Circular RNA hsa_circ_0006522 correlates with unfavorable prognosis and promotes the proliferative, migrative, and invasive abilities of breast cancer cells

Introduction

Breast cancer is one of the most prevalent malignancies among women (1), and its incidence has witnessed a sharp increase in recent years (2). According to the latest epidemiological statistics, there are approximately 2.5 million breast cancer patients in China, and the incidence of breast cancer has ranked first among female malignancies (2). Although remarkable progress has been achieved in the diagnosis and treatment of breast cancer over the past few years, there are still some patients who unknowingly progress to the metastatic stage after treatment. Therefore, within breast oncology clinical practice, identifying novel molecular interventions capable of enhancing cure probability and extending patient survival timelines constitutes an immediate priority. Accumulating evidence underscores non-coding RNAs among diverse biomolecules as pivotal regulators orchestrating breast cancer pathogenesis, from tumorigenesis to metastatic dissemination (3-5).

Circular RNAs (circRNAs) are a type of non-coding RNA molecule. They are characterized by a covalently closed continuous loop, lacking the 5'-3' polarity and poly A tail (6). Due to their unique structure, circRNAs are highly stable within cells and are not easily degraded by nucleases such as RNase R and actinomycin D (Act D) (7). In the past few decades, circRNAs were initially considered as by-products of splicing errors (8). However, with the rapid development of high-throughput sequencing technology in recent years, an increasing number of circRNAs have been successfully detected. These circRNAs display features of developmental regulation, specific localization, and tissue-specific expression (9). There is a wide variety of circRNAs in mammalian cells, which are specifically expressed during particular stages of tissue development. They play crucial roles in numerous physiological and pathological processes. Their functions are diverse, including acting as a microRNA (miRNA) sponge, regulating gene transcription, interacting with RNA-binding proteins, and participating in protein translation (10-14). Some circRNAs, like ciRS-7, have been confirmed to be closely associated with the occurrence and development of various tumors (15). Studies have demonstrated that circRNAs are linked to the progression of various malignancies, including lung cancer, esophageal cancer, and colorectal cancer (16-18).

Currently, the potential roles of circRNAs in breast cancer are gradually emerging as a novel research hotspot. Emerging studies have demonstrated that circRNAs play an essential role in the occurrence and development of breast cancer (19-35). For example, circUBAP2 is upregulated in cisplatin-resistant triple-negative breast cancer (TNBC) cells. It can enhance the resistance of TNBC cells to cisplatin by sponging miR-300. This action leads to the upregulation of the anti-silencing function 1B (ASF1B) gene expression, activates the PI3K/AKT/mTOR (PAM) signaling pathway, and ultimately enhances the resistance of TNBC cells to cisplatin (36). As a molecular sponge of miR-485-3p, circ_0000129 can weaken the inhibitory effect of miR-485-3p on the spindlin 1 (SPIN1) gene, thereby promoting the progression of breast cancer (37). Overexpression of circPRMT5 can promote the proliferation, invasion, and migration abilities of michigan cancer foundation-7 (MCF-7) cells. Conversely, knocking down circPRMT5 can inhibit the proliferation, invasion, and migration of these cells (38). In the circBase database, circRNA hsa_circ_0006522 is transcribed from exon 3 of the host gene EFR3 homolog A (EFR3A), which is located on chromosome 8. Currently, no studies have been reported on the relationship between hsa_circ_0006522 and breast cancer. In this study, we discovered that the high expression of hsa_circ_0006522 was closely associated with lymphatic metastasis and poor prognosis in breast cancer patients. Further in-depth studies suggested that hsa_circ_0006522 promoted the proliferation, migration, and invasion of breast cancer cells. We present this article in accordance with the MDAR reporting checklist (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-313/rc).

Methods

Tissue microarray

Eighty-nine breast cancer tissues derived from HBreD140Su03 tissue microarray were purchased from Shanghai Outdo Biotech Company. All tissue samples and clinical data were collected with informed patient consent and approved by the institutional ethics committee of Shanghai Outdo Biotech Company (No. YB M-05-02). The study was approved by the Fourth Affiliated Hospital of Hebei Medical University’s ethics committee (No. 2023 KY012). The clinicopathological characteristics and survival status of the patients, including age, tumor size, estrogen receptor (ER) status, progesterone receptor (PR) status, human epidermal growth factor receptor 2 (HER2) status, programmed cell death ligand 1 (PD-L1) status, were obtained from the follow-up data. All malignant clinical data samples were primary female breast cancer (from 29 to 87 years old), and none of the patients had received any therapeutic interventions, including chemotherapy or radiotherapy.

Vector construction and cell transfection

The full-length of hsa_circ_0006522 sequence was inserted into the pLC5-ciR vector (Geenseed Biotech, Guangzhou, China) to construct overexpression vector, and small interfering RNAs (siRNAs) targeting back splice junction of hsa_circ_0006522 (siRNA-1, siRNA-2, siRNA-3) were synthesized (Geenseed Biotech, Guangzhou, China) for knockdown of hsa_circ_0006522.

Fluorescence in situ hybridization (FISH)

FISH was performed using probes specific for the hsa_circ_0006522 sequences. The Cy3-labeled hsa_circ_0006522 probe were purchased from RiboBio Corporation (Guangzhou, China). For FISH in tissues, the tissue microarray slides were deparaffinized in xylene and ethanol solutions. In the following day, the microarray was washed with PBS and fixed with 4% paraformaldehyde. After prehybridization, the microarray was hybridized with hsa_circ-0006522 probe overnight at 37 ℃. Hsa_circ_0006522 fluorescence was visualized with Cy3-Streptavidin Conjugate (ZyMAX™ Grade, Invitrogen, Carlsbad, USA). Post-staining nuclei with 4',6-diamidino-2-phenylindole (DAPI) (10-minute incubation), sequential images of tissue samples were acquired on a Zeiss LSM 900 confocal microscope.

Cell culture

Human breast cancer cell lines MDA-MB-231 and MDA-MB-453 were cultured in DMEM media (GIBCO, Grand Island, USA), supplemented with 10% fetal bovine serum (FBS) (GIBCO), and 1% penicillin-streptomycin solution. BT-549 cells were grown in RPMI1640 media (GIBCO), supplemented with 10% FBS, and 1% penicillin-streptomycin solution. MCF-7 cells were grown in MEM media (GIBCO), supplemented with 10% FBS (GIBCO), and 1% penicillin-streptomycin solution. Cell culture was maintained at 37 ℃ under a humidified atmosphere containing 5% CO₂. Their research resource identifiers (RRIDs) are CVCL_0062, CVCL_0418, CVCL_1092, and CVCL_0031.

RNA extraction and actinomycin D assay

Total RNA derived from breast cancer tissues and cells was extracted using TRIzol reagent (Invitrogen), according to the manufacturer’s instructions. For the actinomycin D assay, breast cancer cells were treated with actinomycin D prior to RNA extraction.

Quantitative real-time polymerase chain reaction (qRT-PCR) and the nucleic acid agarose gel electrophoresis

RNA was reverse-transcribed into complementary DNA (cDNA) using GoScript Reverse Transcription System kit (Promega, Madison, USA). The GoTaq qPCR Master Mix (Promega) was used for qRT-PCR. The circRNA and messenger RNA (mRNA) levels were normalized by glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or U6. The fold change in the relative expression of RNAs was calculated using the 2^−ΔΔCt method. The ordinary RT-PCR products were separated using 2% agarose gel and the gray scale of the band was then detected using ultraviolet (UV) irradiation. The primer sequences used for qRT-PCR were listed below: GAPDH: forward primer 5'-CGCTGAGTACGTCGTGGAGTC-3', reverse primer, 5'-GCTGATGATCTTGAGGCTGTTGTC-3'; U6: forward primer 5'-CTCGCTTCGGCAGCACA-3', reverse primer 5'-AACGCTTCACGAATTTGCGT-3'; hsa_circ_0006522: forward primer 5'-AGACCAGTTTTTGCGCATTT-3', reverse primer 5'-GGCCATCTTTTGGATCTTCA-3'; EFR3A: forward primer 5'-ATCCAAAAGATGGCCTTGTG-3', reverse primer 5'-CAACATCCCTGCTCAACCTT-3'. The experiment was repeated three times.

Cell Counting Kit-8 (CCK-8) assay

For cell viability assessment via CCK-8 assay, approximately 4×10³ transfected cells were cultured in 96-well plates and treated with Cell Counting Kit-8 reagent (MCE, Shanghai, China) with 3 replicates per group. On days 0, 1, 2, 3 and 4, 10 µL of CCK-8 solution was added to each well and incubated at 37 ℃ for 2 h. The cell viability was measured by measuring the absorbance at 450 nm. The experiment was repeated three times.

Colony formation assay

Cells were seeded in six-well plates at a density of 2×10³ cells per well in triplicate and were then cultured for 10 days. Once colonies were macroscopically visible, cells were fixed in 4% paraformaldehyde at room temperature and stained with crystal violet solution. Finally, we photographed the cell clones of each well with a camera and counted them under a microscope. The experiment was repeated three times.

Wound healing assay

Breast cancer cells were seeded into a 6-well plate at a density of 2×10⁴ cells/well and cultured for 24 h to allow monolayer formation. Scratches were then made using a 10-µL tip. The wound areas were photographed at 0 and 24 h post-scratch. The wound closure rate was subsequently calculated. The experiment was repeated three times.

Transwell migration and invasion assay

Breast cancer cells were incubated in a serum-free basal medium for 6–8 h to eliminate residual serum. For migration assays, add 800 µL of complete culture medium containing serum to the lower chamber of the Transwell chamber, and inoculate the cell suspension at a density of 2×10⁵ cells/insert into the upper chamber. For invasion assays, inserts were coated with Matrigel basement membrane matrix (BD Biosciences, San Jose, CA, USA). After 24–48 h, fix the cells on the surface of the lower chamber with 4% paraformaldehyde and stain with 1% crystal violet. Finally, the migration and invasion cells were quantified across five randomly selected fields under a microscope at ×100 magnification. The experiment was repeated three times.

Bioinformatics analysis

Circular RNA interactome online database and miRanda were used to predict and annotate the miRNA sponge function. The downstream targets of the three miRNAs were identified based on the TargetScan database. The common downstream targets of the three miRNAs were identified based on the ggplot2 (3.4.4) and VennDiagram (1.7.3) of R software. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Gene Ontology (GO) analyses of hsa_circ_0006522/miRNA pathways were performed by clusterProfiler (4.4.4) and ggplot2 (3.4.4) of R software.

Western blot

We extracted the cell total protein using RadioImmunoPrecipitation Assay (RIPA) lysis buffer coupled with 1% phenylmethylsulfonyl fluoride (PMSF) to inhibit protease activity. The obtained protein was denatured using high temperature and then separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). We then transferred the protein on the gel to the polyvinylidene difluoride (PVDF) membrane (Millipore, Burlington, USA). At room temperature, 5% skim milk powder was incubated for 1 h, and the membrane was cleaned three times with Tris-buffered saline with Tween^® 20 (TBST). The rabbit polyclonal primary antibodies including Vimentin (Proteintech, Chicago, USA, Cat No. 10366-1-AP), E-cadherin (Proteintech, Cat No. 20874-1-AP), N-cadherin (Proteintech, Cat No. 22018-1-AP), and GAPDH (Proteintech, USA, Cat No. 10494-1-AP) were incubated overnight at 4 ℃. After TBST washing, the secondary antibody (CST, Beverly, USA) bound to the corresponding horseradish peroxidase was incubated at 37 ℃ for 1 h and developed using enhanced chemiluminescence (ECL) Plus solution (Solarbio, Beijing, China). The experiment was repeated three times.

Statistical analysis

SPSS software (version 22.0; SPSS Inc., USA) was used to evaluate the statistical significance of the results. Quantitative data were analyzed using t-test. The clinical qualitative data were analyzed using Chi-squared test or Fisher exact probability method. Survival analysis was evaluated by using the Kaplan-Meier log-rank test and Cox regression analysis. All statistical analyses were performed using two-sided tests. Statistical significance was set at P<0.05.

Results

Hsa_circ_0006522 is mainly located in the cytoplasm of breast cancer cells and correlates with lymphatic metastasis and clinical stage

Two specific PCR primer sets targeting ERF3A were meticulously designed and synthesized. Specifically, divergent primers were employed for the specific amplification of circular transcripts, whereas convergent primers were used for the amplification of related linear transcripts. We chose the cDNA obtained through reverse transcription from two breast cancer cell lines (MDA-MB-231 and MDA-MB-453) and the genomic DNA (gDNA) extracted from the genomes of these two cell lines as templates for the PCR reaction. Meanwhile, GAPDH was used as an internal reference gene to guarantee the accuracy and comparability of the experimental results. The results showed that the circular transcripts of EFR3A were only detected in cDNA samples through divergent primer amplification. When convergent primers were used for amplification, the linear transcript of EFR3A could be successfully amplified in both cDNA and gDNA samples (Figure 1A). The connection sequence of the circular transcript hsa_circ_0006522 was verified using Sanger sequencing (Figure 1B). Furthermore, to assess the stability of hsa_circ_0006522, we treated it with actinomycin D. Based on the experimental results after treatment with actinomycin D, hsa_circ_0006522 mRNA exhibited higher stability compared to EFR3A mRNA (Figure 1C).

Figure 1 Characterization of hsa_circ_0006522. (A) Agarose gel electrophoresis of PCR products verifying hsa_circ_0006522 circularization in breast cancer cells using divergent and convergent primers. (B) Sanger sequencing confirming the back-splice junction of hsa_circ_0006522. (C) Relative RNA levels of hsa_circ_0006522 and EFR3A mRNA under treatment with actinomycin D in breast cancer cells. The experiments were repeated three times. ***, P<0.001. mRNA, messenger RNA; PCR, polymerase chain reaction.

To further explore the expression profile of hsa_circ_0006522 in breast cancer tissues and its impact on the malignant progression of breast cancer, we employed FISH analysis technology in combination with tissue microarray to detect the expression of hsa_circ_0006522 in 89 breast cancer tissues. Representative images are shown in Figure 2A. The FISH assay confirmed that hsa_circ_0006522 was mainly localized in the cytoplasm of breast cancer cells. The correlation between the expression level of hsa_circ_0006522 and various clinical characteristics in patients with breast cancer is detailed in Table 1. Briefly, high expression of hsa_circ_0006522 was significantly and positively correlated with lymphatic metastasis (P=0.03) and clinical stage (P=0.02). However, there was no significant correlation with other clinicopathological features such as patient age, tumor size, ER status, PR status, HER2 status, and PD-L1 status. By plotting Kaplan-Meier survival curves for analysis, we found that the overall survival of patients with high hsa_circ_0006522 expression was significantly shorter than that of patients with low expression (P=0.02) (Figure 2B). Stratified analysis across molecular subtypes (triple-negative, HER2-positive, Luminal) revealed that hsa_circ_0006522 expression levels exhibited trends consistent with the overall cohort in terms of overall survival, but no statistically significant differences were observed (Figure S1A-S1C). Additionally, we further conducted univariate and multivariate Cox regression analyses (Table 2). The analysis results indicated that the expression level of hsa_circ_0006522 could serve as an independent prognostic factor for evaluating the prognosis of breast cancer [hazard ratio (HR) =4.472; 95% confidence interval (CI): 1.050–19.048; P=0.04]. These data suggested that hsa_circ_0006522 may play a crucial role in the pathogenesis and progression of breast cancer.

Figure 2 Representative images of FISH and Kaplan-Meier survival curves. (A) The representation pictures of hsa_circ_0006522 expression in 89 breast cancer tissues by using FISH analysis based on tissue microarray. (B) Patients with higher hsa_circ_0006522 expression had a significantly shorter overall survival than patients with low expression (P=0.02). BC, breast cancer; DAPI, 4',6-diamidino-2-phenylindole; FISH, fluorescence in situ hybridization; HE, hematoxylin and eosin.

The overexpression of hsa_circ_0006522 promotes the proliferation, migration, and invasion of breast cancer cells

To investigate the biological function of hsa_circ_0006522 in breast cancer cells, we initially employed real-time fluorescence qRT-PCR technology to measure the expression levels of hsa_circ_0006522 in four breast cancer cell lines. The findings indicated that the expression level of hsa_circ_0006522 was the lowest in MDA-MB-453 cells, whereas it was the highest in MDA-MB-231 cells (Figure 3A).

Figure 3 The overexpression of hsa_circ_0006522 promotes the proliferation, migration, and invasion of MDA-MB-453 breast cancer cells. (A) The expression of hsa_circ_0006522 in four breast cancer cell lines was detected by qRT-PCR. (B) The expression of hsa_circ_0006522 and linear EFR3A mRNA in MDA-MB-453 cells after transfection with hsa_circ_0006522 or pLC5-ciR empty vector were detected by qRT-PCR. (C,D) Proliferative capacity and colony formation ability of MDA-MB-453 cells transfected with either hsa_circ_0006522 or empty pLC5-ciR vector were quantified via CCK-8 and colony formation assays respectively. (E-G) Cell motility and invasive potential under identical transfection conditions were assessed using wound healing assays (×40), transwell migration chambers (crystal violet staining, ×100) and matrigel-based invasion assays (crystal violet staining, ×100). *, P<0.05; **, P<0.01; ***, P<0.001. CCK-8, Cell Counting Kit-8; mRNA, messenger RNA; qRT-PCR, quantitative real-time polymerase chain reaction.

Based on these results, we chose MDA-MB-453 cells and BT-549 as our research subjects and transfected an overexpression plasmid containing hsa_circ_0006522 into these cells. Subsequently, qRT-PCR technology was utilized to assess the transfection efficiency of hsa_circ_0006522 in MDA-MB-453 cells. The experimental outcomes revealed that after transfection with the hsa_circ_0006522 plasmid, the expression level of hsa_circ_0006522 increased significantly, while the expression level of EFR3A mRNA remained relatively unchanged (Figure 3B). CCK8 assay demonstrated that after transfection with hsa_circ_0006522, the proliferative ability of MDA-MB-453 cells was markedly increased (Figure 3C). Colony formation assay demonstrated that, compared to the control group, the colony-formation ability of hsa_circ_0006522-transfected group was significantly improved (Figure 3D). Wound healing assay indicated that after transfection with hsa_circ_0006522, the migration ability of MDA-MB-453 cells was significantly strengthened, suggesting that overexpression of hsa_circ_0006522 can promote cell migration (Figure 3E). Besides, transwell migration and invasion assay further confirmed that overexpression of hsa_circ_0006522 enhanced the migration and invasive capabilities of MDA-MB-453 cells (Figure 3F,3G). The same results were obtained using BT-549 cells (Figure S2A-S2F). In summary, the results of this study demonstrated that the overexpression of hsa_circ_0006522 significantly promoted the proliferation, migration, and invasion of breast cancer cells.

The knockdown of hsa_circ_0006522 suppresses the proliferation, migration, and invasion of breast cancer cells

On the other hand, we transfected siRNA specifically targeting the junction site of hsa_circ_0006522 into MDA-MB-231 cells. Initially, we assessed the interference efficiency of multiple siRNAs in MDA-MB-231 cells using qRT-PCR (Figure 4A). The results indicated that siRNA-2 exhibited the most effective interference and was thus chosen for subsequent experiments. Subsequently, we employed qRT-PCR to measure the knockdown efficiency of hsa_circ_0006522 in MDA-MB-231 cells. The results showed that, in comparison to the transfection of si-NC, the transfection of siRNA targeting hsa_circ_0006522 significantly decreased the expression level of hsa_circ_0006522. Meanwhile, there was no significant alteration in the expression level of EFR3A mRNA (Figure 4B). CCK-8 and colony formation assays revealed that the proliferation ability of MDA-MB-231 cells was notably inhibited after transfection with si-hsa_circ_0006522 (Figure 4C,4D). Moreover, wound healing and transwell assays indicated that the transfection of si-hsa_circ_0006522 significantly suppressed the migration and invasion capabilities of MDA-MB-231 cells (Figure 4E-4G). The same results were obtained using MCF-7 cells (Figure S3A-S3F). In summary, the aforementioned findings revealed that the knockdown of hsa_circ_0006522 suppressed the proliferation, migration, and invasion of breast cancer cells.

Figure 4 The knockdown of hsa_circ_0006522 suppresses the proliferation, migration, and invasion of MDA-MB-231 breast cancer cells. (A) The efficiency of siRNA was evaluated by qRT-PCR in MDA-MB-231. (B) qRT-PCR analysis quantified the knockdown efficiency of hsa_circ_0006522-targeting siRNA in MDA-MB-231 cells. (C,D) Proliferative capacity and colony formation ability of MDA-MB-231 cells transfected with either hsa_circ_0006522 siRNA or NC siRNA were quantified via CCK-8 and colony formation assays respectively (crystal violet staining). (E-G) Cell motility and invasive potential under identical transfection conditions were assessed using wound healing assays (×40), transwell migration chambers (crystal violet staining, ×100) and Matrigel-based invasion assays (crystal violet staining, ×100). *, P<0.05; **, P<0.01. CCK-8, Cell Counting Kit-8; NC, negative control; qRT-PCR, quantitative real-time polymerase chain reaction; siRNA, small interfering RNA.

Annotation of hsa_circ_0006522 biological function

The online website circular RNA interactome (https://circinteractome.nia.nih.gov/index.html) was used to predict putative miRNAs interact with has_circ_0006522. Based on the search results, we selected miRNAs with context+ score percentile more than 98, which include hsa-miR-217, hsa-miR-548b-3p and hsa-miR-769-3p (Table S1). Among these miRNAs, hsa-miR-769-3p had the highest context+ score (−0.411). The binding sites between has_circ_0006522 and miRNAs are shown in Figure 5A. These three miRNAs regulate 461, 3767, and 2603 downstream target protein genes, respectively. Intersection analysis via Venn diagram identified shared targets among all three miRNAs (Figure 5B). GO and KEGG results demonstrated that the hsa_circ_0006522-miRNA axis coordinates downstream gene regulation to mediate multifunctional effects across diverse biological processes and signaling cascades, such as adherens junction, cellular senescence, phosphatidylinositol signaling system (Figure 5C). Since epithelial-mesenchymal transition (EMT) confers the ability of stationary epithelial cells to migrate and invade, this study evaluated whether EMT-labeled proteins (E-cadherin, vimentin, N-cadherin) were altered following hsa_circ_0006522 knockdown and overexpression in MCF-7 and BT-549 breast cancer cells. However, no significant differences were observed in western blot results (Figure 5D).

Figure 5 Annotation of hsa_circ_0006522 biological function. (A) Computational prediction of hsa_circ_0006522-miRNA interactions was performed using circRNA interactome databases and miRanda. (B) Common downstream targets shared by hsa-miR-217, hsa-miR-548b-3p, and hsa-miR-769-3p were identified through Venn analysis. (C) Functional enrichment profiling (GO and KEGG pathways) was conducted for hsa_circ_0006522-regulated miRNAs. (D) Western blot analysis of EMT proteins in MCF-7 and BT-549 cells. Molecule weight: Vimentin (54 kDa), E-cadherin (120 kDa), N-cadherin (130 kDa), and GAPDH (36 kDa). BP, biological process; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; MF, molecullar function.

Discussion

Our study establishes hsa_circ_0006522 as a novel oncogenic circRNA in breast cancer pathogenesis. Three salient findings emerge from this investigation: First, elevated hsa_circ_0006522 expression strongly correlates with lymphatic metastasis (P=0.003) and advanced clinical stage (P=0.007), establishing it as an independent prognostic factor (HR =4.472, P=0.04). Second, functional assays demonstrate its critical role in driving malignant phenotypes - overexpression of hsa_circ_0006522 can significantly stimulate the proliferation, migration, and invasion of breast cancer cells. Conversely, knocking down hsa_circ_0006522 can yield the opposite outcome. Third, bioinformatics analysis predicts miRNA sponge activity through hsa-miR-217/548b-3p/769-3p binding, potentially regulating key pathways like PI3K-AKT signaling.

Notably, our findings align with emerging patterns of oncogenic circRNA function while expanding the molecular repertoire. Similar to circANKS1B (39) and circCDYL (40), hsa_circ_0006522 demonstrates both prognostic significance and functional involvement in metastasis regulation. The cytoplasmic localization pattern we observed through FISH supports the predicted miRNA sponge mechanism, consistent with cytoplasmic circRNAs like ciRS-7 and circCD44 that regulate post-transcriptional processes (15,29).

Since EMT confers the ability of stationary epithelial cells to migrate and invade, we hypothesize that hsa_circ_0006522 may play a positive regulatory role in the EMT process by modulating the activity of miRNAs. However, no significant differences were observed in western blot results. Absence of significant EMT marker changes following hsa_circ_0006522 modulation may stem from biological and technical considerations. First, hsa_circ_0006522 might drive metastasis through non-EMT mechanisms such as miR-769-3p/PDK4-mediated metabolic adaptation or cytoskeletal remodeling, as seen with other circRNAs promoting invasion via matrix degradation or invadopodia formation. Second, compensatory feedback could buffer EMT marker changes while permitting functional invasion. Third, technical limitations include western blot’s inability to detect subcellular protein relocalization or heterogeneous subpopulations undergoing partial EMT. The mesenchymal MDA-MB-231 cells’ low E-cadherin baseline and epithelial MCF-7 cells’ ERα-mediated EMT resistance might further obscure changes. Future investigations employing extended time courses, single-cell profiling, and in vivo models could clarify these context-dependent regulatory dynamics.

Several limitations should be acknowledged. First, while tissue microarray analysis provides clinical correlation, validation in paired normal-tumor samples could strengthen pathological relevance. Second, the miRNA sponge mechanism remains computationally predicted; RIP assays and luciferase reporter experiments are needed for verification. Third, although our in vitro models demonstrated hsa_circ_0006522’s functional impact, the biological significance in tumor microenvironment (TME) regulation requires validation in immunocompetent animal models. Future studies employing murine syngeneic models—particularly the 4T1 cell line in BALB/c mice and E0771 cells in C57BL/6 mice—could critically extend these findings. Such models would enable simultaneous assessment of hsa_circ_0006522’s effects on overall survival, primary tumor formation kinetics, and EMT dynamics within physiologically relevant stromal interactions.

Despite these limitations, our findings significantly advance understanding of circRNA roles in breast cancer. To our knowledge, this is the first report establishing hsa_circ_0006522 as both a prognostic biomarker and functional mediator of breast cancer progression. The concordance between clinical correlations and experimental phenotypes strongly supports its biological significance. Future studies should explore its potential as a therapeutic target, particularly in advanced-stage patients showing high hsa_circ_0006522 expression.

Conclusions

In summary, the expression of hsa_circ_0006522 was detected in the cancerous tissues of breast cancer patients, and its expression level was closely associated with unfavorable pathological indicators. Functionally, hsa_circ_0006522 can promote the growth, migration, and invasion of breast cancer cells. These findings suggest that hsa_circ_0006522 may be a novel potential biomarker and a therapeutic target for breast cancer.

Acknowledgments

We would like to thank all members of Hebei Key Laboratory of Breast Cancer Molecular Medicine and Key Laboratory of Tumor Gene Diagnosis, Prevention and Therapy of Hebei for their support to this work.

Footnote

Reporting Checklist: The authors have completed the MDAR reporting checklist. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-313/rc

Data Sharing Statement: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-313/dss

Peer Review File: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-313/prf

Funding: This work was supported by the Natural Science Foundation of Hebei (No. H2020206199).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-313/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was approved by The Fourth Affiliated Hospital of Hebei Medical University’s ethics committee (No. 2023 KY012).

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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