Circ_0001756, a novel biomarker, promotes breast cancer progression via miR-584-5p/TRAF6 signaling axis

Circ_0001756, a novel biomarker, promotes breast cancer progression via miR-584-5p/TRAF6 signaling axis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Circ_0001756, a novel biomarker, promotes breast cancer progression via miR-584-5p/TRAF6 signaling axis Jun-ying Wu, Xi-xi Wu, Li-yan Shi, Ling-xia Wang, Zhuo Wang, Ying Cao, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3865902/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Purpose Circular RNAs (circRNAs) appear to exert critical functions in breast cancer (BC). The objective of this study is to explore the usefulness of circRNAs as potential diagnostic and prognostic biomarkers of BC. Methods The Gene Expression Omnibus database was referenced to identify differentially expressed circRNAs in BC. We found that circ_0001756 was associated with the malignant potential of BC. Also, the expression levels of circ_0001756 in BC tissues and cell lines were determined by real-time quantitative polymerase chain reaction analysis. The functions of circ_0001756 were investigated both in vitro and in vivo . The luciferase reporter and rescue assays were used to clarify the molecular mechanisms of circ_0001756. Additionally, the clinical value of circ_0001756 as a serum biomarker and potential correlations with the clinicopathological characteristics of BC patients were investigated. Results Circ_0001756 expression was upregulated in BC tissues and substantially correlated with tumor size and tumor-node-metastasis (TNM) stage. Knockdown of circ_0001756 markedly inhibited the malignant potential of BC both in vitro and in vivo . Mechanistically, circ_0001756 acted as a miR-584-5p sponge to regulate TRAF6 in BC cells. Serum levels of circ_0001756 were significantly higher in pre-operative BC patients than in healthy controls, fibroadenoma patients, and post-operative BC patients. Also, serum circ_0001756 was remarkably correlated with tumor size, patient age, metastasis state, and TNM stage. The combination of the traditional tumor markers carcinoembryonic antigen and cancer antigen 15 − 3 with circ_0001756 significantly improved the diagnostic accuracy of BC. Conclusion Circ_0001756 promotes the malignancy of BC through the miR-584-5p/TRAF6 signaling axis. Additionally, serum circ_0001756 is a promising biomarker for screening and diagnosis of BC. Circ_0001756 Breast cancer MiR-584-5p TRAF6 Metastasis Biomarker Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Breast cancer (BC) has emerged as the most prevalent cancer in the female population, and treatment poses a huge burden to healthcare systems (Ferlay J et al. 2020). Hence, regular screening for early diagnosis is particularly important to improve prognosis and survival. Therefore, it is essential to identify novel markers of BC and elucidate the functions and mechanisms to improve screening and early diagnosis. Circular RNAs (circRNAs) are characterized by a covalently closed continuous loop structure without a 5’-cap or 3’-poly A tail (Salzman J et al. 2012 ) and were initially regarded as aberrant splicing by-products, thus attracting little attention. However, recent advancements in molecular techniques and bioinformatics have clarified the roles of various circRNAs in carcinogenesis and tumor progression. For instance, circRNAs can act as sponges of miRNAs (Hansen TB et al. 2013 ) and some contain intronic sequences that have been implicated in transcription of the parental gene by interacting with RNA polymerase II (Zhang Y et al. 2013 ). Additionally, circRNAs with open reading frames and internal ribosome entry sites have the potential to translate proteins (Zhang Y et al. 2021 ). For instance, a recent study demonstrated that circSMARCA5 directly binds to the promoter of parental genes to induce transcription (Xu X et al. 2020 ). Also, many circRNAs have been associated with the onset and progression of BC. Notably, circPTK2 is reported to facilitate mammary carcinogenesis via the YAP1/Hippo signaling pathway (Wang X et al. 2021 ) and circCDYL has been shown to augment autophagy and promote progression of BC via the miR-1275-ATG7/ULK1-autophagic axis (Liang G et al. 2020 ). However, the functions and mechanisms of numerous circRNAs implicated in the onset and progression of BC remain largely unknown. Circ_0001756, which is derived from exon 2 of the HIPK2 gene, has been shown to regulate astrocyte activation in collaboration with autophagy by sponging MIR124–2HG (Huang R et al. 2017 ). Moreover, circ_0001756 is reported to contribute to the functional recovery of neuronal stem cells after ischemic stroke (Wang G et al. 2020 ). A recent study found that circ_0001756 promoted progression of ovarian cancer via activation of the EGFR/MAPK signaling pathway (Ji J et al. 2022 ). A previous study by our group demonstrated that lipopolysaccharide (LPS) stimulation increased the invasive and metastatic capacities of BC cells via the TLR4/NF-κB signaling pathway (Yang H et al. 2014 ). In this study, bioinformatics analysis was conducted in reference to the Gene Expression Omnibus (GEO) database ( https://www.ncbi.nlm.nih.gov/geo/ ) to identify differentially expressed circRNAs associated with BC. The results confirmed that circ_0001756 expression was relatively upregulated in BC tissues as compared to adjacent non-cancerous tissues. Moreover, LPS stimulation was found to enhance circ_0001756 expression in epithelial-like (MDA-MB-231) BC cells as compared to human BC (MCF-7) cells, implying that circ_0001756 may be associated with metastasis of BC. Furthermore, an investigation to establish correlations between circ_0001756 in BC tissues and clinicopathological factors found that circ_0001756 expression was related to tumor size and tumor-node-metastasis (TNM) stage. Collectively, these findings indicate that circ_0001756 is associated with progression of BC. Therefore, the aim of the present study was to investigate the involvement of the circ_0001756/miR-584-5p/TRAF6 signaling axis in progression of BC and assess the clinical usefulness of circ_0001756 as a potential biomarker. Materials and methods Clinical tissues, serum samples, saliva samples, and BC cell lines The protocol of the human study was approved by the Ethics Committee of the Second Affiliated Hospital of Soochow University (approval no. JD-LK-2021-115-01) and conducted in accordance with the ethical principles for medical research involving human subjects described in the Declaration of Helsinki. Prior to inclusion in this study, written informed consent was obtained from all subjects. Surgically resected cancer tissues and adjacent non-cancerous tissues were obtained from 21 BC patients prior to receiving chemotherapy or radiotherapy and immediately frozen with liquid nitrogen. Serum samples from healthy controls, patients with fibroadenomas, and BC patients both pre- and post-operatively were pretreated and stored at -80°C. Saliva samples were collected and pretreated as described by Zhao et al (Zhao SY et al. 2018 ). Human embryonic kidney (HEK)-293T, MDA-MB-231, and MCF-7 cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and cultured in high-glucose Dulbecco's modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Gibco – Invitrogen Corporation, Carlsbad, CA, USA). Transfection Cells were transfected with short interfering RNA (siRNA), microRNA (miRNA) inhibitors, and miRNA mimics (GenePharma Co., Ltd., Suzhou, China) using Lipofectamine 2000 reagent (Invitrogen Corporation) and harvested after 48 h. The RNA sequences used in this study are listed in Tables S1, S2, and S3. Real-time quantitative PCR (qPCR) Total RNA was exacted from BC cells and tissues using TRIzol reagent (Invitrogen Corporation) and the nuclear and cytoplasmic fractions were separated with a Cytoplasmic & Nuclear RNA Purification Kit (Norgen Biotek Corp. Thorold, ON, Canada). Total RNA was isolated from serum and saliva samples using TRIzol LS reagent (Invitrogen Corporation) in accordance with the manufacturer’s instructions. The isolated RNA was reverse-transcribed into complementary DNA (cDNA) using the HiScript® III 1st Strand cDNA Synthesis Kit (+ gDNA wiper), and the cDNA was quantitated using AceQ® qPCR SYBR Green Master Mix (Vazyme Biotech Co., Ltd., Nanjing, China) with the primers (Sangon Biotech Co., Ltd., China) listed in Table S4 , Relative expression of mRNA was calculated using the 2 -ΔΔCt method with U6 or GAPDH as an internal control. The primers of miRNA for reverse transcription and qPCR analysis were designed using MiRNA Design V 1.01 software ( https://www.vazyme.com/companyfile/653.html ). The sequences were listed in Tables S5 and S6. Dual-luciferase reporter assay HEK-293T cells (5 × 10 5 ) were seeded in the wells of 12-well plates, cultured for 24 h, and then transfected with mutant (MUT) or wild-type (WT) circ_0001756 reporter vectors, and miR-584-5p mimics. After 24 h, firefly luciferase activity was measured using the Dual-Glo® Luciferase Assay System (Promega Corporation, Madison, WI, USA). Renilla luciferase was used as a reference. RNase R treatment The resistance of circular RNA to RNase was confirmed using Ribonuclease R (Epicentre Technologies Corporation, Madison, WI, USA). Briefly, RNA (2 µg) was treated with Ribonuclease R (6 U) at 37°C for 15 min. The untreated control group was incubated under the same conditions. Then, the enzyme was rendered inactive by heating it at 70°C for 10 min. Colony formation assay Transfected cells (1 × 10 3 ) were cultured in the wells of 6-well plates for approximately 14 days. Cell clusters were fixed with 4% paraformaldehyde, stained with crystal violet (0.5% w/v), washed with phosphate-buffered saline, and imaged. Cell counting (CCK-8) assay Transfected cells (2 × 10 3 ) were suspended in 100 µL of DMEM in the wells of 96-well plates. Every 24 h, 10 µL of CCK-8 reagent (Dojindo Laboratories Co., Ltd., Kumamoto, Japan) were added to the wells and the plate was incubated at 37°C for 2 h. Afterward, absorbance at 450 nm was measured with a spectrophotometer (Tecan Group Ltd., Männedorf, Switzerland). Wound healing assay Transfected BC cells (2 × 10 5 ) were plated in the wells of 6-well plates. The confluent monolayers were evenly streaked with a sterile 1-mL pipette tip and then washed with phosphate-buffered saline. Migration of MCF-7 cells at 48 h and MDA-MB-231 cells at 24 h was observed with an inverted microscope. Transwell migration and Matrigel invasion assays The transwell cell migration assay was performed to assess the chemotactic capability of cells. Briefly, transfected BC cells (1 × 10 5 ) were plated into the wells of 24-well migration chambers (Corning, USA) with 200 µL of low serum medium. Then, DMEM supplemented with 10% FBS was added to the lower compartment. After 8–10 h, the cells were fixed and stained with crystal. The transwell invasion assay was performed to assess the invasive capacity of cells through an extracellular matrix. Briefly, the chambers were coated with Matrigel. After 24 h, cells in the chambers were harvested and imaged. The mean number of cells in five random fields were counted using ImageJ software ( https://imagej.net/ij/ ). Western blot analysis BC cells were lysed with radioimmunoprecipitation assay buffer containing a proteinase inhibitor (phenylmethylsulfonyl fluoride). The proteins were separated by gel electrophoresis and then transferred to a polyvinylidene fluoride membrane (EMD Millipore Corporation, Billerica, MA, USA), which was blocked for 1 h with 5% (w/v) skim milk and then incubated overnight at 4°C with primary antibodies against E-cadherin (dilution, 1:500; Cell Signaling Technology, Inc., Danvers, MA, USA), N-cadherin (dilution, 1:1000; Cell Signaling Technology, Inc.), vimentin (dilution, 1:1000; Bioworld, Dublin, OH, USA), TRAF6 (dilution, 1:1000; Cell Signaling Technology, Inc.), and GAPDH (dilution, 1:1000; Cell Signaling Technology, Inc.) followed by horseradish peroxidase-conjugated secondary antibodies (Multisciences (Lianke) Biotech Co., Ltd., Hangzhou, China) for 1 h at room temperature. Afterward, the protein bands were visualized using chemiluminescence reagent and imaged. Immunohistochemical analysis Tissues harvested from nude mice were sliced, dewaxed, rehydrated, mounted on glass slides, and incubated with primary antibodies against epithelial-mesenchymal transition (EMT)-related proteins at 4°C overnight, followed by biotin-labeled secondary antibodies at 37°C for 1 h. Afterward, the tissues were stained with hematoxylin and observed under a microscope. Construction of subcutaneous a xenograft BALB/c-nu mouse model The protocol of the animal study was approved by the Institutional Animal Care and Use Committee of the Second Affiliated Hospital of Soochow University (approval no. JD-LK-2021-115-01) and conducted in accordance with the Guide for the Care and Use of Laboratory Animals. Six-week-old BALB/c nude mice were subcutaneously injected with MDA-MB-231 cells (5 × 10 6 ) via the mammary fat pad. The width and length of the tumors in each group were measured once every 3 days. After 21 days, the nude mice were euthanized by cervical dislocation under 3% isoflurane deep anesthesia, and the xenografts were photographed and weighted. The tumor volume was calculated according to the formula 0.5 × L × W 2 . L denotes the longitudinal diameter, and W denotes the latitudinal diameter. Then, two small pieces of tissue specimens were used to extract RNA and immunohistochemical analysis. The remaining tissues were stored at -80°C. All animals in this study were euthanized at the experimental endpoint. The humane endpoints such as decreased food and water consumption, weight loss reaching 20%, max tumor diameter > 20 mm, and so on were not used during the experiment. Statistical analysis Statistical analyses (Student’s t-test, Wilcoxon rank-sum test, and one-way analysis of variance) were performed using IBM SPSS Statistics for Windows, version 24.0 (IBM Corporation, Armonk, NY, USA). All figures and tables were generated using Prism 8.0 software (GraphPad Software, Inc., San Diego, CA, USA). The Fisher's exact test was used to identify correlations between clinical characteristics and expression levels of circ_0001756. A probability ( p ) value < 0.05 was considered statistically significant. Results Circ_0001756 is verified in BC cells and tissues Initially, the microarray data from the GEO dataset GSE182471 were reviewed to compare the expression patterns of circRNAs in BC tissues (GSM5529874, GSM5529877, and GSM5529878) and adjacent non-cancerous breast tissues (GSM5529869, GSM5529872, and GSM5529873). A corresponding volcano plot was constructed, which is presented in Fig. 1 A. Expression of circ_0001756 was markedly upregulated in BC tissues (fold change > 1.5 and p < 0.05). Subsequent comparison of 21 pairs of BC and adjacent non-cancerous tissues found that the expression levels of circ_0001756 were considerably higher in BC tissues (Fig. 1 B). Moreover, circ_0001756 expression was considerably higher in MDA-MB-231 cells than MCF-7 cells (Fig. 1 C). Stimulation with LPS (4 µg/mL) dramatically upregulated circ_0001756 expression in MDA-MB-231 cells (Fig. S1 ). Correlation analysis of the clinicopathological data of 21 BC patients showed that circ_0001756 expression was positively associated with tumor dimension and TNM stage (Fig. 1 D, E; Table. S7). Collectively, these findings suggest that upregulation of circ_0001756 potentially contributes to progression of BC. Circ_0001756 consists of 1084 nucleotides from exon 2 of the HIPK2 gene that form a circular structure (Fig. S2 ). Sanger sequencing with divergent primers identified the presence of a back-splicing sequence in the conventional PCR product (Fig. S2 ). Since circRNAs are generated by back-splicing of pre-mRNAs, appropriate back-to-back reverse primers were designed. The circRNA-specific primers amplify cDNA-derived products, but nor genomic DNA (gDNA), as confirmed by electrophoresis results (Fig. 1 F). RNase digestion revealed that circ_0001756 was resistant to RNase R treatment (Fig. 1 G-I). The GAPDH linear transcript was used as a positive control for validation. Knockdown (KD) of circ_0001756 inhibited viability of BC cells in vitro To explore the possible functions of circ_0001756 in vitro , BC cells were transfected with siRNA targeting the back-splicing site of circ_0001756. The interference efficiency of the siRNA was evaluated by detecting the expression levels of circ_0001756 (Fig. S3 A, B) and EMT-related indicators (Fig. S3 C, D). Due to higher KD efficiency, si-circ_0001756#2 was selected for subsequent functional experiments. The qPCR results showed that si-circ_0001756#2 only targeted circ_0001756 and had little effect on linear HIPK2 (Fig. S3 E, F). Cell proliferation analysis demonstrated that KD of circ_0001756 reduced growth of BC cells (Fig. 2 A-C). The wound healing and transwell migration assays demonstrated that silencing of circ_0001756 inhibited migration of BC cells (Fig. 2 D-F). Additionally, the transwell invasion assay revealed that downregulation of circ_0001756 significantly reduced the metastatic capacity of BC cells (Fig. 2 G). Moreover, in MDA-MB-231 cells, downregulation of circ_0001756 significantly increased E-cadherin protein levels, but had opposite effects on N-cadherin and vimentin protein levels (Fig. 2 H). Western blot analysis was also performed to determine whether circ_0001756 is involved in apoptosis. The findings demonstrated that knockdown circ_0001756 knockdown had no effect on apoptosis of MDA-MB-231 cells (Fig. S4 ). KD of circ_0001756 inhibited proliferation and EMT of BC cells in vivo To explore the functions of circ_0001756 in vivo , BALB/c-nu mice were subcutaneously injected with MDA-MB-231 cells to induce tumorigenesis. The results showed that KD of circ_0001756 dramatically inhibited growth of subcutaneous tumors (Fig. 3 A). The weight and size of the tumor tissues were reduced in the treatment group as compared to the control group (Fig. 3 B, C). The expression levels of EMT-related genes in subcutaneous tumor tissues were assessed by qPCR analysis. The results revealed that E-cadherin expression was notably increased in the si-circ_0001756#2 group, while the expression levels of N-cadherin and vimentin were substantially reduced (Fig. 3 D). The results of immunohistochemical and qPCR analyses were consistent (Fig. 3 E). Circ_0001756 functions by adsorbing miR-584-5p To elucidate the potential roles of circ_001756 in the onset and progression of BC, a nucleocytoplasmic distribution assay was conducted to determine the intracellular localization of circ_0001756. The results detected circ_0001756 in both the nucleus and cytoplasm of BC cells (Fig. 4 A, B), suggesting that circ_0001756 acts as a "miRNA sponge". In addition, searches of the CircBank ( http://www.circbank.cn ) and CircInteractome ( https://circinteractome.nia.nih.gov ) databases identified nine potential target miRNAs (miR-1178-3p、miR-1261、miR-338-3p、miR-516a-5p、miR-578、miR-581、miR-584-5p、miR-663b、miR-889-3p) of circ_0001756 (Fig. 4 C). Further analysis revealed that miR-584-5p expression levels were considerably lower in BC tissues than adjacent non-cancerous tissues. In addition, KD of circ_0001756 significantly increased miR-584-5p expression levels in BC cells. The dual-luciferase reporter assay confirmed that miR-584-5p mimics reduced luciferase activity (Fig. 4 D, E), indicating that miR-584-5p directly interacts with circ_0001756. To validate the functions of miR-584-5p, BC cells were transfected with miR-584-5p mimics to overexpress miR-584-5p. The transfection efficiency was confirmed by qPCR results in MDA-MB-231 cells (Fig. 4 F). The colony formation assay revealed that miR-584-5p inhibited proliferation of both BC cell lines (Fig. 4 G). The wound healing and transwell assays demonstrated that overexpression of miR-584-5p significantly impaired migration and attenuated the invasive capacity of BC cells (Fig. 4 H–K). Western blot analysis revealed that miR-584-5p mimics increased protein expression of E-cadherin in MDA-MB-231 cells, but had opposite effects on protein expression of N-cadherin and vimentin (Fig. 4 L). To further verify that circ_0001756 functions via sponging miR-584-5p, a series of rescue experiments was conducted. The results showed that miR-584-5p inhibitors reversed the attenuation of cell proliferation, migration, and invasion in MDA-MB-231 cells caused by KD of circ_0001756 (Fig. 4 M, N). Circ_0001756 acts as a competing endogenous RNA (ceRNA) to regulate TRAF6 CircRNAs can regulate downstream target genes by sponging miRNAs. Screening of the TargetScan ( https://www.targetscan.org/vert_80/ ), mirDIP ( https://ophid.utoronto.ca/mirDIP/ ) and miRWALK ( http://mirwalk.umm.uni-heidelberg.de/ ) databases identified 531 potential target genes of miR-584-5p (Fig. 5 A). The results of Gene Ontology biological process (GO-BP) enrichment analysis confirmed that some terms were highly enriched, such as positive regulation of transcription by RNA polymerase II, intracellular protein transport and mitotic cell cycle. Several pathways including “Pathways in cancer”, “PD-L1 expression and PD-1 checkpoint pathway in cancer” and “Toll-like receptor signaling pathway” were revealed as significantly enriched by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway. Then, we selected some genes (ANGPT2, TRAF6, MDM2, KRAS, PRKAA2) for more analysis. According to the expression in BC cells, we found that TRAF6 expression was markedly higher in MDA-MB-231 cells than MCF-7 cells (Fig. 5 B) and was attenuated by miR-584-5p mimics (Fig. 5 C). According to the dual luciferase reporter assays, the sequence of TRAF6 contains a binding site for miR-584-5p (Fig. 5 D, E). In addition, downregulation of TRAF6 markedly impaired the mobility and invasiveness of MDA-MB-231 cells (Fig. 5 F). Western blot analysis showed that downregulation of TRAF6 increased E-cadherin protein levels and decreased protein levels of N-cadherin and vimentin (Fig. 5 G). Collectively, these findings confirm that suppression of TRAF6 reduced proliferation, migration, and invasion of BC cells through EMT-related proteins. Rescue experiments were performed to confirm that TRAF6 is a downstream molecular of miR-584-5p. The findings show that downregulation of miR-584-5p increased mobility and invasiveness in MDA-MB-231 cells, whereas co-transfection with si-TRAF6 and a miR-584-5p inhibitor partly reversed this effect (Fig. 5 H). Clinical application of circ_0001756 in BC To explore the potential clinical value of circ_0001756 as a biomarker, serum levels of circ_0001756 in healthy controls, patients with fibroadenomas, and BC patients both pre- and post-operatively were measured. Intriguingly, circ_0001756 expression was notably elevated in BC patients as compared to the other three experimental groups (Fig. 6 A). Subsequently, receiver operating characteristic (ROC) curves were generated to assess the diagnostic value of circ_00001756. The area of the ROC curve (AUC) of BC patients and healthy controls was 0.833 (95% confidence interval [CI]: 0.762–0.905, p < 0.0001, Fig. 6 B). The AUC of BC and fibroadenoma patients was 0.772 (95% CI: 0.695–0.849, p < 0.0001, Fig. 6 C). The AUC of pre- and post-operative BC patients was 0.954 (95% CI: 0.914–0.993, p < 0.0001, Fig. 6 D). Then, potential correlations of serum levels of circ_0001756 with the clinicopathological characteristics of 86 BC patients were investigated. The results showed that circ_0001756 expression was markedly linked with tumor size, patient age, lymph node metastases, and TNM stage (Table. 1), indicating the potential of circ_0001756 as a serum biomarker for screening, diagnosis, and prognosis of BC. To further evaluate the diagnostic value of circ_0001756, serum levels of the conventional BC markers cancer antigen 15 − 3 (CA 15 − 3) and carcinoembryonic antigen (CEA) were evaluated. The results revealed that the AUC of circ_0001756 (AUC: 0.827, 95% CI: 0.744–0.909, p < 0.0001) was greater than that of CEA and CA 15 − 3. The combination of all three indicators for diagnosis of BC had the highest AUC value (AUC: 0.897, 95% CI: 0.829–0.965, p < 0.0001) with a sensitivity of 77.2% and specificity of 93.6% (Fig. 6 E). These results indicate that circ_0001756 had better diagnostic performance than CA 15 − 3 and CEA. The combination of circ_0001756 with CA 15 − 3 and CEA can improve the accuracy of BC diagnosis. Therefore, circ_0001756 should be considered an ideal index for clinical diagnosis of BC. Table. 1 Correlation analysis between clinical characteristics and expression levels of circ_0001756 in serum of 86 BC patients Characteristics No. of patients Circ_0001756 Expression P- value low high Tumor Size (cm) ≤ 2 39 26 13 0.005 > 2 47 17 30 Age (years) ≤ 50 44 28 16 0.010 > 50 42 15 27 Pathological Type IDC 70 34 36 0.579 DCIS and others 16 9 7 Lymph Node Metastasis 0 59 35 24 0.011 ≥ 1 27 8 19 T stage T0 - I 32 21 11 0.026 II - Ⅲ 54 22 32 HER2 - 55 25 30 0.261 + 31 18 13 IDC: Invasive Ductal Carcinoma DCIS: ductal carcinoma in situ The prevalence, prognosis, and current therapeutic strategies significantly differ among the four major subtypes of BC: luminal A, luminal B, human epidermal growth factor receptor 2 (HER2)-enriched, and triple-negative (TN) (Harbeck N et al. 2019 ). In accordance with the criteria established by the Chinese Society of Clinical Oncology, BC was classified based on expression of the estrogen receptor, progesterone receptor, and HER2. The results of qPCR analysis of these four subtypes showed that circ_0001756 expression was significantly elevated in the luminal A, luminal B, and TN subtypes, but not the HER2-enriched subtype (Fig. 7 A). ROC analysis highlighted the superior diagnostic efficacy of circ_0001756 for the luminal B subtype (AUC: 0.888, specificity = 76.5%, and sensitivity = 98%, Fig. 7 B-D). Various proteins and circRNAs in saliva have been reported as useful biomarkers (Zhao SY et al. 2018 ; Freitas AJA et al. 2022 ). Therefore, the expression levels of circ_0001756 in the saliva of three preoperative BC patients and three healthy women were compared. The results revealed no statistically significant difference in circ_0001756 levels between the two groups (p > 0.05) (Fig. S5 ), which could be attributed to the limited sample size. Hence, further studies are needed to investigate the diagnostic potential of circ_0001756 in serum, saliva, and other bodily fluids. Discussion Since first described by Sanger HL et al in 1975 (1976), circRNAs have garnered significant attention in the field of cancer research. With the rapid development of bioinformatics, the roles of many circRNAs have been gradually elucidated (Hansen TB. 2018 ). Numerous studies have reported aberrant expression of circRNAs in tumor tissues (Wei W et al. 2020 ; Liu T et al. 2020 ) and influence on tumor cell growth, apoptosis, invasion, and metastasis, highlighting potential as novel biomarkers. To identify dysregulated circRNAs in BC, the GEO circRNA microarray dataset was referenced to assess differential expression of circ_0001756. Surprisingly, circ_0001756 expression was significantly correlated with tumor size and TNM stage in BC. Recent studies have shown that circ_0001756 promotes cisplatin resistance and malignant potential of ovarian cancer cells via the circ_0001756/miR-338-3p/CHTOP signaling axis (Cao Y et al. 2021 ). However, the role of circ_0001756 in BC has not been reported. The structure of circRNAs is distinct from that of linear mRNA counterparts (Jeck WR et al. 2013 ). Back-to-back reverse primers were designed specifically for the splicing site of circ_0001756. The specificity of the designed primers and identity of the amplified products were confirmed by agarose gel electrophoresis and qPCR analysis. Previous reports have confirmed that circRNAs can resist digestion by RNase R (Cheng ZF et al. 2003; Szabo L et al. 2016). In the present study, RNase R digestion had relatively little effect on circ_0001756. In addition, since circRNAs are formed by reverse splicing of pre-mRNAs, gDNA contains no complementary sites for the primers. Agarose gel electrophoresis confirmed that circ_0001756 is amplified from cDNA, but not gDNA. Collectively, these findings established a foundation for further study of the role of circ_0001756 in BC. The results of this study show that circ_0001756 is aberrantly expressed in different BC cells, and expression in BC tissues is significantly associated with TNM stage and tumor size. Moreover, low expression of circ_0001756 was related to decreased viability of BC cells. Furthermore, EMT, a fundamental process in tumor biology, plays a pivotal role in the invasive and metastatic capacities of tumor cells (de Farias Morais HG et al. 2022 ; Azimi I et al. 2020 ; Bo H et al. 2013 ). In vitro assays revealed that KD of circ_0001756 increased E-cadherin expression and inhibited expression of N-cadherin and vimentin. In vivo , downregulation of circ_0001756 inhibited tumorigenesis of BC cells in nude mice. Collectively, these results demonstrated that circ_0001756 promoted EMT and metastasis of BC cells. To investigate the potential mechanism of circ_0001756 in BC, RNA was isolated from the nuclear and cytoplasmic fractions of BC cells. The results confirmed that circ_0001756 was distributed in both fractions of BC cells and had the potential to function as a miRNA sponge. The results confirmed that circ_0001756 was distributed in both the cytoplasm and nucleus of BC cells, it had the potential to function as a miRNA sponge. Bioinformatics and dual-luciferase reporter assays found that miR-584-5p was the main target of circ_0001756. Previous studies have reported that circPITX1 regulates malignancy of human glioblastoma via the miR-584-5p/KPNB1 signaling pathway (Cao Y et al. 2021 ) and circSMYD4 modulates the viability of liver cancer cells by adsorbing miR-584-5p (Zhang Y et al. 2020 ). However, the impact of circ_0001756 on the malignant potential of BC cells and possible involvement of miR-584-5p has not been explored. Therefore, in vitro experiments were conducted to investigate the impact of miR-584-5p on BC cells. The results showed that overexpression of miR-584-5p significantly reduced proliferation and metastasis of BC cells. This finding was also confirmed by western blot analysis, which demonstrated that miR-584-5p mimics impeded EMT of MDA-MB-231 cells. Rescue studies showed that the regulatory effects of circ_0001756 in BC cells were partially reversed by the inhibition of miR-584-5p. According to bioinformatics analysis and more added experiments, TRAF6 was selected as a target gene of miR-584-5p. KD of TRAF6 attenuated growth and mobility of BC cells. Rescue studies confirmed that the biological function of miR-584-5p was dependent on targeting TRAF6. The results of this study confirmed that the circ_0001756/miR-584-5p/TRAF6 signaling axis was associated with the development of BC. Several studies have confirmed the association of circRNAs with the pathological type, TNM stage, and lymph node metastasis of BC (Feng Y et al. 2019 ), indicating the potential of circRNAs as novel biomarkers (Li Y et al. 2015 ). In the present study, serum levels of circ_0001756 were relatively increased in BC patients, demonstrating high sensitivity and specificity as a novel biomarker for screening, diagnosis, and prognosis of BC. Remarkably, circ_0001756 was superior to CA 15 − 3 and CEA for screening of BC. The results of ROC analysis highlighted that combining circ_0001756 with CEA and CA 15 − 3 enhanced diagnostic capability. Correlation analysis of clinicopathological data and serum levels of circ_0001756 found that circ_0001756 expression was strongly associated with tumor size, patient age, metastasis status, and TNM stage. As an unexpected finding, downregulation of circ_0001756 expression in the serum of BC patients after surgery implied that circ_0001756 can be used as a reliable indicator of the efficacy of BC surgery. Moreover, circ_0001756 was useful for screening of various BC subtypes with the exception of the HER2-enriched type. Given these findings, circ_0001756 may be an ideal biomarker for BC. As a limitation to this study, we did not acquire enough serum available in animal experiments to verify the expression level of circ_0001756 in the serum of BC-bearing mice. Next, our study will focus on the levels of expression of circ_0001756 in the tumor microenvironment and tumor-immersed immune cells, as well as the saliva samples, to confirm the diagnostic value in BC. Conclusion These findings demonstrated that circ_0001756 promotes carcinogenesis of BC through the circ_0001756/miR-584-5p/TRAF6 signaling axis. Hence, circ_0001756 in serum is a promising biomarker for screening, diagnosis, and prognosis of BC. Abbreviations AUC area under the ROC curve BC breast cancer CCK-8 Cell Counting Kit-8 ceRNA competing endogenous RNA DAB 3,3′-diaminobenzidine DMEM Dulbecco's modified Eagle’s medium EMT epithelial-mesenchymal transition FBS fetal bovine serum gDNA genomic DNA GEO Gene Expression Omnibus HER2 human epidermal growth factor receptor 2 HIPK2 homeodomain-interacting protein kinase-2 KD knockdown LPS lipopolysaccharide miRNA microRNA MUT mutant siRNA short interfering RNA TN triple negative TNM tumor-node-metastasis TRAF6 tumor necrosis factor receptor associated factor 6 WT wild-type Declarations Acknowledgements Not applicable Funding This study was financially supported by the National Natural Science Foundation of China (grant no. 81702078 and 81901599), the Natural Science Foundation of Jiangsu Province (grant no. BK20170356), the Suzhou Science, Education and Health Youth Science and Technology Project (grant no.KJXW2020019), Suzhou City, the Seventh Batch of the “Gusu Talent Program” Youth Top Talent Project, the Project of Nuclear Technology Medical Application Supported by Discipline Construction (grant no. XKTJ-HRC2021001), the Project of State Key Laboratory of Radiation Medicine and Protection, Soochow University (grant no. GZK1202137), the Science and Technology Program of Suzhou (grant no. SKY2021045, SKJY2021097), the Maternal and Child Health Association Project of Jiangsu province (grant no. FYX202123). “National Tutorial System” Training Project for Young Health Backbone Talents in Suzhou (grant no. QNGG2023008). Availability of data and materials All data generated in this study are available upon request from the corresponding author. Author contributions JYW and XXW performed the experiments, analyzed the data, and wrote the manuscript. LYS, LXW, YC, ZW, and XJS helped to perform the experiments. HY and BW designed and supervised the study. All of the authors read and approved the final manuscript. JYW and XXW contributed equally to this work. Ethics approval and consent to participate The protocol of the human study was approved by the Ethics Committee of the Second Affiliated Hospital of Soochow University (approval no. JD-LK-2021-115-01) and conducted in accordance with the ethical principles for medical research involving human subjects described in the Declaration of Helsinki. Prior to inclusion in this study, written informed consent was obtained from all subjects. The protocol of the animal study was approved by the Institutional Animal Care and Use Committee of the Second Affiliated Hospital of Soochow University (approval no. JD-LK-2021-115-01) and conducted in accordance with the Guide for the Care and Use of Laboratory Animals. 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Supplementary Files FigS1.tif Fig. S1 Expression of circ_0001756 in MDA-MB-231 cells was determined by qPCR analysis after stimulating with LPS. FigS2.tif Fig. S2 Schematic of circ_0001756formation via circularization of exon 2 of the HIPK2 gene. The back-splice junction sequences were confirmed by Sanger sequencing. FigS3.tif Fig. S3 Evaluation the interference efficiency of two siRNAs targeting circ_0001756. A and BKD efficiency of two siRNAs from circ_0001756 was verified in MDA-MB-231 and MCF-7 cells. C and D Expression patterns of EMT-related markers (E-cadherin, N-cadherin, and vimentin) after transfection with si-circ_0001756 was determined by qPCR analysis. E and FExpression patterns of circ_0001756 and mHIPK2 in BC cells transfected with si-circ_0001756 was determined by qPCR analysis. FigS4.tif Fig. S4 Expression profiles of apoptosis-related proteins (caspase3, PARP) in MDA-MB-231 cells after transfection with si-circ_0001756 by western blot analysis. FigS5.tif Fig. S5 Saliva expression levels of circ_0001756 in pre-operative BC patients and healthy controls (n=3). TableS17.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3865902","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":267569835,"identity":"821ddf2b-adce-45f7-a93b-827ca2a55709","order_by":0,"name":"Jun-ying Wu","email":"","orcid":"","institution":"The Second Affiliated Hospital of Soochow University","correspondingAuthor":false,"prefix":"","firstName":"Jun-ying","middleName":"","lastName":"Wu","suffix":""},{"id":267569836,"identity":"a6e5bfb4-0c7e-46a4-bfc6-0076d853b80e","order_by":1,"name":"Xi-xi Wu","email":"","orcid":"","institution":"The Second Affiliated Hospital of Soochow University","correspondingAuthor":false,"prefix":"","firstName":"Xi-xi","middleName":"","lastName":"Wu","suffix":""},{"id":267569837,"identity":"5448ede3-6aba-4289-a0d9-22a469b252a3","order_by":2,"name":"Li-yan Shi","email":"","orcid":"","institution":"The First Affiliated Hospital of Soochow University","correspondingAuthor":false,"prefix":"","firstName":"Li-yan","middleName":"","lastName":"Shi","suffix":""},{"id":267569838,"identity":"499155fd-ceef-4bed-8a08-56734a533601","order_by":3,"name":"Ling-xia Wang","email":"","orcid":"","institution":"The Second Affiliated Hospital of Soochow University","correspondingAuthor":false,"prefix":"","firstName":"Ling-xia","middleName":"","lastName":"Wang","suffix":""},{"id":267569839,"identity":"4f39ec4b-31b0-4e74-a3c9-8d4488a5d989","order_by":4,"name":"Zhuo Wang","email":"","orcid":"","institution":"The Second Affiliated Hospital of Soochow University","correspondingAuthor":false,"prefix":"","firstName":"Zhuo","middleName":"","lastName":"Wang","suffix":""},{"id":267569840,"identity":"8ecb8ba8-1308-4d51-b851-0ef947d545e1","order_by":5,"name":"Ying Cao","email":"","orcid":"","institution":"The Second Affiliated Hospital of Soochow University","correspondingAuthor":false,"prefix":"","firstName":"Ying","middleName":"","lastName":"Cao","suffix":""},{"id":267569841,"identity":"f2eeb26a-ac3b-484e-b958-b3ca2099fbfc","order_by":6,"name":"Xue-jun Shao","email":"","orcid":"","institution":"The Children’s Hospital of Soochow University","correspondingAuthor":false,"prefix":"","firstName":"Xue-jun","middleName":"","lastName":"Shao","suffix":""},{"id":267569842,"identity":"3059dd00-9d90-4d35-9753-857d2bb9ebee","order_by":7,"name":"Bo Wang","email":"","orcid":"","institution":"The Second Affiliated Hospital of Soochow University","correspondingAuthor":false,"prefix":"","firstName":"Bo","middleName":"","lastName":"Wang","suffix":""},{"id":267569843,"identity":"229b4b8c-090f-42bb-bd39-ebf1b7c01b2f","order_by":8,"name":"Huan Yang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA10lEQVRIiWNgGAWjYFCCA2wMjA0gBvOBAx8qSNPClnhwxhnirIFp4TE+zNtChHr5xjNmD37usMk3n5Hz4QBvA4M8v9gB/FoYG86YG/aeSbOccyN3wwHJHQyGM2cn4NfCzHDGTJqx7bCBhARQi+EZhgSD2wS0sEG0/AdqyXlwILGNCC08EC0HQFoYDhwkRosEw7Eyyd62ZAMJnmcGBxvOSBD2i/yMw9skfrbZGUiwJz/+/KfCRp5fmoAWBokTBhCGQALEVsKAv/0BlHGACNWjYBSMglEwIgEACaxITAurkf0AAAAASUVORK5CYII=","orcid":"","institution":"The Second Affiliated Hospital of Soochow University","correspondingAuthor":true,"prefix":"","firstName":"Huan","middleName":"","lastName":"Yang","suffix":""}],"badges":[],"createdAt":"2024-01-15 08:48:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3865902/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3865902/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":49826695,"identity":"718b5e43-2ad4-499a-9cbd-1a1584fc6862","added_by":"auto","created_at":"2024-01-18 15:54:14","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":648323,"visible":true,"origin":"","legend":"\u003cp\u003eVerification of\u003cstrong\u003e \u003c/strong\u003ecirc_0001756 in BC cells and tissues. \u003cstrong\u003eA\u003c/strong\u003e Volcano plot showing dysregulation of circRNAs in BC tissues as compared to adjacent normal tissues, as determined by analysis of the GEO dataset (fold change \u0026gt; 1.5, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). The red spots in the upper right and upper left represent upregulated and downregulated circRNAs, respectively. \u003cstrong\u003eB\u003c/strong\u003e Expression of circ_0001756 was confirmed in 21 pairs of BC tissues and adjacent normal tissues (*\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). \u003cstrong\u003eC\u003c/strong\u003e The expression profiles of circ_0001756 in MDA-MB-231 and MCF-7 were clarified by qPCR\u003cstrong\u003e \u003c/strong\u003eanalysis\u003cstrong\u003e \u003c/strong\u003e(**\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01). \u003cstrong\u003eD\u003c/strong\u003e and \u003cstrong\u003eE\u003c/strong\u003eExpression of circ_0001756 in BC tissues was positively correlated with tumor size (*\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05) and TNM stage (**\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01). \u003cstrong\u003eF\u003c/strong\u003e The existence of circ_0001756 from cDNA and gDNA of MDA-MB-231 cells was detected by conventional PCR analysis and agarose gel electrophoresis. Divergent primers amplified circ_0001756 from cDNA but not gDNA. \u003cstrong\u003eG\u003c/strong\u003e, \u003cstrong\u003eH\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003eand\u003cstrong\u003e I\u003c/strong\u003e Expression of circ_0001756 and GAPDH after RNase R treatment were detected by agarose gel electrophoresis followed by conventional PCR and qPCR analyses (***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001). The data are presented as the mean ± standard deviation (SD), *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-3865902/v1/03215d2dcf97fc57b5e6011d.png"},{"id":49826077,"identity":"f6e21a65-0745-47ee-b153-d13635cc132a","added_by":"auto","created_at":"2024-01-18 15:46:14","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":975846,"visible":true,"origin":"","legend":"\u003cp\u003eKD of circ_0001756 inhibited proliferation, migration, and invasion of BC \u003cem\u003ein vitro\u003c/em\u003e. \u003cstrong\u003eA\u003c/strong\u003e, \u003cstrong\u003eB\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003eand\u003cstrong\u003e C\u003c/strong\u003e The CCK-8 and colony formation assays were used to assess viability and proliferation of BC cells transfected with siRNA. \u003cstrong\u003eD\u003c/strong\u003e, \u003cstrong\u003eE\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003eand\u003cstrong\u003e F\u003c/strong\u003e The wound healing and migration assays were conducted to detect migration of BC cells transfected with siRNA. \u003cstrong\u003eG\u003c/strong\u003e The transwell Matrigel invasion assay was used to detect invasive capacity of BC cells transfected with siRNA. \u003cstrong\u003eH\u003c/strong\u003eExpression profiles of EMT-related proteins (E-cadherin, N-cadherin, and vimentin) in MDA-MB-231 cells after transfection with si-circ_0001756 by western blot analysis. GAPDH was used as a housekeeping gene. The data are presented as the mean ± SD, *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-3865902/v1/101f7085847bef605bcfd1ea.png"},{"id":49826696,"identity":"50ba7c53-8056-4263-89d7-969be59e78f4","added_by":"auto","created_at":"2024-01-18 15:54:14","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":642700,"visible":true,"origin":"","legend":"\u003cp\u003eKD of circ_0001756 inhibits the proliferation, migration, and invasion of BC \u003cem\u003ein vivo\u003c/em\u003e. \u003cstrong\u003eA\u003c/strong\u003e Representative images of xenograft tumors of the si-NC and si-circ_0001756 groups. \u003cstrong\u003eB\u003c/strong\u003e and \u003cstrong\u003eC\u003c/strong\u003e Downregulation of circ_0001756 decreased the weight and volume of xenograft tumors. \u003cstrong\u003eD\u003c/strong\u003e and \u003cstrong\u003eE\u003c/strong\u003eThe expression levels of E-cadherin, N-cadherin, and vimentin in tumors of nude mice were detected by qPCR and immunohistochemical analyses (×200 and ×400, scale bar, 50 μm). GAPDH was used as a housekeeping. The data are presented as the mean ± SD, *\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-3865902/v1/c05d5b1cdc1aade498748b32.png"},{"id":49826078,"identity":"517f8789-7a61-455d-b3c1-65ee4348a1bf","added_by":"auto","created_at":"2024-01-18 15:46:14","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1094619,"visible":true,"origin":"","legend":"\u003cp\u003eCirc_0001756 acts as a miR-584-5p sponge.\u003cstrong\u003e A \u003c/strong\u003eand\u003cstrong\u003e B\u003c/strong\u003e Presence of circ_0001756 in the nuclear and cytoplasmic fractions of BC cells. U6 and GAPDH were used as controls for the nuclear and cytoplasmic fractions, respectively. \u003cstrong\u003eC\u003c/strong\u003e Venn diagram showing the overlap of target miRNAs of circ_0001756 was predicted with the CircBank and CircInteractome databases. \u003cstrong\u003eD\u003c/strong\u003eSchematic model of WT and MUT sequences of putative binding sites of circ_0001756. \u003cstrong\u003eE\u003c/strong\u003e The dual-luciferase reporter assays of miR-584-5p and circ_0001756 (*\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). F. Expression of miR-584-5p in MDA-MB-231 cells after transfection with miR-584-5p mimics was determined by qPCR analysis. \u003cstrong\u003eG\u003c/strong\u003eThe viability of MDA-MB-231 cells with transfection of miR-584-5p mimics and mimics NC was measured by colony formation assays (**\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01). \u003cstrong\u003eH\u003c/strong\u003e, \u003cstrong\u003eI,\u003c/strong\u003e and \u003cstrong\u003eJ\u003c/strong\u003e The migration of MCF-7 and MDA-MB-231 cells was measured by wound healing and transwell migration assays (**\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01). \u003cstrong\u003eK\u003c/strong\u003eThe invasive ability of MDA-MB-231 cells was measured by the Matrigel invasion assay (*\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). \u003cstrong\u003eL\u003c/strong\u003eWestern blot detected the expression of E-cadherin, N-cadherin, and vimentin in MDA-MB-231 cells transfected with miR-584-5p mimicsand mimics NC. \u003cstrong\u003eM\u003c/strong\u003e and \u003cstrong\u003eN\u003c/strong\u003e The rescue experiment between miR-584-5p and circ_0001756 in proliferation, migration, and invasion of MDA-MB-231 cells were measured by colony formation, transwell migration, and invasion assays. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-3865902/v1/d59a0d47fcddf60dd5844279.png"},{"id":49826084,"identity":"094da892-59e7-4421-9b27-e51d762dfa81","added_by":"auto","created_at":"2024-01-18 15:46:14","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":819027,"visible":true,"origin":"","legend":"\u003cp\u003eCirc_0001756 acts as a ceRNA to regulate TRAF6. \u003cstrong\u003eA\u003c/strong\u003e Venn diagram detailing the exploration of miR-584-5p downstream targets predicted by TargetScan, mirDIP and miRWalk. \u003cstrong\u003eB\u003c/strong\u003e The expression level of TRAF6 in two BC cells (***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001). \u003cstrong\u003eC\u003c/strong\u003e The expression level of TRAF6 in miR-584-5p mimics and mimics NC groups (***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001). \u003cstrong\u003eD\u003c/strong\u003e Schematic model of WT and MUT sequences of putative binding sites in the 3’UTR of TRAF6. \u003cstrong\u003eE\u003c/strong\u003e The dual-luciferase reporter analysis of TRAF6 and miR-584-5p in 293T cells (**\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01). \u003cstrong\u003eF\u003c/strong\u003e Transwell migration and invasion analyses of MDA-MB-231 cells transfected with si-TRAF6 (***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001). \u003cstrong\u003eG\u003c/strong\u003eExpression levels of E-cadherin, N-cadherin, and vimentin in MDA-MB-231 cells transfected with si-TRAF6 was determined by western blot analysis. \u003cstrong\u003eH\u003c/strong\u003e Rescue experiments identified TRAF6 as a downstream target of miR-584-5p. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-3865902/v1/10d2ad29e81287399a79a81f.png"},{"id":49826080,"identity":"aa22fa8b-6f3b-49d9-9cdb-281a2ea8e697","added_by":"auto","created_at":"2024-01-18 15:46:14","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":133767,"visible":true,"origin":"","legend":"\u003cp\u003eClinical application of circ_0001756 in BC. \u003cstrong\u003eA\u003c/strong\u003e Serum expression levels of circ_0001756 in healthy controls, fibroadenoma patients, and pre- and post-operative BC patients (***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). \u003cstrong\u003eB\u003c/strong\u003e ROC curve analysis of BC patients and healthy controls (AUC: 0.833, 95% CI: 0.762–0.905, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). \u003cstrong\u003eC\u003c/strong\u003e ROC curve analysis of BC and fibroadenoma patients (AUC: 0.772, 95% CI: 0.695–0.849, \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.0001). \u003cstrong\u003eD\u003c/strong\u003e AUC of pre- and post-operative BC patients (AUC: 0.954, 95% CI: 0.914–0.993, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001). \u003cstrong\u003eE\u003c/strong\u003eAUC of the combination of CA 15-3, CEA, and circ_0001756 as serum biomarkers of BC. The combination of CA 15-3, CEA, and circ_0001756 had the highest AUC value for diagnosis of BC (AUC: 0.897, 95% CI: 0.829–0.965, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001) *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-3865902/v1/51ad7a7fa31975629e0af13a.png"},{"id":49826085,"identity":"aacfb491-aad9-4a50-8c07-bb5d3519267c","added_by":"auto","created_at":"2024-01-18 15:46:14","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":93426,"visible":true,"origin":"","legend":"\u003cp\u003eClinical application of circ_0001756 as biomarkers for the major subtypes of BC. \u003cstrong\u003eA\u003c/strong\u003e Serum expression levels of circ_0001756 for the luminal A, luminal B, HER2-enriched, and TN subtypes (***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). \u003cstrong\u003eB\u003c/strong\u003e AUC of the luminal A and healthy control groups (AUC: 0.833, 95% CI: 0.762–0.905, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). \u003cstrong\u003eC\u003c/strong\u003eAUC of the luminal B and healthy control groups (AUC: 0.772, 95% CI: 0.695–0.849, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001). \u003cstrong\u003eD\u003c/strong\u003eAUC of the TN and healthy control groups (AUC: 0.954, 95% CI: 0.914–0.993, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"Fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-3865902/v1/b4460d4501ee4b6188d3975b.png"},{"id":53239565,"identity":"0853c39c-f885-45ea-8bc6-73b9e12075ee","added_by":"auto","created_at":"2024-03-22 09:41:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2203938,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3865902/v1/3ab38d12-51cb-4348-aa01-21ab69fc4ea7.pdf"},{"id":49826081,"identity":"0b565e8e-1765-4c9b-8aa3-9951d5a019ad","added_by":"auto","created_at":"2024-01-18 15:46:14","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":121034,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. \u003c/strong\u003eS1 Expression of circ_0001756 in MDA-MB-231 cells was determined by qPCR analysis after stimulating with LPS.\u003c/p\u003e","description":"","filename":"FigS1.tif","url":"https://assets-eu.researchsquare.com/files/rs-3865902/v1/30ca91c9b4edcd0410460094.tif"},{"id":49826698,"identity":"72ee183c-a843-4b9d-a474-18c67abf7aa0","added_by":"auto","created_at":"2024-01-18 15:54:14","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1744704,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig.\u003c/strong\u003e S2 Schematic of circ_0001756formation via circularization of exon 2 of the HIPK2 gene. The back-splice junction sequences were confirmed by Sanger sequencing.\u003c/p\u003e","description":"","filename":"FigS2.tif","url":"https://assets-eu.researchsquare.com/files/rs-3865902/v1/d677985d1630468f59f6c408.tif"},{"id":49826087,"identity":"3738547d-5fe9-46d8-9567-00f85bd2bc7a","added_by":"auto","created_at":"2024-01-18 15:46:14","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":1719840,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig.\u003c/strong\u003e S3 Evaluation the interference efficiency of two siRNAs targeting circ_0001756. \u003cstrong\u003eA\u003c/strong\u003e and \u003cstrong\u003eB\u003c/strong\u003eKD efficiency of two siRNAs from circ_0001756 was verified in MDA-MB-231 and MCF-7 cells. \u003cstrong\u003eC\u003c/strong\u003e and \u003cstrong\u003eD\u003c/strong\u003e Expression patterns of EMT-related markers (E-cadherin, N-cadherin, and vimentin) after transfection with si-circ_0001756 was determined by qPCR analysis. \u003cstrong\u003eE\u003c/strong\u003e and \u003cstrong\u003eF\u003c/strong\u003eExpression patterns of circ_0001756 and mHIPK2 in BC cells transfected with si-circ_0001756 was determined by qPCR analysis.\u003c/p\u003e","description":"","filename":"FigS3.tif","url":"https://assets-eu.researchsquare.com/files/rs-3865902/v1/e35484bd04d8e3641b71fdda.tif"},{"id":49826082,"identity":"b1fa04ab-be43-49dc-b259-b30af86084bb","added_by":"auto","created_at":"2024-01-18 15:46:14","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":69644,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig.\u003c/strong\u003e S4 Expression profiles of apoptosis-related proteins (caspase3, PARP) in MDA-MB-231 cells after transfection with si-circ_0001756 by western blot analysis.\u003c/p\u003e","description":"","filename":"FigS4.tif","url":"https://assets-eu.researchsquare.com/files/rs-3865902/v1/24f7a1f1ef12be85a7f08b48.tif"},{"id":49826088,"identity":"64efb8a2-1d55-419a-b56a-a76df94c9527","added_by":"auto","created_at":"2024-01-18 15:46:14","extension":"tif","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":91160,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig.\u003c/strong\u003e S5 Saliva expression levels of circ_0001756 in pre-operative BC patients and healthy controls (n=3).\u003c/p\u003e","description":"","filename":"FigS5.tif","url":"https://assets-eu.researchsquare.com/files/rs-3865902/v1/ea174a16c7463980129573ce.tif"},{"id":49826697,"identity":"22a54f7f-81bf-4f90-a391-3481185bc265","added_by":"auto","created_at":"2024-01-18 15:54:14","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":26480,"visible":true,"origin":"","legend":"","description":"","filename":"TableS17.docx","url":"https://assets-eu.researchsquare.com/files/rs-3865902/v1/fdcf68b17d2d2663ce2fca2d.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Circ_0001756, a novel biomarker, promotes breast cancer progression via miR-584-5p/TRAF6 signaling axis","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eBreast cancer (BC) has emerged as the most prevalent cancer in the female population, and treatment poses a huge burden to healthcare systems (Ferlay J et al. 2020). Hence, regular screening for early diagnosis is particularly important to improve prognosis and survival. Therefore, it is essential to identify novel markers of BC and elucidate the functions and mechanisms to improve screening and early diagnosis.\u003c/p\u003e \u003cp\u003eCircular RNAs (circRNAs) are characterized by a covalently closed continuous loop structure without a 5\u0026rsquo;-cap or 3\u0026rsquo;-poly A tail (Salzman J et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) and were initially regarded as aberrant splicing by-products, thus attracting little attention. However, recent advancements in molecular techniques and bioinformatics have clarified the roles of various circRNAs in carcinogenesis and tumor progression. For instance, circRNAs can act as sponges of miRNAs (Hansen TB et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) and some contain intronic sequences that have been implicated in transcription of the parental gene by interacting with RNA polymerase II (Zhang Y et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Additionally, circRNAs with open reading frames and internal ribosome entry sites have the potential to translate proteins (Zhang Y et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). For instance, a recent study demonstrated that circSMARCA5 directly binds to the promoter of parental genes to induce transcription (Xu X et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Also, many circRNAs have been associated with the onset and progression of BC. Notably, circPTK2 is reported to facilitate mammary carcinogenesis via the YAP1/Hippo signaling pathway (Wang X et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) and circCDYL has been shown to augment autophagy and promote progression of BC via the miR-1275-ATG7/ULK1-autophagic axis (Liang G et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). However, the functions and mechanisms of numerous circRNAs implicated in the onset and progression of BC remain largely unknown.\u003c/p\u003e \u003cp\u003eCirc_0001756, which is derived from exon 2 of the HIPK2 gene, has been shown to regulate astrocyte activation in collaboration with autophagy by sponging MIR124\u0026ndash;2HG (Huang R et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Moreover, circ_0001756 is reported to contribute to the functional recovery of neuronal stem cells after ischemic stroke (Wang G et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). A recent study found that circ_0001756 promoted progression of ovarian cancer via activation of the EGFR/MAPK signaling pathway (Ji J et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). A previous study by our group demonstrated that lipopolysaccharide (LPS) stimulation increased the invasive and metastatic capacities of BC cells via the TLR4/NF-κB signaling pathway (Yang H et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). In this study, bioinformatics analysis was conducted in reference to the Gene Expression Omnibus (GEO) database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/geo/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/geo/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to identify differentially expressed circRNAs associated with BC. The results confirmed that circ_0001756 expression was relatively upregulated in BC tissues as compared to adjacent non-cancerous tissues. Moreover, LPS stimulation was found to enhance circ_0001756 expression in epithelial-like (MDA-MB-231) BC cells as compared to human BC (MCF-7) cells, implying that circ_0001756 may be associated with metastasis of BC. Furthermore, an investigation to establish correlations between circ_0001756 in BC tissues and clinicopathological factors found that circ_0001756 expression was related to tumor size and tumor-node-metastasis (TNM) stage. Collectively, these findings indicate that circ_0001756 is associated with progression of BC. Therefore, the aim of the present study was to investigate the involvement of the circ_0001756/miR-584-5p/TRAF6 signaling axis in progression of BC and assess the clinical usefulness of circ_0001756 as a potential biomarker.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003eClinical tissues, serum samples, saliva samples, and BC cell lines\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eThe protocol of the human study was approved by the Ethics Committee of the Second Affiliated Hospital of Soochow University (approval no. JD-LK-2021-115-01) and conducted in accordance with the ethical principles for medical research involving human subjects described in the Declaration of Helsinki. Prior to inclusion in this study, written informed consent was obtained from all subjects. Surgically resected cancer tissues and adjacent non-cancerous tissues were obtained from 21 BC patients prior to receiving chemotherapy or radiotherapy and immediately frozen with liquid nitrogen. Serum samples from healthy controls, patients with fibroadenomas, and BC patients both pre- and post-operatively were pretreated and stored at -80\u0026deg;C. Saliva samples were collected and pretreated as described by Zhao et al (Zhao SY et al. \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e). Human embryonic kidney (HEK)-293T, MDA-MB-231, and MCF-7 cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and cultured in high-glucose Dulbecco\u0026apos;s modified Eagle\u0026rsquo;s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Gibco \u0026ndash; Invitrogen Corporation, Carlsbad, CA, USA).\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003eTransfection\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eCells were transfected with short interfering RNA (siRNA), microRNA (miRNA) inhibitors, and miRNA mimics (GenePharma Co., Ltd., Suzhou, China) using Lipofectamine 2000 reagent (Invitrogen Corporation) and harvested after 48 h. The RNA sequences used in this study are listed in Tables S1, S2, and S3.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003eReal-time quantitative PCR (qPCR)\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eTotal RNA was exacted from BC cells and tissues using TRIzol reagent (Invitrogen Corporation) and the nuclear and cytoplasmic fractions were separated with a Cytoplasmic \u0026amp; Nuclear RNA Purification Kit (Norgen Biotek Corp. Thorold, ON, Canada). Total RNA was isolated from serum and saliva samples using TRIzol LS reagent (Invitrogen Corporation) in accordance with the manufacturer\u0026rsquo;s instructions. The isolated RNA was reverse-transcribed into complementary DNA (cDNA) using the HiScript\u0026reg; III 1st Strand cDNA Synthesis Kit (+\u0026thinsp;gDNA wiper), and the cDNA was quantitated using AceQ\u0026reg; qPCR SYBR Green Master Mix (Vazyme Biotech Co., Ltd., Nanjing, China) with the primers (Sangon Biotech Co., Ltd., China) listed in Table \u003cspan class=\"InternalRef\"\u003eS4\u003c/span\u003e, Relative expression of mRNA was calculated using the 2\u003csup\u003e-\u0026Delta;\u0026Delta;Ct\u003c/sup\u003e method with U6 or GAPDH as an internal control. The primers of miRNA for reverse transcription and qPCR analysis were designed using MiRNA Design V 1.01 software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.vazyme.com/companyfile/653.html\u003c/span\u003e\u003c/span\u003e). The sequences were listed in Tables S5 and S6.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003eDual-luciferase reporter assay\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eHEK-293T cells (5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e) were seeded in the wells of 12-well plates, cultured for 24 h, and then transfected with mutant (MUT) or wild-type (WT) circ_0001756 reporter vectors, and miR-584-5p mimics. After 24 h, firefly luciferase activity was measured using the Dual-Glo\u0026reg; Luciferase Assay System (Promega Corporation, Madison, WI, USA). Renilla luciferase was used as a reference.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003eRNase R treatment\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eThe resistance of circular RNA to RNase was confirmed using Ribonuclease R (Epicentre Technologies Corporation, Madison, WI, USA). Briefly, RNA (2 \u0026micro;g) was treated with Ribonuclease R (6 U) at 37\u0026deg;C for 15 min. The untreated control group was incubated under the same conditions. Then, the enzyme was rendered inactive by heating it at 70\u0026deg;C for 10 min.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003eColony formation assay\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eTransfected cells (1 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e) were cultured in the wells of 6-well plates for approximately 14 days. Cell clusters were fixed with 4% paraformaldehyde, stained with crystal violet (0.5% w/v), washed with phosphate-buffered saline, and imaged.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003eCell counting (CCK-8) assay\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eTransfected cells (2 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e) were suspended in 100 \u0026micro;L of DMEM in the wells of 96-well plates. Every 24 h, 10 \u0026micro;L of CCK-8 reagent (Dojindo Laboratories Co., Ltd., Kumamoto, Japan) were added to the wells and the plate was incubated at 37\u0026deg;C for 2 h. Afterward, absorbance at 450 nm was measured with a spectrophotometer (Tecan Group Ltd., M\u0026auml;nnedorf, Switzerland).\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003eWound healing assay\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eTransfected BC cells (2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e) were plated in the wells of 6-well plates. The confluent monolayers were evenly streaked with a sterile 1-mL pipette tip and then washed with phosphate-buffered saline. Migration of MCF-7 cells at 48 h and MDA-MB-231 cells at 24 h was observed with an inverted microscope.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003eTranswell migration and Matrigel invasion assays\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eThe transwell cell migration assay was performed to assess the chemotactic capability of cells. Briefly, transfected BC cells (1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e) were plated into the wells of 24-well migration chambers (Corning, USA) with 200 \u0026micro;L of low serum medium. Then, DMEM supplemented with 10% FBS was added to the lower compartment. After 8\u0026ndash;10 h, the cells were fixed and stained with crystal. The transwell invasion assay was performed to assess the invasive capacity of cells through an extracellular matrix. Briefly, the chambers were coated with Matrigel. After 24 h, cells in the chambers were harvested and imaged. The mean number of cells in five random fields were counted using ImageJ software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://imagej.net/ij/\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003eWestern blot analysis\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eBC cells were lysed with radioimmunoprecipitation assay buffer containing a proteinase inhibitor (phenylmethylsulfonyl fluoride). The proteins were separated by gel electrophoresis and then transferred to a polyvinylidene fluoride membrane (EMD Millipore Corporation, Billerica, MA, USA), which was blocked for 1 h with 5% (w/v) skim milk and then incubated overnight at 4\u0026deg;C with primary antibodies against E-cadherin (dilution, 1:500; Cell Signaling Technology, Inc., Danvers, MA, USA), N-cadherin (dilution, 1:1000; Cell Signaling Technology, Inc.), vimentin (dilution, 1:1000; Bioworld, Dublin, OH, USA), TRAF6 (dilution, 1:1000; Cell Signaling Technology, Inc.), and GAPDH (dilution, 1:1000; Cell Signaling Technology, Inc.) followed by horseradish peroxidase-conjugated secondary antibodies (Multisciences (Lianke) Biotech Co., Ltd., Hangzhou, China) for 1 h at room temperature. Afterward, the protein bands were visualized using chemiluminescence reagent and imaged.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003eImmunohistochemical analysis\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eTissues harvested from nude mice were sliced, dewaxed, rehydrated, mounted on glass slides, and incubated with primary antibodies against epithelial-mesenchymal transition (EMT)-related proteins at 4\u0026deg;C overnight, followed by biotin-labeled secondary antibodies at 37\u0026deg;C for 1 h. Afterward, the tissues were stained with hematoxylin and observed under a microscope.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003eConstruction of subcutaneous a xenograft BALB/c-nu mouse model\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eThe protocol of the animal study was approved by the Institutional Animal Care and Use Committee of the Second Affiliated Hospital of Soochow University (approval no. JD-LK-2021-115-01) and conducted in accordance with the Guide for the Care and Use of Laboratory Animals. Six-week-old BALB/c nude mice were subcutaneously injected with MDA-MB-231 cells (5 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e) via the mammary fat pad. The width and length of the tumors in each group were measured once every 3 days. After 21 days, the nude mice were euthanized by cervical dislocation under 3% isoflurane deep anesthesia, and the xenografts were photographed and weighted. The tumor volume was calculated according to the formula 0.5 \u0026times; \u003cem\u003eL\u003c/em\u003e \u0026times; \u003cem\u003eW\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e. \u003cem\u003eL\u003c/em\u003e denotes the longitudinal diameter, and \u003cem\u003eW\u003c/em\u003e denotes the latitudinal diameter. Then, two small pieces of tissue specimens were used to extract RNA and immunohistochemical analysis. The remaining tissues were stored at -80\u0026deg;C. All animals in this study were euthanized at the experimental endpoint. The humane endpoints such as decreased food and water consumption, weight loss reaching 20%, max tumor diameter\u0026thinsp;\u0026gt;\u0026thinsp;20 mm, and so on were not used during the experiment.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003eStatistical analysis\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eStatistical analyses (Student\u0026rsquo;s t-test, Wilcoxon rank-sum test, and one-way analysis of variance) were performed using IBM SPSS Statistics for Windows, version 24.0 (IBM Corporation, Armonk, NY, USA). All figures and tables were generated using Prism 8.0 software (GraphPad Software, Inc., San Diego, CA, USA). The Fisher\u0026apos;s exact test was used to identify correlations between clinical characteristics and expression levels of circ_0001756. A probability (\u003cem\u003ep\u003c/em\u003e) value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003eCirc_0001756 is verified in BC cells and tissues\u003c/h2\u003e\n \u003cp\u003eInitially, the microarray data from the GEO dataset GSE182471 were reviewed to compare the expression patterns of circRNAs in BC tissues (GSM5529874, GSM5529877, and GSM5529878) and adjacent non-cancerous breast tissues (GSM5529869, GSM5529872, and GSM5529873). A corresponding volcano plot was constructed, which is presented in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA. Expression of circ_0001756 was markedly upregulated in BC tissues (fold change\u0026thinsp;\u0026gt;\u0026thinsp;1.5 and \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Subsequent comparison of 21 pairs of BC and adjacent non-cancerous tissues found that the expression levels of circ_0001756 were considerably higher in BC tissues (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB). Moreover, circ_0001756 expression was considerably higher in MDA-MB-231 cells than MCF-7 cells (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC). Stimulation with LPS (4 \u0026micro;g/mL) dramatically upregulated circ_0001756 expression in MDA-MB-231 cells (Fig. \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e). Correlation analysis of the clinicopathological data of 21 BC patients showed that circ_0001756 expression was positively associated with tumor dimension and TNM stage (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD, E; Table. S7). Collectively, these findings suggest that upregulation of circ_0001756 potentially contributes to progression of BC.\u003c/p\u003e\n\u003c/div\u003e\n\u003cp\u003eCirc_0001756 consists of 1084 nucleotides from exon 2 of the HIPK2 gene that form a circular structure (Fig. \u003cspan class=\"InternalRef\"\u003eS2\u003c/span\u003e). Sanger sequencing with divergent primers identified the presence of a back-splicing sequence in the conventional PCR product (Fig. \u003cspan class=\"InternalRef\"\u003eS2\u003c/span\u003e). Since circRNAs are generated by back-splicing of pre-mRNAs, appropriate back-to-back reverse primers were designed. The circRNA-specific primers amplify cDNA-derived products, but nor genomic DNA (gDNA), as confirmed by electrophoresis results (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eF). RNase digestion revealed that circ_0001756 was resistant to RNase R treatment (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eG-I). The GAPDH linear transcript was used as a positive control for validation.\u003c/p\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003eKnockdown (KD) of circ_0001756 inhibited viability of BC cells in vitro\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eTo explore the possible functions of circ_0001756 \u003cem\u003ein vitro\u003c/em\u003e, BC cells were transfected with siRNA targeting the back-splicing site of circ_0001756. The interference efficiency of the siRNA was evaluated by detecting the expression levels of circ_0001756 (Fig. \u003cspan class=\"InternalRef\"\u003eS3\u003c/span\u003eA, B) and EMT-related indicators (Fig. \u003cspan class=\"InternalRef\"\u003eS3\u003c/span\u003eC, D). Due to higher KD efficiency, si-circ_0001756#2 was selected for subsequent functional experiments. The qPCR results showed that si-circ_0001756#2 only targeted circ_0001756 and had little effect on linear HIPK2 (Fig. \u003cspan class=\"InternalRef\"\u003eS3\u003c/span\u003eE, F). Cell proliferation analysis demonstrated that KD of circ_0001756 reduced growth of BC cells (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA-C). The wound healing and transwell migration assays demonstrated that silencing of circ_0001756 inhibited migration of BC cells (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD-F). Additionally, the transwell invasion assay revealed that downregulation of circ_0001756 significantly reduced the metastatic capacity of BC cells (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eG). Moreover, in MDA-MB-231 cells, downregulation of circ_0001756 significantly increased E-cadherin protein levels, but had opposite effects on N-cadherin and vimentin protein levels (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eH). Western blot analysis was also performed to determine whether circ_0001756 is involved in apoptosis. The findings demonstrated that knockdown circ_0001756 knockdown had no effect on apoptosis of MDA-MB-231 cells (Fig. \u003cspan class=\"InternalRef\"\u003eS4\u003c/span\u003e).\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n \u003ch2\u003eKD of circ_0001756 inhibited proliferation and EMT of BC cells in vivo\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eTo explore the functions of circ_0001756 \u003cem\u003ein vivo\u003c/em\u003e, BALB/c-nu mice were subcutaneously injected with MDA-MB-231 cells to induce tumorigenesis. The results showed that KD of circ_0001756 dramatically inhibited growth of subcutaneous tumors (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA). The weight and size of the tumor tissues were reduced in the treatment group as compared to the control group (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB, C). The expression levels of EMT-related genes in subcutaneous tumor tissues were assessed by qPCR analysis. The results revealed that E-cadherin expression was notably increased in the si-circ_0001756#2 group, while the expression levels of N-cadherin and vimentin were substantially reduced (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eD). The results of immunohistochemical and qPCR analyses were consistent (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eE).\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n \u003ch2\u003eCirc_0001756 functions by adsorbing miR-584-5p\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eTo elucidate the potential roles of circ_001756 in the onset and progression of BC, a nucleocytoplasmic distribution assay was conducted to determine the intracellular localization of circ_0001756. The results detected circ_0001756 in both the nucleus and cytoplasm of BC cells (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA, B), suggesting that circ_0001756 acts as a \u0026quot;miRNA sponge\u0026quot;. In addition, searches of the CircBank (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.circbank.cn\u003c/span\u003e\u003c/span\u003e) and CircInteractome (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://circinteractome.nia.nih.gov\u003c/span\u003e\u003c/span\u003e) databases identified nine potential target miRNAs (miR-1178-3p、miR-1261、miR-338-3p、miR-516a-5p、miR-578、miR-581、miR-584-5p、miR-663b、miR-889-3p) of circ_0001756 (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC). Further analysis revealed that miR-584-5p expression levels were considerably lower in BC tissues than adjacent non-cancerous tissues. In addition, KD of circ_0001756 significantly increased miR-584-5p expression levels in BC cells. The dual-luciferase reporter assay confirmed that miR-584-5p mimics reduced luciferase activity (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eD, E), indicating that miR-584-5p directly interacts with circ_0001756.\u003c/p\u003e\n \u003c/div\u003e\n \u003cp\u003eTo validate the functions of miR-584-5p, BC cells were transfected with miR-584-5p mimics to overexpress miR-584-5p. The transfection efficiency was confirmed by qPCR results in MDA-MB-231 cells (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eF). The colony formation assay revealed that miR-584-5p inhibited proliferation of both BC cell lines (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eG). The wound healing and transwell assays demonstrated that overexpression of miR-584-5p significantly impaired migration and attenuated the invasive capacity of BC cells (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eH\u0026ndash;K). Western blot analysis revealed that miR-584-5p mimics increased protein expression of E-cadherin in MDA-MB-231 cells, but had opposite effects on protein expression of N-cadherin and vimentin (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eL).\u003c/p\u003e\n \u003cp\u003eTo further verify that circ_0001756 functions via sponging miR-584-5p, a series of rescue experiments was conducted. The results showed that miR-584-5p inhibitors reversed the attenuation of cell proliferation, migration, and invasion in MDA-MB-231 cells caused by KD of circ_0001756 (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eM, N).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\n \u003ch2\u003eCirc_0001756 acts as a competing endogenous RNA (ceRNA) to regulate TRAF6\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eCircRNAs can regulate downstream target genes by sponging miRNAs. Screening of the TargetScan (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.targetscan.org/vert_80/\u003c/span\u003e\u003c/span\u003e), mirDIP (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://ophid.utoronto.ca/mirDIP/\u003c/span\u003e\u003c/span\u003e) and miRWALK (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://mirwalk.umm.uni-heidelberg.de/\u003c/span\u003e\u003c/span\u003e) databases identified 531 potential target genes of miR-584-5p (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA). The results of Gene Ontology biological process (GO-BP) enrichment analysis confirmed that some terms were highly enriched, such as positive regulation of transcription by RNA polymerase II, intracellular protein transport and mitotic cell cycle. Several pathways including \u0026ldquo;Pathways in cancer\u0026rdquo;, \u0026ldquo;PD-L1 expression and PD-1 checkpoint pathway in cancer\u0026rdquo; and \u0026ldquo;Toll-like receptor signaling pathway\u0026rdquo; were revealed as significantly enriched by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway. Then, we selected some genes (ANGPT2, TRAF6, MDM2, KRAS, PRKAA2) for more analysis. According to the expression in BC cells, we found that TRAF6 expression was markedly higher in MDA-MB-231 cells than MCF-7 cells (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB) and was attenuated by miR-584-5p mimics (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC). According to the dual luciferase reporter assays, the sequence of TRAF6 contains a binding site for miR-584-5p (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD, E). In addition, downregulation of TRAF6 markedly impaired the mobility and invasiveness of MDA-MB-231 cells (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eF). Western blot analysis showed that downregulation of TRAF6 increased E-cadherin protein levels and decreased protein levels of N-cadherin and vimentin (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eG). Collectively, these findings confirm that suppression of TRAF6 reduced proliferation, migration, and invasion of BC cells through EMT-related proteins.\u003c/p\u003e\n \u003c/div\u003e\n \u003cp\u003eRescue experiments were performed to confirm that TRAF6 is a downstream molecular of miR-584-5p. The findings show that downregulation of miR-584-5p increased mobility and invasiveness in MDA-MB-231 cells, whereas co-transfection with si-TRAF6 and a miR-584-5p inhibitor partly reversed this effect (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eH).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\n \u003ch2\u003eClinical application of circ_0001756 in BC\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eTo explore the potential clinical value of circ_0001756 as a biomarker, serum levels of circ_0001756 in healthy controls, patients with fibroadenomas, and BC patients both pre- and post-operatively were measured. Intriguingly, circ_0001756 expression was notably elevated in BC patients as compared to the other three experimental groups (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA). Subsequently, receiver operating characteristic (ROC) curves were generated to assess the diagnostic value of circ_00001756. The area of the ROC curve (AUC) of BC patients and healthy controls was 0.833 (95% confidence interval [CI]: 0.762\u0026ndash;0.905, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eB). The AUC of BC and fibroadenoma patients was 0.772 (95% CI: 0.695\u0026ndash;0.849, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eC). The AUC of pre- and post-operative BC patients was 0.954 (95% CI: 0.914\u0026ndash;0.993, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eD). Then, potential correlations of serum levels of circ_0001756 with the clinicopathological characteristics of 86 BC patients were investigated. The results showed that circ_0001756 expression was markedly linked with tumor size, patient age, lymph node metastases, and TNM stage (Table. 1), indicating the potential of circ_0001756 as a serum biomarker for screening, diagnosis, and prognosis of BC. To further evaluate the diagnostic value of circ_0001756, serum levels of the conventional BC markers cancer antigen 15\u0026thinsp;\u0026minus;\u0026thinsp;3 (CA 15\u0026thinsp;\u0026minus;\u0026thinsp;3) and carcinoembryonic antigen (CEA) were evaluated. The results revealed that the AUC of circ_0001756 (AUC: 0.827, 95% CI: 0.744\u0026ndash;0.909, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) was greater than that of CEA and CA 15\u0026thinsp;\u0026minus;\u0026thinsp;3. The combination of all three indicators for diagnosis of BC had the highest AUC value (AUC: 0.897, 95% CI: 0.829\u0026ndash;0.965, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) with a sensitivity of 77.2% and specificity of 93.6% (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eE). These results indicate that circ_0001756 had better diagnostic performance than CA 15\u0026thinsp;\u0026minus;\u0026thinsp;3 and CEA. The combination of circ_0001756 with CA 15\u0026thinsp;\u0026minus;\u0026thinsp;3 and CEA can improve the accuracy of BC diagnosis. Therefore, circ_0001756 should be considered an ideal index for clinical diagnosis of BC.\u003c/p\u003e\n \u003c/div\u003e\n \u003cp\u003e\u003cstrong\u003eTable. 1\u0026nbsp;\u003c/strong\u003eCorrelation analysis between clinical characteristics and expression levels of circ_0001756 in serum of 86 BC patients\u003c/p\u003e\n \u003ctable id=\"Taba\" border=\"1\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eCharacteristics\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eNo. of patients\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eCirc_0001756 Expression\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cem\u003eP-\u003c/em\u003evalue\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003elow\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ehigh\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eTumor Size (cm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026le;\u0026thinsp;2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" rowspan=\"2\"\u003e\n \u003cp\u003e0.005\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt;\u0026thinsp;2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eAge (years)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026le;\u0026thinsp;50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" rowspan=\"2\"\u003e\n \u003cp\u003e0.010\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt;\u0026thinsp;50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e27\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ePathological Type\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eIDC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" rowspan=\"2\"\u003e\n \u003cp\u003e0.579\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDCIS and others\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eLymph Node Metastasis\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" rowspan=\"2\"\u003e\n \u003cp\u003e0.011\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026ge;\u0026thinsp;1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eT stage\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT0 - I\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" rowspan=\"2\"\u003e\n \u003cp\u003e0.026\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eII - Ⅲ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eHER2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" rowspan=\"2\"\u003e\n \u003cp\u003e0.261\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eIDC: Invasive Ductal Carcinoma\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eDCIS: ductal carcinoma in situ\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003eThe prevalence, prognosis, and current therapeutic strategies significantly differ among the four major subtypes of BC: luminal A, luminal B, human epidermal growth factor receptor 2 (HER2)-enriched, and triple-negative (TN) (Harbeck N et al. \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e). In accordance with the criteria established by the Chinese Society of Clinical Oncology, BC was classified based on expression of the estrogen receptor, progesterone receptor, and HER2. The results of qPCR analysis of these four subtypes showed that circ_0001756 expression was significantly elevated in the luminal A, luminal B, and TN subtypes, but not the HER2-enriched subtype (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eA). ROC analysis highlighted the superior diagnostic efficacy of circ_0001756 for the luminal B subtype (AUC: 0.888, specificity\u0026thinsp;=\u0026thinsp;76.5%, and sensitivity\u0026thinsp;=\u0026thinsp;98%, Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eB-D).\u003c/p\u003e\n \u003cp\u003eVarious proteins and circRNAs in saliva have been reported as useful biomarkers (Zhao SY et al. \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e; Freitas AJA et al. \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e). Therefore, the expression levels of circ_0001756 in the saliva of three preoperative BC patients and three healthy women were compared. The results revealed no statistically significant difference in circ_0001756 levels between the two groups (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Fig. \u003cspan class=\"InternalRef\"\u003eS5\u003c/span\u003e), which could be attributed to the limited sample size. Hence, further studies are needed to investigate the diagnostic potential of circ_0001756 in serum, saliva, and other bodily fluids.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eSince first described by Sanger HL et al in 1975 (1976), circRNAs have garnered significant attention in the field of cancer research. With the rapid development of bioinformatics, the roles of many circRNAs have been gradually elucidated (Hansen TB. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Numerous studies have reported aberrant expression of circRNAs in tumor tissues (Wei W et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Liu T et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) and influence on tumor cell growth, apoptosis, invasion, and metastasis, highlighting potential as novel biomarkers.\u003c/p\u003e \u003cp\u003eTo identify dysregulated circRNAs in BC, the GEO circRNA microarray dataset was referenced to assess differential expression of circ_0001756. Surprisingly, circ_0001756 expression was significantly correlated with tumor size and TNM stage in BC. Recent studies have shown that circ_0001756 promotes cisplatin resistance and malignant potential of ovarian cancer cells via the circ_0001756/miR-338-3p/CHTOP signaling axis (Cao Y et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, the role of circ_0001756 in BC has not been reported.\u003c/p\u003e \u003cp\u003eThe structure of circRNAs is distinct from that of linear mRNA counterparts (Jeck WR et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Back-to-back reverse primers were designed specifically for the splicing site of circ_0001756. The specificity of the designed primers and identity of the amplified products were confirmed by agarose gel electrophoresis and qPCR analysis. Previous reports have confirmed that circRNAs can resist digestion by RNase R (Cheng ZF et al. 2003; Szabo L et al. 2016). In the present study, RNase R digestion had relatively little effect on circ_0001756. In addition, since circRNAs are formed by reverse splicing of pre-mRNAs, gDNA contains no complementary sites for the primers. Agarose gel electrophoresis confirmed that circ_0001756 is amplified from cDNA, but not gDNA. Collectively, these findings established a foundation for further study of the role of circ_0001756 in BC.\u003c/p\u003e \u003cp\u003eThe results of this study show that circ_0001756 is aberrantly expressed in different BC cells, and expression in BC tissues is significantly associated with TNM stage and tumor size. Moreover, low expression of circ_0001756 was related to decreased viability of BC cells. Furthermore, EMT, a fundamental process in tumor biology, plays a pivotal role in the invasive and metastatic capacities of tumor cells (de Farias Morais HG et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Azimi I et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Bo H et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). \u003cem\u003eIn vitro\u003c/em\u003e assays revealed that KD of circ_0001756 increased E-cadherin expression and inhibited expression of N-cadherin and vimentin. \u003cem\u003eIn vivo\u003c/em\u003e, downregulation of circ_0001756 inhibited tumorigenesis of BC cells in nude mice. Collectively, these results demonstrated that circ_0001756 promoted EMT and metastasis of BC cells.\u003c/p\u003e \u003cp\u003eTo investigate the potential mechanism of circ_0001756 in BC, RNA was isolated from the nuclear and cytoplasmic fractions of BC cells. The results confirmed that circ_0001756 was distributed in both fractions of BC cells and had the potential to function as a miRNA sponge. The results confirmed that circ_0001756 was distributed in both the cytoplasm and nucleus of BC cells, it had the potential to function as a miRNA sponge. Bioinformatics and dual-luciferase reporter assays found that miR-584-5p was the main target of circ_0001756. Previous studies have reported that circPITX1 regulates malignancy of human glioblastoma via the miR-584-5p/KPNB1 signaling pathway (Cao Y et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) and circSMYD4 modulates the viability of liver cancer cells by adsorbing miR-584-5p (Zhang Y et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). However, the impact of circ_0001756 on the malignant potential of BC cells and possible involvement of miR-584-5p has not been explored.\u003c/p\u003e \u003cp\u003eTherefore, in vitro experiments were conducted to investigate the impact of miR-584-5p on BC cells. The results showed that overexpression of miR-584-5p significantly reduced proliferation and metastasis of BC cells. This finding was also confirmed by western blot analysis, which demonstrated that miR-584-5p mimics impeded EMT of MDA-MB-231 cells. Rescue studies showed that the regulatory effects of circ_0001756 in BC cells were partially reversed by the inhibition of miR-584-5p.\u003c/p\u003e \u003cp\u003eAccording to bioinformatics analysis and more added experiments, TRAF6 was selected as a target gene of miR-584-5p. KD of TRAF6 attenuated growth and mobility of BC cells. Rescue studies confirmed that the biological function of miR-584-5p was dependent on targeting TRAF6. The results of this study confirmed that the circ_0001756/miR-584-5p/TRAF6 signaling axis was associated with the development of BC.\u003c/p\u003e \u003cp\u003eSeveral studies have confirmed the association of circRNAs with the pathological type, TNM stage, and lymph node metastasis of BC (Feng Y et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), indicating the potential of circRNAs as novel biomarkers (Li Y et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In the present study, serum levels of circ_0001756 were relatively increased in BC patients, demonstrating high sensitivity and specificity as a novel biomarker for screening, diagnosis, and prognosis of BC. Remarkably, circ_0001756 was superior to CA 15\u0026thinsp;\u0026minus;\u0026thinsp;3 and CEA for screening of BC. The results of ROC analysis highlighted that combining circ_0001756 with CEA and CA 15\u0026thinsp;\u0026minus;\u0026thinsp;3 enhanced diagnostic capability. Correlation analysis of clinicopathological data and serum levels of circ_0001756 found that circ_0001756 expression was strongly associated with tumor size, patient age, metastasis status, and TNM stage. As an unexpected finding, downregulation of circ_0001756 expression in the serum of BC patients after surgery implied that circ_0001756 can be used as a reliable indicator of the efficacy of BC surgery. Moreover, circ_0001756 was useful for screening of various BC subtypes with the exception of the HER2-enriched type. Given these findings, circ_0001756 may be an ideal biomarker for BC.\u003c/p\u003e \u003cp\u003eAs a limitation to this study, we did not acquire enough serum available in animal experiments to verify the expression level of circ_0001756 in the serum of BC-bearing mice. Next, our study will focus on the levels of expression of circ_0001756 in the tumor microenvironment and tumor-immersed immune cells, as well as the saliva samples, to confirm the diagnostic value in BC.\u003c/p\u003e "},{"header":"Conclusion","content":" \u003cp\u003eThese findings demonstrated that circ_0001756 promotes carcinogenesis of BC through the circ_0001756/miR-584-5p/TRAF6 signaling axis. Hence, circ_0001756 in serum is a promising biomarker for screening, diagnosis, and prognosis of BC.\u003c/p\u003e "},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAUC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003earea under the ROC curve\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ebreast cancer\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCCK-8\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCell Counting Kit-8\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eceRNA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecompeting endogenous RNA\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDAB\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e3,3\u0026prime;-diaminobenzidine\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDMEM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDulbecco's modified Eagle\u0026rsquo;s medium\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eEMT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eepithelial-mesenchymal transition\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFBS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003efetal bovine serum\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003egDNA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003egenomic DNA\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGEO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGene Expression Omnibus\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHER2\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehuman epidermal growth factor receptor 2\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHIPK2\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehomeodomain-interacting protein kinase-2\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eKD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eknockdown\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLPS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003elipopolysaccharide\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003emiRNA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emicroRNA\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMUT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emutant\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003esiRNA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eshort interfering RNA\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTN\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etriple negative\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTNM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etumor-node-metastasis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTRAF6\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etumor necrosis factor receptor associated factor 6\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eWT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ewild-type\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was financially supported by the National Natural Science Foundation of China (grant no. 81702078 and 81901599), the Natural Science Foundation of Jiangsu Province (grant no. BK20170356), the Suzhou Science, Education and Health Youth Science and Technology Project (grant no.KJXW2020019), Suzhou City, the Seventh Batch of the \u0026ldquo;Gusu Talent Program\u0026rdquo; Youth Top Talent Project, the Project of Nuclear Technology Medical Application Supported by Discipline Construction (grant no. XKTJ-HRC2021001), the Project of State Key Laboratory of Radiation Medicine and Protection, Soochow University (grant no. GZK1202137), the Science and Technology Program of Suzhou (grant no. SKY2021045, SKJY2021097), the Maternal and Child Health Association Project of Jiangsu province (grant no. FYX202123). \u0026ldquo;National Tutorial System\u0026rdquo; Training Project for Young Health Backbone Talents in Suzhou (grant no. QNGG2023008).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll data generated in this study are available upon request from the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJYW and XXW performed the experiments,\u0026nbsp;analyzed the data,\u0026nbsp;and\u0026nbsp;wrote the manuscript. LYS, LXW, YC, ZW, and XJS\u0026nbsp;helped to perform the experiments. HY and BW designed\u0026nbsp;and supervised the study. All of\u0026nbsp;the authors read and approved the final manuscript. JYW and XXW contributed equally to this work.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe protocol of the human study was approved by the Ethics Committee of the Second Affiliated Hospital of Soochow University (approval no. JD-LK-2021-115-01)\u0026nbsp;and conducted in accordance with the ethical principles for medical research involving human subjects described in the Declaration of Helsinki. Prior to inclusion in this study, written informed consent was obtained from all subjects. The protocol of the animal study was approved by the Institutional Animal Care and Use Committee of the Second Affiliated Hospital of Soochow University (approval no. JD-LK-2021-115-01) and conducted in accordance with the Guide for the Care and Use of Laboratory Animals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no conflict of interest to declare regarding the publication of this article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAzimi I, Robitaille M, Armitage K, So CL, Milevskiy MJG, Northwood K, Lim HF, Thompson EW, Roberts-Thomson SJ, Monteith GR (2020) Activation of the ion channel TRPV4 induces epithelial to mesenchymal transition in breast cancer cells. International journal of molecular sciences 21.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBo H, Zhang S, Gao L, Chen Y, Zhang J, Chang X, Zhu M (2013) Upregulation of Wnt5a promotes epithelial-to-mesenchymal transition and metastasis of pancreatic cancer cells. 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Int J Mol Sci 23(17):9952.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFeng Y, Yang Y, Zhao X, Fan Y, Zhou L, Rong J, Yu Y (2019) Circular RNA circ0005276 promotes the proliferation and migration of prostate cancer cells by interacting with FUS to transcriptionally activate XIAP. Cell death \u0026amp; disease 10:792.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHansen TB, Jensen TI, Clausen BH, Bramsen JB, Finsen B, Damgaard CK, Kjems J (2013) Natural RNA circles function as efficient microRNA sponges. Nature 495:384\u0026ndash;388.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang R, Zhang Y, Han B, Bai Y, Zhou R, Gan G, Chao J, Hu G, Yao H (2017) Circular RNA HIPK2 regulates astrocyte activation via cooperation of autophagy and ER stress by targeting MIR124-2HG. 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RNA 19:141\u0026ndash;157.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiang G, Ling Y, Mehrpour M, Saw PE, Liu Z, Tan W, Tian Z, Zhong W, Lin W, Luo Q et al (2020) Autophagy-associated circRNA circCDVL augments autophagy and promotes breast cancer progression. Molecular cancer 19:65.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu T, Ye P, Ye Y, Lu S, Han B (2020) Circular RNA hsa_circRNA_002178 silencing retards breast cancer progression via microRNA-328-3p-mediated inhibition of COL1A1. Journal of cellular and molecular medicine 24:2189\u0026ndash;2201.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi Y, Zheng Q, Bao C, Li S, Guo W, Zhao J, Chen D, Gu J, He X, Huang S (2015) Circular RNA is enriched and stable in exosomes: A promising biomarker for cancer diagnosis. Cell research 25:981\u0026ndash;984.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSalzman J, Gawad C, Wang PL, Lacayo N, Brown PO (2012) Circular RNAs are the predominant transcript isoform from hundreds of human genes in diverse cell types. PloS one 7(2):e30733.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSanger HL, Klotz G, Riesner D, Gross HJ, Kleinschmidt AK (1976) Viroids are single-stranded covalently closed circular RNA molecules existing as highly base-paired rod-like structures. Proc Natl Acad Sci USA 73:3852\u0026ndash;3856.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSzabo L, Salzman J (2016) Detecting circular RNAs: Bioinformatic and experimental challenges. Nature reviews. Genetics 17:679\u0026ndash;692.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang X, Ji C, Hu J, Deng X, Zheng W, Yu Y, Hua K, Zhou X, Fang L (2021) Hsa_circ_0005273 facilitates breast cancer tumorigenesis by regulating YAP1-hippo signaling pathway. Journal of experimental \u0026amp; clinical cancer research: CR 40:29.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang G, Han B, Shen L, Wu S, Yang L, Liao J, Wu F, Li M, Leng S, Zang F et al (2020) Silencing of circular RNA HIPK2 in neural stem cells enhances functional recovery following ischaemic stroke. EBioMedicine 52:102660.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWei W, Mo X, Yan L, Huang M, Yang Y, Jin Q, Zhong H, Cao W, Wu K, Wu L et al (2020) Circular RNA profiling reveals that circRNA_104433 regulates cell growth by targeting miR-497-5p in gastric cancer. Cancer management and research 12:15\u0026ndash;30.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu X, Zhang J, Tian Y, Gao Y, Dong X, Chen W, Yuan X, Yin W, Xu J, Chen K et al (2020) CircRNA inhibits DNA damage repair by interacting with host gene. Molecular cancer 19:128.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang H, Wang B, Wang T, Xu L, He C, Wen H, Yan J, Su H, Zhu X (2014) Toll-like receptor 4 prompts human breast cancer cells invasiveness via lipopolysaccharide stimulation and is overexpressed in patients with lymph node metastasis. PloS one 9(10):e109980.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang Y, Zhang XO, Chen T, Xiang JF, Yin QF, Xing YH, Zhu S, Yang L, Chen LL (2013) Circular intronic long noncoding RNAs. Molecular cell 51:792\u0026ndash;806.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang Y, Jiang J, Zhang J, Shen H, Wang M, Guo Z, Zang X, Shi H, Gao J, Cai H et al (2021) CircDIDO1 inhibits gastric cancer progression by encoding a novel DIDO1-529aa protein and regulating PRDX2 protein stability. Molecular cancer 20:101.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao SY, Wang J, Ouyang SB, Huang ZK, Liao L (2018) Salivary Circular RNAs Hsa_Circ_0001874 and Hsa_Circ_0001971 as Novel Biomarkers for the Diagnosis of Oral Squamous Cell Carcinoma. Cell Physiol Biochem 47(6):2511\u0026ndash;2521.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang Y, Wang H, Li C, Gao L, Zheng Y, Chang W, Lu C, Zhao X (2020) CircSMYD4 regulates proliferation, migration and apoptosis of hepatocellular carcinoma cells by sponging miR-584-5p. Cancer cell international 20:556.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Circ_0001756, Breast cancer, MiR-584-5p, TRAF6, Metastasis, Biomarker","lastPublishedDoi":"10.21203/rs.3.rs-3865902/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3865902/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003eCircular RNAs (circRNAs) appear to exert critical functions in breast cancer (BC). The objective of this study is to explore the usefulness of circRNAs as potential diagnostic and prognostic biomarkers of BC.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThe Gene Expression Omnibus database was referenced to identify differentially expressed circRNAs in BC. We found that circ_0001756 was associated with the malignant potential of BC. Also, the expression levels of circ_0001756 in BC tissues and cell lines were determined by real-time quantitative polymerase chain reaction analysis. The functions of circ_0001756 were investigated both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. The luciferase reporter and rescue assays were used to clarify the molecular mechanisms of circ_0001756. Additionally, the clinical value of circ_0001756 as a serum biomarker and potential correlations with the clinicopathological characteristics of BC patients were investigated.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eCirc_0001756 expression was upregulated in BC tissues and substantially correlated with tumor size and tumor-node-metastasis (TNM) stage. Knockdown of circ_0001756 markedly inhibited the malignant potential of BC both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. Mechanistically, circ_0001756 acted as a miR-584-5p sponge to regulate TRAF6 in BC cells. Serum levels of circ_0001756 were significantly higher in pre-operative BC patients than in healthy controls, fibroadenoma patients, and post-operative BC patients. Also, serum circ_0001756 was remarkably correlated with tumor size, patient age, metastasis state, and TNM stage. The combination of the traditional tumor markers carcinoembryonic antigen and cancer antigen 15\u0026thinsp;\u0026minus;\u0026thinsp;3 with circ_0001756 significantly improved the diagnostic accuracy of BC.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eCirc_0001756 promotes the malignancy of BC through the miR-584-5p/TRAF6 signaling axis. Additionally, serum circ_0001756 is a promising biomarker for screening and diagnosis of BC.\u003c/p\u003e","manuscriptTitle":"Circ_0001756, a novel biomarker, promotes breast cancer progression via miR-584-5p/TRAF6 signaling axis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-18 15:46:09","doi":"10.21203/rs.3.rs-3865902/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"18148d01-bf8a-4f42-a882-73b01b9cc07a","owner":[],"postedDate":"January 18th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-04-03T05:28:08+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-18 15:46:09","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3865902","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3865902","identity":"rs-3865902","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}