downregulation enhances bone metastasis in breast cancer [ 26 ] , its upregulation promotes cell proliferation, apoptosis, and migration in TNBC [ 27 ] . DLC1 is a well-established tumor suppressor gene that regulates cell morphology, adhesion, and migration through modulation of small GTP-binding proteins [ 28 ] . It functions to inhibit tumor cell proliferation and migration [ 29 ] , and its loss of function is strongly linked to the initiation and progression of various cancers, including liver, breast, gastric, and prostate cancers [ 30 – 33 ] . Gong et al [ 34 ] . demonstrated that DLC1 suppressed prostate cancer cell proliferation via inhibition of the Rho kinase pathway. Similarly, Yang et al [ 35 ] . showed that DLC1 overexpression significantly inhibited the proliferation of cutaneous squamous cell carcinoma. In breast cancer, Ren et al [ 36 ] . reported a negative correlation between DLC1 expression and osseous metastasis, and poor prognosis. bone metastases and poor prognosis Previous studies have shown that in TNBC, miR-429 is upregulated while DLC1 expression is downregulated, and the levels of both are associated with prognosis. miR-429 promotes TNBC progression by inhibiting DLC1 [ 37 ] . Analysis of online databases suggested that circDUSP1 may interact with multiple miR-429 binding sites, indicating its potential role as a sponge for miR-429. Based on the ceRNA hypothesis, a connection between circDUSP1 and miR-429, and DLC1 in the malignant progression of TNBC is proposed. To test this, we first confirmed that circDUSP1 expression was significantly reduced in TNBC tissues and cells. We then constructed overexpression and knockdown models of circDUSP1 and performed a series of in vivo and in vitro experiments. The results revealed that circDUSP1 overexpression inhibited TNBC cell proliferation, migration, and invasion, while circDUSP1 knockdown promoted these processes. These data suggest that circDUSP1 is crucial in TNBC progression by regulating downstream signaling pathways that enhance malignancy. To further investigate the interaction between circDUSP1 and miR-429, rescue assays with miR-429 mimics and inhibitors were conducted. The results confirmed that circDUSP1 regulated miR-429 expression, and upregulation of miR-429 partially reversed the tumor suppressor effect of circDUSP1, supporting its role in inhibiting miR-429 via sponge action. Additional experiments demonstrated that circDUSP1 functioned by competitively binding to miR-429, thereby upregulating the expression of DLC1, a miR-429 target gene. Activation of this pathway plays a critical role in the proliferation, migration, and invasion of TNBC cells. This study emphasized the central role of the circDUSP1-miR-429-DLC1 signaling axis in regulating TNBC cell proliferation, migration, and invasion, suggesting it as a key mechanism underlying TNBC progression and metastasis. These results offer deeper insights into the molecular pathways driving TNBC. Targeting this axis—such as by inhibiting miR-429 expression or restoring the function of circDUSP1 and DLC1—could effectively suppress TNBC cell proliferation, migration, and invasion, presenting a potential therapeutic strategy. Furthermore, circDUSP1 holds promise as a diagnostic biomarker and for monitoring treatment efficacy in TNBC, underscoring its significant clinical potential. Although this study elucidated the potential mechanism by which circDUSP1 regulated TNBC cell processes via the miR-429/DLC1 signaling axis, several limitations remain. First, while the role of the circDUSP1/miR-429/DLC1 axis in cell proliferation has been confirmed in both cell and animal models, its impact on TNBC metastasis requires further investigation at the animal level. Second, although preliminary experiments have validated these markers, larger clinical cohorts are necessary to assess their clinical viability as therapeutic targets. Finally, the precise mechanism underlying circDUSP1 and the miR-429/DLC1 axis remains poorly understood, and the involvement of other potential pathways in regulating TNBC cell behavior warrants additional exploration. In summary, this study highlights the critical involvement of the circDUSP1-miR-429-DLC1 axis in TNBC cell proliferation, migration, and invasion, based on the expression of circDUSP1 in TNBC and its interactions with miR-429 and DLC1 (Fig. 7 ). These results provide valuable insights into the molecular mechanisms of TNBC and suggest potential molecular targets for targeted therapy. Future research will aim to further investigate the clinical applicability of circDUSP1 in TNBC and optimize treatment strategies accordingly. Declarations Acknowledgments We sincerely thanks to all the participants in this study. We thank the Affiliated Hospital of Zunyi Medical University for providing samples of breast tissue.Thanks to FIG Graw2.0 for providing us with the mapping website. confirming that informed consent was obtained from all participants and/or their legal guardians. Research involving human research participants must have been performed in accordance with the Declaration of Helsinki. For articles describing human transplantation studies, extra information must be provided (see below). Author C ontribution s C.J., X.T., and S.L. performed cell proliferation, migration, and invasion assays; J.Z. conducted bioinformatics analysis of circRNA-miRNA networks; J.L. and T.X. established the xenograft mouse models and performed histological evaluations; C.J. and X.T. prepared figures and tables; J.W. secured funding and supervised the project; C.J. and J.W. wrote the original draft; All authors (C.J., X.T., S.L., J.Z., J.L., T.X., J.W.) reviewed and edited the manuscript. Data A vailability The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding author. Figure 1E and Figure 7 are drawn and exported through the online website (FIG Graw2.0,https://www.figdraw.com/）,and the export copyright ID is TOWUWb7932, SPOYO79c06. The datasets generated and / or analyzed during the current study are available at the GenBank, the GenBank accession numbers :BankIt2929803 1HSA PV176472、BankIt2929803 3HSA PV176473. Co mpeting I nterest s The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Funding Statement The study was supported by Guizhou Provincial Science and Technology Projects (No. Qiankehejichu[2020]1Y429). Ethics approval and consent to participate Confirm that informed consent was obtained from the patients for the collection of all samples, and the review approval was obtained from the Ethics committee of Zunyi Medical University (Approval #KLLY-2021-028) and strictly complied with the ethical requirements of the Declaration of Helsinki. We performed the animal studies following the ARRIVE guidelines. All the animal studies were performed following the Guidelines for the Care and Use of Laboratory Animals and were approved by the Ethics Committee of the Affiliated Hospital of Zunyi Medical University (Approval #zyfy-an-2025-0304). Corresponding author Correspondence to Jinjing Wang. Registry and the Registration No N/A. Declaration of generative AI N/A. References Wahba, H. A. & El-Hadaad, H. A. Current approaches in treatment of TNBC [J]. Cancer biology Med. 12 (2), 106–116 (2015). Bianchini, G. et al. Treatment landscape of TNBC - expanded options, evolving needs [J]. 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The oncogenic miR-429 promotes TNBC progression by degrading DLC1 [J]. Aging 15 (18), 9809–9821 (2023). Tables Table 1 Primer sequences for qRT-PCR Gene Primer sequence (5′-3′) circDUSP1 F：5’-CACCATCTGCCTTGCTTACCTTATG- 3’ R：5’-GCTTCGCCTCTGCTTCACAAAC- 3’ circDUSP1 Divergent primers 5’-CTGGACGAGGCCTTTGAGTT- 3’ miR-429 F：5’-CGCGCGTAATACTGTCTGGTAA- 3’ R：5’-AGTGCAGGGTCCGAGGTATT- 3’ miR-429 stem-loop primers 5’-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACACGGTT- 3’ DLC1 F：5’-CGAGATCTTCCTGAGCCACTAAT- 3’ R：5’-GCTGTGACATCGCTCAGGAAATA- 3’ DUSP1 F：5’-ACCACCACCGTGTTCAACTTC- 3’ R：5’-TGGGAGAGGTCGTAATGGGG- 3’ GAPDH F：5’-GAAGGTGAAGGTCGGAGTC- 3’ R：5’-GAAGATGGTGATGGGATTTC- 3’ U6 F：5’-CCAGTTGTAGGTCGTTCTCAAG- 3’ R：5’-CCAGTTGTAGGTCGTTCTCAAG- 3’ U6stem-loop primers 5’-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAAAATATGG- 3’ KRT19P3 F：5’-CAGTGAGAGGCAGAATCAGG- 3’ R：5’-TTGGAGGTGGACAGGCTATT- 3’ Table 2 FISH probe synthesis sequence Gene Primer sequence (5′-3′) circDUSP1 5’-CCAGCATTCTTGATGGAGTTTGAAA- 3’ miR-429 5’-CCAGCATTCTTGATGGAGTTTGAAA- 3’ Additional Declarations No competing interests reported. Supplementary Files WB.pdf Cite Share Download PDF Status: Published Journal Publication published 20 Jul, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 13 Jun, 2025 Reviews received at journal 12 Jun, 2025 Reviews received at journal 10 Jun, 2025 Reviewers agreed at journal 10 Jun, 2025 Reviewers agreed at journal 07 Jun, 2025 Reviewers agreed at journal 03 May, 2025 Reviewers agreed at journal 28 Apr, 2025 Reviewers invited by journal 28 Apr, 2025 Editor assigned by journal 28 Apr, 2025 Editor invited by journal 07 Apr, 2025 Submission checks completed at journal 07 Apr, 2025 First submitted to journal 03 Mar, 2025 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. 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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-6147334","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":449559661,"identity":"20f9eee4-3671-47df-8b89-b636a5455838","order_by":0,"name":"Canhui Jian","email":"","orcid":"","institution":"Zunyi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Canhui","middleName":"","lastName":"Jian","suffix":""},{"id":449559662,"identity":"56f1177a-40ed-465f-8a06-afb38e6cb602","order_by":1,"name":"Xiaoxue Tian","email":"","orcid":"","institution":"Zunyi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xiaoxue","middleName":"","lastName":"Tian","suffix":""},{"id":449559663,"identity":"8c563bc7-a1b4-4212-8f3c-1f8e60ebe2e2","order_by":2,"name":"Shuai Luo","email":"","orcid":"","institution":"Zunyi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Shuai","middleName":"","lastName":"Luo","suffix":""},{"id":449559664,"identity":"64fab48a-42c5-41dd-9fb5-aebc02195bfa","order_by":3,"name":"Jiafei Zeng","email":"","orcid":"","institution":"Zunyi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jiafei","middleName":"","lastName":"Zeng","suffix":""},{"id":449559665,"identity":"47ccc596-d22a-4b7b-b121-2a5c322852f9","order_by":4,"name":"Jin Li","email":"","orcid":"","institution":"Zunyi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jin","middleName":"","lastName":"Li","suffix":""},{"id":449559666,"identity":"14f10201-4d9b-421a-bd1d-1ac675788f7c","order_by":5,"name":"Ting Xu","email":"","orcid":"","institution":"Zunyi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Ting","middleName":"","lastName":"Xu","suffix":""},{"id":449559667,"identity":"2b2345a4-f080-4288-8b14-caa628aeb574","order_by":6,"name":"Jinjing Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABCUlEQVRIie3Qv0vEMBTA8VcC5xJ1rUvzL7wS8Mei/0pCwVvuwMnFA1MO7FJwvc1/oX/CK4G6iHOHGwThJsHCLTfcYKQgLmkdHfKdkuHDewlAKPQPuyqWRB0CF6DcdUIJsBGCvNH16gaS1PREjpN4Ji3vQAL1RJuxxc5AIXG81HkxQ9jdrqdPxWHdwWLtJReGFMWY6YJ/YFS+buaVPcpiaDb+MbUhQmQ6X10rFj3YecU4xpGxfmIjNwfvtWl7MhVLLndDBBsGboyV0Gb0TRRYfjo45aScuN3wOUnLd6rdW9LKkXPV+Mmx+Nxu9/s7Lg50/uZ+TIjHF9l2i4G3/I5+TupvIBQKhUKevgBTz2B0oE39hAAAAABJRU5ErkJggg==","orcid":"","institution":"Zunyi Medical University","correspondingAuthor":true,"prefix":"","firstName":"Jinjing","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2025-03-03 14:53:29","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6147334/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6147334/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-11621-7","type":"published","date":"2025-07-20T15:57:35+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":81702015,"identity":"3274ba05-d2bc-4b46-a9eb-c93993792c43","added_by":"auto","created_at":"2025-04-30 13:08:59","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2118238,"visible":true,"origin":"","legend":"\u003cp\u003ecircDUSP1 is down-regulated in TNBC and can interact with miR-429, and is highly stable. (A) Venn diagram illustrating the online prediction outcomes from circRNADisease.V2.0, starBase, and circBank, identifying circRNAs with low expression in breast cancer that bind to miR-429. (B) starBase database prediction indicating the binding of circDUSP1 to miR-429. (C) Quantitative real-time polymerase chain reaction (qRT-PCR) analysis showing the relative expression of circDUSP1 in triple-negative breast cancer (TNBC) tissues compared to adjacent tissues, revealing a significant decrease in circDUSP1 expression in cancerous tissues. (D) qRT-PCR assessment of circDUSP1 expression levels in the human normal breast epithelial cell line (MCF-10A) and TNBC cell lines (MDA-MB-231, MDA-MB-468), demonstrating a notable reduction of circDUSP1 in TNBC cell lines. (E) Illustration depicting the structure of circDUSP1, formed through back-splicing of the mRNA encoded by the DUSP1 gene, with validation of the splice site through Sanger sequencing.(F) Agarose gel electrophoresis of qRT-PCR products revealed the presence of GAPDH, DUSP1, and circDUSP1. CircDUSP1 was exclusively detected in the cDNA samples and absent in the gDNA samples. (G) Following RNase R treatment of total RNA extracted from MDA-MB-231 and MDA-MB-468 cells, qRT-PCR analysis was conducted to determine the relative expression levels of circDUSP1 and DUSP1. CircDUSP1 exhibited resistance to degradation by the RNase R 3'-5' exoribonuclease, as its expression levels remained stable. (H) qRT-PCR analysis was employed to assess the relative expression levels of circDUSP1, GAPDH, and KRT19P3 in the nucleus and cytoplasm of MDA-MB-231 and MDA-MB-468 cells. CircDUSP1 was predominantly localized in the cytoplasmic fraction. (I) Fluorescence in situ hybridization (FISH) results demonstrated that circDUSP1, labeled with Cy3 probe, was primarily localized in the cytoplasm rather than the DAPI-stained cell nucleus (600; Scale bar: 20 μm).\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-6147334/v1/f40b46835b1784f5eaef18a4.png"},{"id":81702166,"identity":"fd541c20-28de-4b9a-8b96-2cb7b76b88d1","added_by":"auto","created_at":"2025-04-30 13:09:10","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3323594,"visible":true,"origin":"","legend":"\u003cp\u003eIn vitro experiments demonstrated that circDUSP1 knockdown enhanced the proliferation, migration, and invasion of TNBC cells, while its overexpression inhibited these processes. (A) Two breast cancer cell lines with stable circDUSP1 overexpression and knockdown were successfully constructed, with transfection efficiency confirmed by qRT-PCR. (B) The impact of circDUSP1 on cell proliferation was assessed using the CCK-8 assay. (C-D) Colony formation assays evaluated circDUSP1's influence on cell colony-forming ability, with clone numbers quantified via ImageJ. (E-F) EdU assays measured the effect of circDUSP1 on cell proliferation (scale bar: 100 μm), with proliferative cells quantified by ImageJ. (G-H) Wound healing assayassessed circDUSP1's effect on wound healing (scale bar: 100 μm), with the degree of healing quantified by ImageJ. (I-L) Transwell assays evaluated circDUSP1's impact on cell migration and invasion, with migrated and invaded cells quantified by ImageJ (scale bar: 100 μm).\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-6147334/v1/7926f682ebac6007c889330d.png"},{"id":81702017,"identity":"03367d93-7871-48f4-abc4-61ddd06a1946","added_by":"auto","created_at":"2025-04-30 13:08:59","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4606888,"visible":true,"origin":"","legend":"\u003cp\u003eIn vivo experiments demonstrated that circDUSP1 knockdown enhanced TNBC cell growth, while its overexpression inhibited growth. (A) Assessment of circDUSP1's impact on tumor growth in nude mice; (B) Tumor volume changes every 5 days post-knockdown or overexpression; (C) Effects on tumor weight; (D-F) qRT-PCR analysis of circDUSP1, miR-429, and DLC1 expression levels in tumors following circDUSP1 knockdown or overexpressio; (G) Representative HE-stained tumor sections and IHC staining to assess changes in the ki-67 proliferation index and DLC1 expression; (H-I) Quantification of ki-67 positivity via ImageJ and average optical density of DLC1 IHC staining.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-6147334/v1/0c16e76c4e0884cedd6085d3.png"},{"id":81701850,"identity":"44b59491-ea85-425c-898b-f8dbea360425","added_by":"auto","created_at":"2025-04-30 13:08:42","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1417463,"visible":true,"origin":"","legend":"\u003cp\u003ecircDUSP1 functions as a miR-429 sponge. (A) The Circular RNA Interactome database predicts circDUSP1 binding sites for miR-429. (B) FISH analysis reveals co-localization of circDUSP1 (Cy3-labeled) and miR-429 (FAM-labeled) in the cytoplasm, with DAPI staining the nucleus (magnification: 600×; scale bar: 20 μm). (C) Relative luciferase activity of circDUSP1-WT was assessed following co-transfection with miR-429 mimics, MIMics-NC, and psi-CHECK2-circDUSP1 wild type (WT)/mutant (MUT) plasmids in 293T cells. (D-E) RNA pull-down assays demonstrate significant enrichment of circDUSP1 and miR-429 using a circDUSP1-specific probe. (F) qRT-PCR was employed to measure miR-429 expression changes following circDUSP1 overexpression and knockdown.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-6147334/v1/fc7780b188b4b34231d30537.png"},{"id":81702218,"identity":"30f01fe3-1db7-4162-8aec-af1c428b6acf","added_by":"auto","created_at":"2025-04-30 13:09:22","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1166523,"visible":true,"origin":"","legend":"\u003cp\u003eRegulatory interactions among circDUSP1, miR-429, and DLC1 were investigated. (A) qRT-PCR assessed DLC1 mRNA levels following circDUSP1 overexpression and knockdown. (B-C) Western blot analysis confirmed and quantified DLC1 protein expression under similar conditions. (D-F) qRT-PCR evaluated expression changes of circDUSP1, miR-429, and DLC1 in cells co-transfected with circDUSP1 overexpression or knockdown and miR-429 mimics or inhibitors. (G-H) Western blotting examined DLC1 protein expression in cells co-transfected with circDUSP1 and miR-429 mimics or inhibitors.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-6147334/v1/6b81fe57680af7eba477c0ec.png"},{"id":81702074,"identity":"e74038bc-311b-437b-8bb5-6790f198761b","added_by":"auto","created_at":"2025-04-30 13:09:07","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":4104323,"visible":true,"origin":"","legend":"\u003cp\u003eThe rescue experiment demonstrated that co-transfection of circDUSP1 with miR-429 revealed miR-429 mimics counteracted the suppressive effect of circDUSP1 overexpression on TNBC cell proliferation, migration, and invasion. Conversely, the miR-429 inhibitor negated the enhancement of these cellular processes following circDUSP1 knockdown. (A) CCK-8 assays assessed the impact of circDUSP1 and miR-429 on cell proliferation; (B-C) Colony formation assays evaluated their effects, with clone numbers quantified using ImageJ; (D-E) EdU assays measured cell proliferation, with proliferative cells quantified by ImageJ (100x magnification, scale bar: 100 μm); (F-G) Wound healing assay assessed the impact on wound healing post-co-conversion, quantified by ImageJ (100x magnification, scale bar: 100 μm); (H-K) Transwell assays evaluated cell migration and invasion following co-conversion, with migrated and invaded cells quantified using ImageJ (100x magnification, scale bar: 100 μm).\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-6147334/v1/9e81a9e49b7bd2d065c00c85.png"},{"id":81701849,"identity":"159a471e-8313-474f-a731-c64019de210b","added_by":"auto","created_at":"2025-04-30 13:08:42","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":677546,"visible":true,"origin":"","legend":"\u003cp\u003eCircDUSP1 promotes TNBC progression by modulating miR-429/DLC11 axis. The graphic model of this study was shown. Briefly, circDUSP1 downregulated DLC 1 expression by sponging miR-429, thereby promoting the proliferation, migration, and invasion in the TNBC cells.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-6147334/v1/4fed13251b6390be2e132ea9.png"},{"id":87219367,"identity":"c9a7bf48-831a-40ea-99ee-9945e8079245","added_by":"auto","created_at":"2025-07-21 16:04:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":16636683,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6147334/v1/620f7025-2aca-4ff4-b01d-1d964eca86a0.pdf"},{"id":81702091,"identity":"78f809e7-5944-4b97-ace2-4d58f229246b","added_by":"auto","created_at":"2025-04-30 13:09:07","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2520297,"visible":true,"origin":"","legend":"","description":"","filename":"WB.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6147334/v1/e688fae255782b30beb15d8f.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The Role of the circDUSP1-Mediated miR-429/DLC1 Signaling Axis in the Proliferation, Migration, and Invasion of TNBC Cells","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBreast cancer ranks among the most prevalent malignancies affecting women globally, with its incidence steadily rising, posing a significant threat to women's health. TNBC is a distinct and heterogeneous subtype lacking the three primary targets: estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2)\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. Consequently, patients do not benefit from existing endocrine and targeted therapies. Chemotherapy remains the primary treatment for TNBC\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e, yet its effectiveness is limited, and prognosis remains poor. Recently, the combination of cytotoxic drugs with immune checkpoint inhibitors and antibody-drug conjugates has emerged as a promising treatment avenue.\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e However, no targeted drugs for TNBC have received approval from the U.S. Food and Drug Administration (FDA). Thus, investigating the molecular mechanisms of TNBC and identifying new biomarkers and targets are crucial for enhancing patient survival and prognosis, offering innovative treatment strategies.\u003c/p\u003e \u003cp\u003eCircular RNA (circRNA), a novel class of non-coding RNA, plays a crucial regulatory role in cancer development. Formed from pre-mRNA via back splicing, circRNAs possess a closed-loop structure and are abundant in miRNA binding sites\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. Dysregulation of circRNA can promote tumorigenesis and metastasis, making it a promising target for cancer therapy\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. Numerous studies have linked circRNA to tumor cell proliferation, apoptosis, and metastasis\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. Notably, circRNAs exhibit high stability and specificity and can modulate miRNA activity through the competing endogenous RNA (ceRNA) mechanism\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e, influencing downstream gene expression\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. circDUSP1 (hsa_circ_0075043), originating from exon 4 of the dual-specificity phosphatase 1 (DUSP1) gene, is a circular transcript of 1043 nucleotides formed by back splicing. Research indicates that circDUSP1 expression is reduced in TNBC (TNBC) and inversely correlates with TNBC tumorigenesis and progression\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMicroRNAs (miRNAs) are a class of conserved endogenous non-coding single-stranded RNAs, approximately 18\u0026ndash;25 nucleotides in length, prevalent in eukaryotes and highly conserved across species. miRNAs regulate post-transcriptional gene expression by binding to the 3' untranslated region (3'UTR) of target mRNAs through base pairing. They function as oncogenes or tumor suppressors\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e, influencing tumor cell survival, proliferation, invasion, metastasis, apoptosis, and drug response\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. Consequently, miRNAs hold significant potential for early tumor diagnosis, prognosis, and therapeutic target development. Our previous research indicates that miR-429 is markedly overexpressed in TNBC, enhancing TNBC cell proliferation, migration, and invasion by targeting DLC1.\u003c/p\u003e \u003cp\u003eDLC 1 (Deleted in Liver Cancer 1) is a crucial tumor suppressor gene\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e within the RhoGTP enzyme-activating protein (RhoGAP) family, known for inhibiting RhoGTP enzyme activity\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. As a GTP enzyme-activating protein, DLC 1 is pivotal in processes like cell migration and proliferation by terminating RhoGTP enzyme signaling and regulating cytoskeletal reorganization and RhoGTPase pathways\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. Beyond its role as a tumor biomarker, DLC 1 is a promising therapeutic target with significant clinical implications. Our previous research demonstrated that DLC 1 is underexpressed in TNBC and is regulated by miR-429. Based on the ceRNA mechanism hypothesis, circDUSP1 might upregulate DLC 1 by sponging miR-429. Currently, the specific interactions among circDUSP1, miR-429, and DLC 1 remain unexplored. This study aims to elucidate the mechanisms involving circDUSP1, miR-429, and DLC 1 in TNBC using molecular techniques, offering new molecular targets for TNBC prediction and treatment.\u003c/p\u003e \u003cp\u003eThe study demonstrates that circDUSP1 expression is reduced in TNBC, facilitating the proliferation, migration, and invasion of TNBC cells in vitro and enhancing tumor growth in vivo. Mechanistic investigations reveal that circDUSP1 acts as a sponge for miR-429, thereby upregulating the tumor suppressor gene DLC1. Consequently, circDUSP1 may serve as both a prognostic biomarker and a potential therapeutic target for TNBC.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eTissue samples\u003c/h2\u003e \u003cp\u003eFreshly resected tissue specimens were obtained from three patients diagnosed with TNBC, with pathologic confirmation, at the Affiliated Hospital of Zunyi Medical University. The samples included three pairs of cancerous and adjacent normal breast tissues. All patients were newly diagnosed with breast cancer and had not undergone prior radiotherapy or chemotherapy. Informed consent was obtained from each participant before surgery, and the study received approval from the hospital\u0026rsquo;s Ethics Committee. Post-surgical samples were immediately stored in liquid nitrogen.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCell culture\u003c/h3\u003e\n\u003cp\u003eHuman mammary epithelial cells (MCF-10A), TNBC cells (MDA-MB-231 and MDA-MB-468), and 293T cells were sourced from Procell. MCF-10A cells were cultured in a specialized MCF-10A medium (Procell, China), while MDA-MB-231 and MDA-MB-468 cells were maintained in DMEM containing 10% FBS (Procell, China). Cell lines were cultured in an incubator with 5% CO2 at 37\u0026deg;C.s were incubated at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\n\u003ch3\u003eCell transfection\u003c/h3\u003e\n\u003cp\u003eLentiviral transfection was performed using knockdown lentiviruses (Lv-NC-sh, Lv-hsa-circ-0075043-sh) and overexpression lentiviruses (Lv-NC-oe, Lv-hsa-circ-0075043-oe), synthesized by Fenghui Biotechnology Co., Ltd. Twenty-four hours before transfection, adherent cells were plated in 6-well plates at 1\u0026times;105 cells per well and incubated overnight under standard conditions (37\u0026deg;C, 5% CO2). After reaching 50% confluence, the medium was replaced with 2 mL fresh medium (0.5 \u0026micro;g/mL polybrene), and lentiviral solution was added. The cells were then incubated under the same conditions for 48 hours. Afterward, the viral-containing medium was replaced with fresh culture medium, and successful transfection was confirmed by the appearance of green fluorescence in over 80% of the cells under a fluorescence microscope. At this point, the medium was replaced with culture medium supplemented with 2 \u0026micro;g/mL puromycin, with daily observation and medium replacement. Once all cells in the blank control group had died, the infected cells were expanded to establish a stable transfected cell line.\u003c/p\u003e \u003cp\u003eThe RNA of hsa-miR-429 mimics, hsa-miR-429 inhibitors, and their respective negative controls (NC) were synthesized by Sangon Biotech Co., Ltd. (Shanghai). TNBC cell lines with stable knockdown or overexpression of hsa-circ-0075043 (circDUSP1) were seeded into 6-well plates and incubated for 24 hours. Subsequently, two 1.5 ml sterile EP tubes were prepared: 100 \u0026micro;l of serum-free medium was added to each, with 100 nm RNA placed in one tube and 12 \u0026micro;l of enhanced transfection reagent (Biodragon, China) in the other. After 5 minutes of incubation, the contents of both tubes were combined and allowed to stand for 20 minutes. The resulting mixture was then added to the appropriate (0.2 ml) wells of the 6-well plate for continued incubation. Cells were subsequently harvested for expression analysis and functional assays.\u003c/p\u003e\n\u003ch3\u003eQuantitative real-time PCR (qRT-PCR)\u003c/h3\u003e\n\u003cp\u003eTotal RNA was extracted using the Beyotime kit (China), then reverse-transcribed into cDNA with Takara's PrimeScript Reverse Transcription Kit (China). cDNA amplification was carried out using either the TB Green PCR Kit (Takara, China) or the TB Green Master Mix Kit (Takara, China). GAPDH (circDUSP1 and DLC1) and U6 (miR-429) served as internal controls to assess the expressions of circDUSP1, miR-429 and DLC1 mRNA. The 2-ΔΔCt method was employed to obtain relative expression. Table\u0026nbsp;1 showed primer sequences.\u003c/p\u003e\n\u003ch3\u003eRNase R digestion\u003c/h3\u003e\n\u003cp\u003eTotal RNA was extracted using the RNA extraction kit, and the RNA was divided into two equal aliquots (1 \u0026micro;g/aliquot). One aliquot was left untreated, while RNase R (Epicentre, USA) was added to the other, with incubation at 37\u0026deg;C for 20 minutes for digestion. Both undigested and digested RNA samples were analyzed via qRT-PCR to quantify the expression levels of circDUSP1 and DUSP1. Genes from the control group were used as internal references, and relative gene expression was assessed by the 2-ΔCT method.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eNucleocytoplasmic separation assay\u003c/h2\u003e \u003cp\u003eCell pellets were harvested, and nuclear and cytoplasmic fractions were isolated using a nuclear protein extraction kit (Solarbio, China). Total RNA was subsequently extracted, and the expression of circDUSP1 in both the nucleus and cytoplasm was quantified via qRT-PCR. KRT19P3 and GAPDH were employed as internal controls for the nucleus and cytoplasm, respectively. The target gene's relative expression was obtained in each compartment (2-ΔCT method).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eExtraction of cell genomic DNA (gDNA)\u003c/h3\u003e\n\u003cp\u003eg-DNA was extracted by an animal tissue/cell g-DNA extraction kit (Solarbio, China). CircDUSP1, DUSP1, and GAPDH expression were analyzed via qRT-PCR. Divergent primers for circDUSP1 were designed and synthesized by Sangon Biotech Co., Ltd. (Shanghai), with primer sequences provided in Table\u0026nbsp;1.\u003c/p\u003e\n\u003ch3\u003eAgarose gel electrophoresis\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eAgarose gel electrophoresis\u003c/div\u003e \u003cp\u003eThe cDNA and gDNA amplification products from the preceding experiment were analyzed via agarose gel electrophoresis. A 40 mL solution of 3% agarose gel was prepared and allowed to cool naturally to approximately 60\u0026deg;C. Nucleic acid dye was then added at a 1:5000 ratio, mixed thoroughly, and the solution poured into a gel casting tray with a comb, where it was left to solidify at room temperature. After the gel solidified, the comb was removed, and the gel was transferred to an electrophoresis chamber filled with 1\u0026times;TBE buffer. The 6\u0026times; loading buffer was diluted to 1\u0026times;, and the PCR products were further diluted 1:1 with the buffer. Both the marker and the diluted samples were loaded into the gel wells. Electrophoresis was initiated immediately after loading. Upon completion, the gel was visualized and photographed using a UV gel imaging system.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eFISH\u003c/h2\u003e \u003cp\u003eCircDUSP1 and miR-429 FISH probes (Shanghai) were designed and synthesized by GenePharma Pharmaceutical Co., Ltd. (Shanghai), with the probe sequences detailed in Table\u0026nbsp;2. Localization of circDUSP1 in TNBC cells was assessed using the RNA FISH kit (GenePharma). Briefly, fixed and permeabilized cell slides were prehybridized at 55\u0026deg;C for 2 hours. The probe and hybridization solution were denatured in a PCR instrument, and the denatured mixture was applied to the cell slides for 24 hours of hybridization. Following this, cell nuclei were stained with DAPI and observed under a fluorescence microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCCK-8 assay\u003c/h2\u003e \u003cp\u003eStable transfected TNBC cell lines (2 \u0026times; 103 cells/well) were seeded in 96-well plates, following by assessment of cell proliferation through CCK-8 reagent (GLPBIO, China) at 0, 24, 48, 72, and 96 hours. The fresh medium containing 10% CCK-8 solution was utilized. Following a 90-minute incubation at 37\u0026deg;C in the dark, absorbance at 450 nm was measured with an ELISA reader.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eColony Formation Assay\u003c/h2\u003e \u003cp\u003eStably transfected TNBC cells lines were seeded into 6-well plates at a density of 600 cells per well, followed by the addition of 2 mL complete culture medium with thorough mixing. The plates were then incubated in a humidified cell culture incubator. The culture medium was replaced with fresh complete medium every 3 days. Visible colonies emerged 13 days post-seeding. The colonies were gently washed with PBS and fixed with 4% paraformaldehyde for 15 minutes. After fixation, the colonies were stained with crystal violet solution for 10 minutes, followed by PBS washes to remove residual dye. Microscopic images were captured for documentation, and the number of colonies was quantitatively analyzed using Image J software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eEdU proliferation assay\u003c/h2\u003e \u003cp\u003eStably transfected TNBC cells were seeded into 24-well plates at 1 \u0026times; 105 cells/well. After 24 hours of standard culture, prewarmed EdU working solution (BeyoClick\u0026trade; EdU-594 Cell Proliferation Detection Kit, Beyotime, China) was added, and cells were incubated for 12 hours. Following EdU labeling, cells were fixed, permeabilized, and treated with an appropriate volume of Click reaction solution. Cells were incubated in the dark for 30 minutes, after which the nuclei were counterstained with DAPI. Fluorescence microscopy was used for observation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eWound healing assay\u003c/h2\u003e \u003cp\u003eStably transfected TNBC cells were cultured in 6-well plates for 24 hours. Using a sterile 10-\u0026micro;L pipette tip to create a scratch on the cell monolayer. PBS washing was performed, supplied with serum-free medium, and incubated for another 24 hours. Scratch area was monitored at 0 and 24 hours under a microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eTranswell assay\u003c/h2\u003e \u003cp\u003eStably transfected TNBC cells were digested, centrifuged, and resuspended in serum-free DMEM. The cell suspension was added to the upper chamber of an 8 \u0026micro;m Transwell, with DMEM containing 12% FBS in the lower chamber. After 24 hours of incubation, the upper chamber was removed, and non-migrated cells were gently wiped off with a cotton swab. Migrated cells on the membrane underside were fixed with 4% paraformaldehyde, stained with 0.1% crystal violet for 5 minutes, imaged under a microscope, and quantified using ImageJ software.\u003c/p\u003e \u003cp\u003eFor the invasion assay, a 0.5 mg/ml matrix gel (pore size 8 \u0026micro;m, Corning, USA) was pre-coated on the upper chamber of the Transwell. Other steps followed the same procedure as in the migration assay after the gel had solidified.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eDual-luciferase reporter gene assay\u003c/h2\u003e \u003cp\u003eThe psi-CHECK2-circDUSP1-WT and psi-CHECK2-circDUSP1-Mut vectors were synthesized by Sangon Biotech Co., Ltd. (Shanghai). Approximately 6 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e 293T cells were plated in a 24-well plate and cultured for 24 hours. Subsequently, 50 \u0026micro;L of DMEM was mixed with 0.4 \u0026micro;g of WT or MUT plasmid and 1 \u0026micro;L of 20 pmol/\u0026micro;L hsa-miR-429 mimics or NC, incubating for 30 minutes (solution A). Separately, 48 \u0026micro;L of DMEM was combined with 2 \u0026micro;L of transfection reagent (solution B). Mix both solutions, incubate for 30 minutes, and add to cells. After 48 hours of incubation, luciferase activity was measured using the Dual-Luciferase Reporter Gene Detection Kit (Sangon Biotech, Shanghai, E608001).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eRNA-pull down assay\u003c/h2\u003e \u003cp\u003eBiotin-labeled circDUSP1 and control probes were synthesized by GenePharma Pharmaceutical Co., Ltd. (Shanghai). Following cell lysis with lysis buffer, the RNA-protein binding reaction mixture was prepared, and the RNA complex was incubated with the biotin-labeled circDUSP1 probe at room temperature for 1 hour. The biotin-labeled RNA complex was then incubated with Streptavidin Magnetic Beads (BEAVER, Suzhou, China) for an additional hour. The magnetic beads bound to the biotin-labeled RNA complex were isolated using a magnetic stand, followed by elution with elution buffer. The RNA complex was collected, and total RNA was extracted for subsequent detection of circDUSP1 and miR-429 enrichment via qRT-PCR.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot\u003c/h2\u003e \u003cp\u003eTotal protein from TNBC cells was extracted by RIPA lysis buffer (EpiZyme, PC101, Shanghai, China). Protein concentration was tested with the BCA kit (EpiZyme, PC101, Shanghai, China). Proteins were resolved by 7.5% SDS-PAGE (EpiZyme, PG114, Shanghai, China) and transferred to PVDF membranes (0.45 \u0026micro;m, Merck, GER). The membranes were blocked for 30 minutes at room temperature using a rapid blocking solution. Overnight incubation with rabbit primary antibodies targeting DLC1 and β-actin was performed at 4\u0026deg;C, followed by incubation with secondary HRP-linked antibodies (1:50000, HUABIO, China) by DLC1 antibody (1: 1000, Abcam, USA) and β-actin (HUABIO, 1: 40000, China) for 1 hour. Afterward, the membrane was developed using ECL solution and imaged with a gel imager. Data analysis was conducted with ImageJ.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eIn vivo tumor growth assay\u003c/h2\u003e \u003cp\u003eThree-week-old female BALB/C nude mice were purchased from Chongqing Tengxin Huafu Laboratory Animal Sales Co., Ltd.. MDA-MB-231 cells with stable overexpression or knockdown of circDUSP1 were injected subcutaneously into the axillary region of each mouse (5 mice per group), with 100 \u0026micro;L of cell suspension (containing 3 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells) administered per mouse. Tumor volume was measured weekly. After 30 days, all mice were euthanized via tail vein injection of 3% pentobarbital sodium solution. Upon confirmation of unconsciousness and absence of pain response, complete euthanasia was ensured by cervical dislocation. And the tumors were excised for measurement of tumor weight.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eHE and IHC immunohistochemical staining\u003c/h2\u003e \u003cp\u003eHE staining: Paraffin-embedded sections were dewaxed, rehydrated, and stained with hematoxylin to visualize nuclei and eosin for cytoplasmic staining. Sections were then dehydrated and mounted.\u003c/p\u003e \u003cp\u003eImmunohistochemical staining involved rehydrating dewaxed paraffin sections and performing antigen retrieval with sodium citrate buffer. Endogenous peroxidase activity was inhibited by a 15-minute incubation with 3% hydrogen peroxide. Then, block sections by 10% goat serum for 30 minutes. Primary and secondary antibodies were applied in sequence. DBA chromogen was added for 2 minutes, followed by a 3-minute rinse under running water. The sections were counterstained by hematoxylin, dehydrated, and mounted using graded ethanol. DLC1 cytoplasmic staining (1:100, Abcam, USA) was considered positive. The average optical density of DLC1 was quantified using ImageJ 6.4 software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eStatistic analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis utilized GraphPad Prism 9.5, presenting data as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Group comparisons employed t-tests or one-way ANOVA, with statistical significance set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Significance levels were indicated as follows: * for p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ** for p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, *** for p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, **** for p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, and ns for p\u0026thinsp;\u0026ge;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003ecircDUSP1 level was decreased in TNBC tissues and cells\u003c/h2\u003e \u003cp\u003eInitial studies revealed elevated expression of miR-429 in TNBC tissues and cells, with evidence supporting a targeted regulatory relationship between miR-429 and DLC1 that influenced TNBC cell processes. To investigate the upstream regulatory mechanism of miR-429 in TNBC through DLC1 targeting, online databases such as circRNADisease.V2.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://cgga.org.cn:9091/circRNADisease/\u003c/span\u003e\u003cspan address=\"http://cgga.org.cn:9091/circRNADisease/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), starBase, and circBank were utilized to identify circRNAs that were underexpressed in breast cancer and could potentially interact with miR-429. A Venn diagram intersection analysis revealed four candidate circRNAs: circCDYL, circDUSP1, circPTK2, and circSMAD2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Notably, circDUSP1 (circBase ID: hsa_circ_0075043) had been documented to be significantly downregulated in TNBC and linked to patient prognosis. StarBase predictions confirmed a binding site between circDUSP1 and miR-429 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), leading to the selection of circDUSP1 as the focal point for subsequent investigations.\u003c/p\u003e \u003cp\u003eThis study analyzed tissue samples from three pairs of TNBC patients using qRT-PCR. The results demonstrated significantly lower levels of circDUSP1 in tumor tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Furthermore, circDUSP1 expression was downregulated in TNBC cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD), consistent with previous reports. According to the circBase database, the full-length sequence of circDUSP1 spans 1043 bp and is derived from the 5' and 3' ends of exon 4 of the DUSP1 gene on chromosome 5 through reverse splicing. Specific primers targeting the reverse splicing junction of circDUSP1 were designed, and the qRT-PCR products underwent Sanger sequencing, confirming the reverse splicing formation of circDUSP1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). cDNA and gDNA were extracted from MDA-MB-231 and MDA-MB-468 cells, respectively, and amplified by qRT-PCR, followed by agarose gel electrophoresis. The results revealed that circDUSP1 was only detected in the cDNA samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF), confirming that the reverse-spliced product of circDUSP1 originated from pre-RNA and was absent from the gDNA. RNase R digestion experiments further confirmed that circDUSP1 exhibited higher resistance to RNase R and greater stability than linear DUSP1 mRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). Nuclear-cytoplasmic separation and FISH assays revealed that circDUSP1 was primarily localized in the cytoplasm (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH-I). Results demonstrate that circDUSP1 is downregulated and exhibits exceptional stability in TNBC.\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003ecircDUSP1 suppressed proliferation and metastasis of TNBC cells in vitro\u003c/h2\u003e \u003cp\u003eTo assess the impact of circDUSP1 on the biological behavior of TNBC cells, in vitro functional assays were conducted. MDA-MB-231 and MDA-MB-468 cells were transfected with stable circDUSP1 overexpression lentivirus (circDUSP1) and stable circDUSP1 knockdown lentivirus (si-circDUSP1), respectively. The efficiency of circDUSP1 overexpression and knockdown was confirmed by qRT-PCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). CCK8, colony formation, and EdU were employed to evaluate the effect of circDUSP1 on cell proliferation. Results showed that circDUSP1 knockdown promoted cell proliferation, while its overexpression suppressed proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). Furthermore, Transwell and scratch assays revealed that circDUSP1 overexpression reduced cell migration and invasion, whereas circDUSP1 knockdown exerted the opposite effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG-\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eL). Data indicate that circDUSP1 inhibits the proliferation, migration, and invasion of TNBC cells in vitro.\u003c/p\u003e\u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003ecircDUSP1 suppressed tumor growth in TNBC in vivo\u003c/h2\u003e \u003cp\u003eTo assess the impact of circDUSP1 on tumor growth in vivo, MDA-MB-231 cells stably overexpressing or knocking down circDUSP1 were subcutaneously injected into female nude mice. Tumor volume and weight were significantly reduced in the circDUSP1 overexpression group, while they were markedly increased in the circDUSP1 knockdown group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Immunohistochemical analysis of the Ki-67 proliferation index revealed low Ki-67 expression in tumors from the circDUSP1 overexpression group, whereas tumors from the circDUSP1 knockdown group exhibited significantly higher Ki-67 positivity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Examination of DLC1 expression via immunohistochemistry showed a notable increase in DLC1 levels in tumors from the circDUSP1 overexpression group, while DLC1 expression was significantly reduced in the knockdown group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Additionally, qRT-PCR analysis of circDUSP1, miR-429, and DLC1 expression in the tumor tissues confirmed that in the overexpression group, circDUSP1 expression was elevated, miR-429 was downregulated, and DLC1 mRNA levels were upregulated, whereas the knockdown group showed the opposite trend (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG-\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI). These results support the conclusion that circDUSP1 suppresses tumor growth in vivo. In summary, both in vitro and in vivo data confirm the tumor suppressor role of circDUSP1 in TNBC cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003eIn TNBC, circDUSP1 functioned as a molecular sponge by binding miR-429\u003c/h2\u003e \u003cp\u003eThe binding site of circDUSP1 and miR-429 was first predicted using the online tool Circular RNA Interactome, and a schematic diagram of this interaction was constructed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Subcellular localization of circDUSP1 and miR-429 in TNBC cells was then confirmed through FISH experiments. The circDUSP1 probe was labeled with Cy3 (red), while the miR-429 probe was labeled with FAM (green). Results showed that in MDA-MB-231 and MDA-MB-468 cells, circDUSP1 and miR-429 co-localized in the cytoplasm (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), supporting the ceRNA mechanism. To further confirm circDUSP1\u0026rsquo;s role as a sponge for miR-429, dual-luciferase reporter plasmids with wild-type and mutant circDUSP1 were constructed. The binding effect was assessed by measuring luciferase activity. miR-429 showed no significant effect on the luciferase activity of the mutant circDUSP1-3'UTR, indicating that miR-429 inhibited luciferase activity by binding to circDUSP1-3'UTR (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). To validate this interaction, RNA pull-down assays using biotin-labeled circDUSP1 were performed. The results demonstrated that circDUSP1-specific probes significantly enriched circDUSP1 and miR-429 in both cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). Additionally, qRT-PCR results showed that stable overexpression of circDUSP1 led to a downregulation of miR-429, while circDUSP1 knockdown resulted in miR-429 upregulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). Collectively, these data confirm that circDUSP1 regulates miR-429 through the ceRNA mechanism.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003ecircDUSP1 regulated proliferation, migration, and invasion of TNBC cells via the miR-429/DLC1 axis\u003c/h2\u003e \u003cp\u003ePrior work established that miR-429 was overexpressed in TNBC tissues and cells, promoting these cell processes, while inhibiting the tumor-suppressive function of DLC1. Preliminary data also indicated an interaction between circDUSP1 and miR-429. To investigate the regulatory relationship among circDUSP1, miR-429, and DLC1, the impact of circDUSP1 on DLC1 expression was assessed via qRT-PCR and Western blot. Overexpression of circDUSP1 caused a significant increase in mRNA and protein levels of DLC1, while silencing circDUSP1 resulted in a marked reduction in DLC1 expression at both the mRNA and protein levels (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). In vivo experiments also corroborated these observations, revealing corresponding increases or decreases in DLC1 mRNA levels in tumor tissues with stable overexpression or knockdown of circDUSP1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). These consistent results from both in vitro and in vivo experiments further supported that circDUSP1 regulated DLC1 expression.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo examine the functional role of circDUSP1 in TNBC cells, rescue assays were conducted by co-transfecting miR-429 mimics and inhibitors alongside circDUSP1 overexpression and knockdown vectors into MDA-MB-231 and MDA-MB-468 cells. Expression levels of circDUSP1, miR-429, and DLC1 mRNA were then assessed by qRT-PCR (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD-\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). The results indicated that miR-429 mimics suppressed the upregulation of circDUSP1 and DLC1 expression induced by circDUSP1 overexpression, while simultaneously increasing miR-429 expression. Conversely, miR-429 inhibitors restored the expression of circDUSP1 and DLC1 following circDUSP1 knockdown, accompanied by a reduction in miR-429 levels. Western blot analysis further confirmed that miR-429 mimics reduced DLC1 protein levels, whereas miR-429 inhibitors reversed this effect (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG-\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH).\u003c/p\u003e \u003cp\u003eA series of rescue assays (CCK-8, colony formation, etc.) demonstrated that miR-429 mimics significantly reduced the inhibitory effects of circDUSP1 overexpression on these TNBC cell processes. In contrast, miR-429 inhibitors counteracted the promoting effects of circDUSP1 knockdown on these processes (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eK). These data provide additional evidence that circDUSP1 functions as a miR-429 sponge. In summary, circDUSP1 binds miR-429 via sponging, inhibiting TNBC cell processes, with miR-429 partially reversing the tumor-suppressive effects of circDUSP1.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eRescue assays demonstrated that co-transfection of circDUSP1 and miR-429 counteracted the inhibitory effects of circDUSP1 overexpression on TNBC cell processes, while miR-429 inhibition reversed the promoting effect of circDUSP1 knockdown on TNBC cell growth, migration, and invasion.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eTNBC is an aggressive and recurrent subtype of breast cancer\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e, with treatment primarily involving a combination of chemotherapy, surgery, and radiotherapyy\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. However, due to its high heterogeneity and the absence of effective targeted therapies, treatment efficacy remains limited, and drug resistance frequently develops\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. These challenges continue to complicate TNBC management. Therefore, a thorough investigation of the molecular mechanisms driving TNBC onset and progression, along with the identification of potential therapeutic targets, is essential for improving treatment outcomes and prognosis. Recent studies have highlighted the significant roles of circRNA and miRNA, emerging non-coding RNAs\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. In various cancers. Our previous research demonstrated that miR-429 promoted TNBC cell proliferation and invasion by targeting DLC1. Expanding on the ceRNA hypothesis, this study further examined the upstream regulatory mechanisms of miR-429 in modulating DLC1 in TNBC, identified circDUSP1 as a key regulator, and explored its impact on TNBC cell proliferation, migration, and invasion via the miR-429/DLC1 signaling axis, providing insights into its potential role in TNBC biology.\u003c/p\u003e \u003cp\u003e.. .. .\u003c/p\u003e \u003cp\u003eCircRNAs are a class of non-coding RNA molecules that have emerged as key regulators in the onset, progression, and metastasis of various cancers\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. Among them, circDUSP1, a novel circRNA, has been identified as a tumor suppressor in TNBC\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. DUSP1, the host gene of circDUSP1, functions as a critical regulatory factor in the MAPK signaling pathway, modulating the activities of ERK1/2, JNK1/2, and p38 through dephosphorylation8\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. This regulation is integral to processes such as cell proliferation, differentiation, and apoptosis\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. DUSP1 participates in the pathogenesis of multiple cancers; for instance, it suppresses the progression of ESCC by inhibiting the ERK signaling pathway, while its downregulation in prostate cancer leads to MAPK pathway activation, promoting metastatic spread\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. Although a functional relationship between circDUSP1 and DUSP1 exists, the precise mechanisms by which circDUSP1 modulates TNBC cell behavior through alternative pathways remain incompletely understood.\u003c/p\u003e \u003cp\u003eThe functions of circRNAs are primarily mediated through interactions with miRNAs\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e, a class of non-coding RNAs that inhibit target mRNA expression by binding to them\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. In the context of cancer initiation and progression, miRNAs have been classified as either oncogenes or tumor suppressors and are increasingly utilized as biomarkers for cancer diagnosis and prognosis. miR-429, a member of a specific miRNA family, is critical in the pathogenesis of various cancers. It is widely involved in regulating critical processes such as cell growth, migration, invasion, and EMT\u003csup\u003e[\u003cspan additionalcitationids=\"CR23 CR24\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. Studies indicate that the role of miR-429 in different cancers can be either pro-oncogenic or anti-oncogenic, influenced by the tumor\u0026rsquo;s genetic background and microenvironment. In TNBC, miR-429 exerts a dual function: while its downregulation enhances bone metastasis in breast cancer\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e, its upregulation promotes cell proliferation, apoptosis, and migration in TNBC\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eDLC1 is a well-established tumor suppressor gene that regulates cell morphology, adhesion, and migration through modulation of small GTP-binding proteins\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. It functions to inhibit tumor cell proliferation and migration\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e, and its loss of function is strongly linked to the initiation and progression of various cancers, including liver, breast, gastric, and prostate cancers\u003csup\u003e[\u003cspan additionalcitationids=\"CR31 CR32\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. Gong et al\u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e. demonstrated that DLC1 suppressed prostate cancer cell proliferation via inhibition of the Rho kinase pathway. Similarly, Yang et al\u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e. showed that DLC1 overexpression significantly inhibited the proliferation of cutaneous squamous cell carcinoma. In breast cancer, Ren et al\u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e. reported a negative correlation between DLC1 expression and osseous metastasis, and poor prognosis. bone metastases and poor prognosis\u003c/p\u003e \u003cp\u003ePrevious studies have shown that in TNBC, miR-429 is upregulated while DLC1 expression is downregulated, and the levels of both are associated with prognosis. miR-429 promotes TNBC progression by inhibiting DLC1\u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e. Analysis of online databases suggested that circDUSP1 may interact with multiple miR-429 binding sites, indicating its potential role as a sponge for miR-429. Based on the ceRNA hypothesis, a connection between circDUSP1 and miR-429, and DLC1 in the malignant progression of TNBC is proposed. To test this, we first confirmed that circDUSP1 expression was significantly reduced in TNBC tissues and cells. We then constructed overexpression and knockdown models of circDUSP1 and performed a series of in vivo and in vitro experiments. The results revealed that circDUSP1 overexpression inhibited TNBC cell proliferation, migration, and invasion, while circDUSP1 knockdown promoted these processes. These data suggest that circDUSP1 is crucial in TNBC progression by regulating downstream signaling pathways that enhance malignancy. To further investigate the interaction between circDUSP1 and miR-429, rescue assays with miR-429 mimics and inhibitors were conducted. The results confirmed that circDUSP1 regulated miR-429 expression, and upregulation of miR-429 partially reversed the tumor suppressor effect of circDUSP1, supporting its role in inhibiting miR-429 via sponge action. Additional experiments demonstrated that circDUSP1 functioned by competitively binding to miR-429, thereby upregulating the expression of DLC1, a miR-429 target gene. Activation of this pathway plays a critical role in the proliferation, migration, and invasion of TNBC cells.\u003c/p\u003e \u003cp\u003eThis study emphasized the central role of the circDUSP1-miR-429-DLC1 signaling axis in regulating TNBC cell proliferation, migration, and invasion, suggesting it as a key mechanism underlying TNBC progression and metastasis. These results offer deeper insights into the molecular pathways driving TNBC. Targeting this axis\u0026mdash;such as by inhibiting miR-429 expression or restoring the function of circDUSP1 and DLC1\u0026mdash;could effectively suppress TNBC cell proliferation, migration, and invasion, presenting a potential therapeutic strategy. Furthermore, circDUSP1 holds promise as a diagnostic biomarker and for monitoring treatment efficacy in TNBC, underscoring its significant clinical potential.\u003c/p\u003e \u003cp\u003eAlthough this study elucidated the potential mechanism by which circDUSP1 regulated TNBC cell processes via the miR-429/DLC1 signaling axis, several limitations remain. First, while the role of the circDUSP1/miR-429/DLC1 axis in cell proliferation has been confirmed in both cell and animal models, its impact on TNBC metastasis requires further investigation at the animal level. Second, although preliminary experiments have validated these markers, larger clinical cohorts are necessary to assess their clinical viability as therapeutic targets. Finally, the precise mechanism underlying circDUSP1 and the miR-429/DLC1 axis remains poorly understood, and the involvement of other potential pathways in regulating TNBC cell behavior warrants additional exploration.\u003c/p\u003e \u003cp\u003eIn summary, this study highlights the critical involvement of the circDUSP1-miR-429-DLC1 axis in TNBC cell proliferation, migration, and invasion, based on the expression of circDUSP1 in TNBC and its interactions with miR-429 and DLC1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). These results provide valuable insights into the molecular mechanisms of TNBC and suggest potential molecular targets for targeted therapy. Future research will aim to further investigate the clinical applicability of circDUSP1 in TNBC and optimize treatment strategies accordingly.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe sincerely thanks to all the participants in this study. We thank the Affiliated Hospital of Zunyi Medical University for providing samples of breast tissue.Thanks to FIG Graw2.0 for providing us with the mapping website.\u0026nbsp;confirming that informed consent was obtained from all participants and/or their legal guardians. Research involving human research participants must have been performed in accordance with the Declaration of Helsinki. For articles describing human transplantation studies, extra information must be provided (see below).\u003c/p\u003e\n\u003cp id=\"_Toc19562\"\u003e\u003cstrong\u003eAuthor\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;C\u003c/strong\u003e\u003cstrong\u003eontribution\u003c/strong\u003e\u003cstrong\u003es\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eC.J., X.T., and S.L. performed cell proliferation, migration, and invasion assays;\u003c/p\u003e\n\u003cp\u003eJ.Z. conducted bioinformatics analysis of circRNA-miRNA networks;\u003c/p\u003e\n\u003cp\u003eJ.L. and T.X. established the xenograft mouse models and performed histological evaluations;\u003c/p\u003e\n\u003cp\u003eC.J. and X.T. prepared figures and tables;\u003c/p\u003e\n\u003cp\u003eJ.W. secured funding and supervised the project;\u003c/p\u003e\n\u003cp\u003eC.J. and J.W. wrote the original draft;\u003c/p\u003e\n\u003cp\u003eAll authors (C.J., X.T., S.L., J.Z., J.L., T.X., J.W.) reviewed and edited the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eA\u003c/strong\u003e\u003cstrong\u003evailability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding author. Figure 1E and Figure 7 are drawn and exported through the online website (FIG Graw2.0,https://www.figdraw.com/）,and the export copyright ID is TOWUWb7932, SPOYO79c06. The datasets generated and / or analyzed during the current study are available at the GenBank, the GenBank accession numbers :BankIt2929803 1HSA PV176472、BankIt2929803 \u0026nbsp;3HSA PV176473.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCo\u003c/strong\u003e\u003cstrong\u003empeting I\u003c/strong\u003e\u003cstrong\u003enterest\u003c/strong\u003e\u003cstrong\u003es\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe\u0026nbsp;study was supported by Guizhou Provincial Science and Technology Projects (No. Qiankehejichu[2020]1Y429).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConfirm that informed consent was obtained from the patients for the collection of all samples,\u0026nbsp;and the review approval was obtained from the Ethics committee of Zunyi Medical University\u0026nbsp;\u0026nbsp;(Approval #KLLY-2021-028) and strictly complied with the ethical requirements of the Declaration of Helsinki.\u0026nbsp;We performed the animal\u0026nbsp;studies\u0026nbsp;following the ARRIVE guidelines.\u0026nbsp;All the animal studies were performed following the Guidelines for the Care and Use of Laboratory Animals and were approved by the Ethics Committee of the Affiliated Hospital of Zunyi Medical University (Approval #zyfy-an-2025-0304).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding author\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence to Jinjing Wang.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRegistry and the Registration No\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;N/A.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of generative AI\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;N/A.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWahba, H. 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Oncodevelopmental Biology Med.\u003c/em\u003e \u003cb\u003e34\u003c/b\u003e (5), 2633\u0026ndash;2643 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eReng, Li, G. Tumor suppressor gene DLC1: Its modifications, interactive molecules, and potential prospects for clinical cancer application [J]. \u003cem\u003eInt. J. Biol. Macromol.\u003c/em\u003e \u003cb\u003e182\u003c/b\u003e, 264\u0026ndash;275 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi, Y. et al. The oncogenic miR-429 promotes TNBC progression by degrading DLC1 [J]. \u003cem\u003eAging\u003c/em\u003e \u003cb\u003e15\u003c/b\u003e (18), 9809\u0026ndash;9821 (2023).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 Primer sequences for qRT-PCR\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGene\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 576px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePrimer sequence (5\u0026prime;-3\u0026prime;)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003ecircDUSP1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 576px;\"\u003e\n \u003cp\u003eF：5\u0026rsquo;-CACCATCTGCCTTGCTTACCTTATG- 3\u0026rsquo;\u003c/p\u003e\n \u003cp\u003eR：5\u0026rsquo;-GCTTCGCCTCTGCTTCACAAAC- 3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003ecircDUSP1 Divergent primers\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 576px;\"\u003e\n \u003cp\u003e5\u0026rsquo;-CTGGACGAGGCCTTTGAGTT- 3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003emiR-429\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 576px;\"\u003e\n \u003cp\u003eF：5\u0026rsquo;-CGCGCGTAATACTGTCTGGTAA- 3\u0026rsquo;\u003c/p\u003e\n \u003cp\u003eR：5\u0026rsquo;-AGTGCAGGGTCCGAGGTATT- 3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003emiR-429 stem-loop primers\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 576px;\"\u003e\n \u003cp\u003e5\u0026rsquo;-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACACGGTT- 3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003eDLC1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 576px;\"\u003e\n \u003cp\u003eF：5\u0026rsquo;-CGAGATCTTCCTGAGCCACTAAT- 3\u0026rsquo;\u003c/p\u003e\n \u003cp\u003eR：5\u0026rsquo;-GCTGTGACATCGCTCAGGAAATA- 3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003eDUSP1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 576px;\"\u003e\n \u003cp\u003eF：5\u0026rsquo;-ACCACCACCGTGTTCAACTTC- 3\u0026rsquo;\u003c/p\u003e\n \u003cp\u003eR：5\u0026rsquo;-TGGGAGAGGTCGTAATGGGG- 3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003eGAPDH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 576px;\"\u003e\n \u003cp\u003eF：5\u0026rsquo;-GAAGGTGAAGGTCGGAGTC- 3\u0026rsquo;\u003c/p\u003e\n \u003cp\u003eR：5\u0026rsquo;-GAAGATGGTGATGGGATTTC- 3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003eU6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 576px;\"\u003e\n \u003cp\u003eF：5\u0026rsquo;-CCAGTTGTAGGTCGTTCTCAAG- 3\u0026rsquo;\u003c/p\u003e\n \u003cp\u003eR：5\u0026rsquo;-CCAGTTGTAGGTCGTTCTCAAG- 3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003eU6stem-loop primers\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 576px;\"\u003e\n \u003cp\u003e5\u0026rsquo;-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAAAATATGG- 3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003eKRT19P3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 576px;\"\u003e\n \u003cp\u003eF：5\u0026rsquo;-CAGTGAGAGGCAGAATCAGG- 3\u0026rsquo;\u003c/p\u003e\n \u003cp\u003eR：5\u0026rsquo;-TTGGAGGTGGACAGGCTATT- 3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eTable 2 FISH probe synthesis sequence\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"676\" class=\"fr-table-selection-hover\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003eGene\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 576px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePrimer sequence (5\u0026prime;-3\u0026prime;)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003ecircDUSP1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 576px;\"\u003e\n \u003cp\u003e5\u0026rsquo;-CCAGCATTCTTGATGGAGTTTGAAA- 3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003emiR-429\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 576px;\"\u003e\n \u003cp\u003e5\u0026rsquo;-CCAGCATTCTTGATGGAGTTTGAAA- 3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"TNBC, triple-negative breast cancer, circDUSP1, miR‐429, DLC1","lastPublishedDoi":"10.21203/rs.3.rs-6147334/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6147334/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTNBC is a highly invasive and heterogeneous breast cancer subtype, lacking specific targeted therapies and linked to high morbidity and mortality rates. In recent years, circular RNA (circRNA), a significant non-coding RNA, has garnered substantial attention in cancer research. Through the competing endogenous RNA (ceRNA) mechanism, circRNAs can modulate miRNA activity, thereby influencing downstream gene expressions. This study investigated the expression profiles and biological roles of circDUSP1 in TNBC, demonstrating that circDUSP1 functions as a molecular sponge, adsorbing miR-429 and subsequently modulating the its target gene expression, DLC1, which suppresses the malignant progression of TNBC. These findings indicate that circDUSP1 holds promise as both a biomarker and a therapeutic target in TNBC.\u003c/p\u003e","manuscriptTitle":"The Role of the circDUSP1-Mediated miR-429/DLC1 Signaling Axis in the Proliferation, Migration, and Invasion of TNBC Cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-30 12:49:15","doi":"10.21203/rs.3.rs-6147334/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-06-13T07:45:03+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-12T12:33:33+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-10T10:43:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"13493903195587213586062741530473063730","date":"2025-06-10T06:49:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"324419942130571543496618486402743612974","date":"2025-06-07T07:49:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"277879595664648911167621017787141267950","date":"2025-05-03T16:43:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"6630669458576718996550894544026131781","date":"2025-04-28T22:20:01+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-28T16:48:31+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-28T16:47:27+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-04-07T15:26:47+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-07T04:12:11+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-03-03T14:51:51+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"253e5082-0b1a-48e8-bf6f-5c98ceee1a6a","owner":[],"postedDate":"April 30th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":47837358,"name":"Biological sciences/Cancer"},{"id":47837359,"name":"Biological sciences/Cell biology"}],"tags":[],"updatedAt":"2025-07-21T16:01:25+00:00","versionOfRecord":{"articleIdentity":"rs-6147334","link":"https://doi.org/10.1038/s41598-025-11621-7","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-07-20 15:57:35","publishedOnDateReadable":"July 20th, 2025"},"versionCreatedAt":"2025-04-30 12:49:15","video":"","vorDoi":"10.1038/s41598-025-11621-7","vorDoiUrl":"https://doi.org/10.1038/s41598-025-11621-7","workflowStages":[]},"version":"v1","identity":"rs-6147334","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6147334","identity":"rs-6147334","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}