Circular RNA hsa_circ_0003472 Promotes Pancreatic Ductal Adenocarcinoma Progression and Gemcitabine Resistance Via the mir-1253/ERCC1 Axis

Abstract

Background

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies with limited therapeutic options. Circular RNAs (circRNAs) have emerged as critical regulators of cancer progression; however, the functional role of hsa_circ_0003472 in PDAC remains unexplored.

Methods

Expression of hsa_circ_0003472 was assessed in 30 paired PDAC and adjacent normal tissues and pancreatic cancer cell lines using quantitative RT-PCR. Loss-of-function experiments were performed to evaluate effects on proliferation (CCK-8, EdU), apoptosis (TUNEL, Western blot), migration, and invasion (Transwell assays). Gemcitabine sensitivity was determined by IC50 analysis. Bioinformatic prediction and dual-luciferase reporter assays identified the downstream regulatory axis. Xenograft mouse models validated findings in vivo.

Results

hsa_circ_0003472 was significantly upregulated in PDAC tissues and cell lines. Silencing hsa_circ_0003472 inhibited proliferation, migration, and invasion while promoting apoptosis and enhancing gemcitabine sensitivity. Mechanistically, hsa_circ_0003472 functioned as a competing endogenous RNA by sponging miR-1253, thereby relieving suppression of excision repair cross-complementing group 1 (ERCC1). Rescue experiments confirmed that the oncogenic effects of hsa_circ_0003472 were mediated through the miR-1253/ERCC1 axis. In vivo, hsa_circ_0003472 knockdown significantly reduced tumor growth and recapitulated molecular changes observed in vitro.

Conclusion

hsa_circ_0003472 promotes PDAC progression and chemoresistance through the miR-1253/ERCC1 regulatory axis, representing a potential therapeutic target for this devastating malignancy.

Introduction

Pancreatic ductal adenocarcinoma (PDAC) represents one of the most aggressive malignancies, with fewer than 10% of patients surviving beyond five years [1]. The challenges in diagnosing and treating PDAC are substantial, contributing to its dismal prognosis. Global cancer registry data indicate that PDAC accounted for 458,918 new cases and 41,242 deaths in 2018 [2]. Both incidence and mortality rates continue to rise worldwide, with projections from the American Cancer Society suggesting that PDAC will become the second leading cause of cancer-related death by 2030 [3]. In China, the disease burden has progressively increased, with a 5-year survival rate of merely 7.2% that continues to decline annually [4].

Surgical resection remains the primary curative treatment for PDAC [5]. However, the absence of specific early symptoms and the propensity for micrometastasis at early stages severely limit opportunities for timely intervention. Consequently, more than 80% of patients present with advanced, unresectable disease at diagnosis, rendering local treatments such as radiation therapy insufficient for controlling systemic spread [6]. These clinical realities underscore the urgent need for novel biomarkers enabling early detection and new therapeutic targets for intervention.

Circular RNAs (circRNAs) are a class of non-coding RNAs characterized by their covalently closed-loop structure, which confers remarkable stability compared with linear RNA molecules [7]. These molecules lack the 5′ cap and 3′ polyadenylated tail typical of linear transcripts [8] and exhibit tissue-specific, temporally regulated, and disease-associated expression patterns [9]. CircRNAs have emerged as important regulatory molecules in cancer biology, functioning primarily as competitive endogenous RNAs (ceRNAs) that sequester microRNAs (miRNAs) and modulate gene expression [10]. This sponge activity enables circRNAs to derepress miRNA target genes, thereby influencing diverse cellular processes including proliferation, apoptosis, and metastasis [11]. Beyond miRNA sequestration, circRNAs can regulate gene expression through direct RNA-RNA base pairing, protein binding, and in some cases, translation into functional peptides [12]. The involvement of circRNAs in transcriptional regulation has linked these molecules to the pathophysiology of numerous disorders [13], with particular relevance to colorectal cancer where they serve as potential biomarkers and regulators [14].

The role of circRNAs in PDAC has attracted increasing attention. Specific circRNAs have been identified as dysregulated in PDAC and shown to influence tumor growth and survival through interactions with cancer-associated miRNAs [15–17]. The stable structure of circRNAs and their presence in body fluids make them attractive candidates for non-invasive diagnostic approaches [18, 19]. Recent investigations have demonstrated that hsa_circ_0050102 facilitates PDAC progression by upregulating PPME1 through modulation of miR-218-5p [20]. However, the potential role of hsa_circ_0003472 in PDAC remains unexplored.

MicroRNA-1253 (miR-1253) has been identified as a tumor suppressor in multiple malignancies, including PDAC, where it modulates cell proliferation, apoptosis, and migration by targeting genes involved in DNA repair mechanisms [21]. Among its targets, excision repair cross-complementing group 1 (ERCC1) is particularly relevant, as this gene promotes chemoresistance by facilitating repair of DNA damage induced by therapeutic agents [22]. ERCC1 is the rate-limiting enzyme in the nucleotide excision repair pathway and mainly participates in the repair process of DNA cross-linking damage. Existing studies have shown that the expression of this gene is associated with tumorigenesis and development, as well as resistance to platinum-based chemotherapy drugs [23]. Downregulation of miR-1253 has been associated with aggressive tumor characteristics and poor prognosis across various cancer types [24]. Given that circRNAs can function as miRNA sponges, we hypothesized that hsa_circ_0003472 might regulate PDAC progression through interactions with miR-1253 and its downstream targets.

In the present study, we investigated the biological role of hsa_circ_0003472 in PDAC. Through comprehensive in vitro and in vivo experiments, we examined its effects on PDAC cell behavior and gemcitabine sensitivity, with particular focus on the miR-1253/ERCC1 regulatory axis. Our findings provide insights into the functional significance of hsa_circ_0003472 and its potential as a therapeutic target in PDAC.

Materials and Methods

Patient Recruitment and Tissue Collection

Paired pancreatic ductal adenocarcinoma (PDAC) and adjacent non-cancerous tissues were obtained from 30 patients who underwent surgical resection. The patient cohort comprised individuals aged 57 to 73 years (mean age 61.12 ± 8.27 years), with an equal distribution of males and females. Inclusion criteria encompassed histologically confirmed PDAC without prior neoadjuvant therapy, while patients with concurrent malignancies or systemic diseases were excluded. All specimens were immediately snap-frozen in liquid nitrogen following resection and stored at − 80 °C until RNA and protein extraction. The study protocol received approval from the Ethics Committee of the Second Affiliated Hospital of Nanjing Medical University (permit number 2022-KY-058-01) and was conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained from all participants prior to surgical intervention.

Reagents and Antibodies

Primary antibodies were obtained from Abcam and included antibodies against ERCC1 (ab129267), GAPDH (ab8245), PCNA (ab29), Bax (ab32503), Bcl-2 (ab182858), caspase-9 (ab32539), caspase-3 (ab32150), MMP-2 (ab254516), MMP-9 (ab76003), and Ki-67 (ab15580). Secondary horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG antibody (A0208) was purchased from Beyotime (Beijing, China).

Cell Culture and Maintenance

Human pancreatic cancer cell lines (CFPAC-1, BxPC-3, PANC-1, and ASPC-1) and the immortalized normal pancreatic ductal epithelial cell line HPNE were utilized in this study. All cell lines were authenticated by short tandem repeat profiling and confirmed negative for mycoplasma contamination prior to experimentation. Cells were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal bovine serum at 37 °C in a humidified atmosphere containing 5% CO₂. Culture medium was refreshed every 48 h, and cells were passaged upon reaching 80–90% confluence using 0.25% trypsin-EDTA.

Lentiviral Transfection

For transfection experiments, cells were seeded at a density of 1 × 10⁵ cells per well in 24-well plates and cultured overnight to achieve appropriate confluence. The following day, culture medium was replaced with 500 µL of fresh medium containing 10 µg/mL polybrene, and 10 µL of lentiviral suspension (1 × 10⁸ TU/mL) was added. Lentiviral constructs included miR-1253 mimic, miR-1253 inhibitor, miR-1253 inhibitor combined with sh-hsa_circ_0003472, sh-circ_0003472, and sh-ERCC1. Empty vector controls (sh-NC and mimic-NC) were utilized as negative controls in all transfection experiments. Following 72 h of infection, cells were harvested for subsequent functional analyses, and RNA and protein were extracted for molecular characterization.

Cell Proliferation Assay

Cell proliferation was assessed using the Cell Counting Kit-8 (CCK-8; Beyotime Biotechnology, Shanghai, China) according to the manufacturer’s protocol. Cells were seeded at a density of 1 × 10⁴ cells per well in 96-well plates in RPMI 1640 medium and maintained at 37 °C with 5% CO₂. At 24 h post-treatment, CCK-8 reagent was added to each well, and plates were incubated for an additional 2 h at 37 °C. For time-course experiments, measurements were performed at 24, 48, and 72 h following treatment. Optical density was measured at 450 nm using a Bio-Rad Microplate Reader (Hercules, CA, USA). All experiments were performed in triplicate, and each experiment was independently repeated three times.

Drug Sensitivity Assay

To evaluate gemcitabine sensitivity, cells were treated with varying concentrations of gemcitabine following transfection with the indicated constructs. Cells were exposed to a series of gemcitabine concentrations (ranging from 0.01 to 100 µM) for 72 h. Cell viability was determined using the CCK-8 assay as described above, and half-maximal inhibitory concentration (IC50) values were calculated from dose-response curves.

Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL) Assay

Apoptosis was quantified using a commercially available TUNEL staining kit (Abcam, Shanghai, China) following the manufacturer’s instructions. Following treatment, cells were fixed and subjected to the TUNEL reaction. Stained cells were visualized using a fluorescent microscope (Olympus, Japan). TUNEL-positive cells were identified by their characteristic fluorescent signal, and the average number of apoptotic cells was counted across ten randomly selected fields by two independent observers blinded to experimental conditions.

Transwell Migration and Invasion Assays

Cell migration and invasion capabilities were evaluated using Transwell chambers (8 μm pore size). For invasion assays, the upper chamber membranes were pre-coated with Matrigel matrix, whereas uncoated membranes were used for migration assays. Following treatment, cells were resuspended in serum-free medium and seeded into the upper chamber, while 700 µL of complete medium containing serum was added to the lower compartment of 24-well plates as a chemoattractant. Following incubation at 37 °C for the designated period, non-migrated cells were removed from the upper surface, and cells that traversed the membrane were fixed, stained, and washed gently with PBS. The number of migrated or invaded cells was quantified under light microscopy across multiple random fields.

Bioinformatic Target Prediction

Potential downstream target genes of miR-1253 were predicted using the miRDB (https://mirdb.org/) and miRWalk (http://mirwalk.umm.uni-heidelberg.de/) databases. Putative circRNA-miRNA interactions between hsa_circ_0003472 and miRNAs were identified by interrogating the circBANK (https://www.circbank.cn/), Circinteractome (https://circinteractome.nia.nih.gov/), and miRanda (http://www.microrna.org/) databases. Candidate target genes were identified through intersection analysis of predicted mRNAs, and regulatory networks were constructed based on the established relationships between circRNA, miRNA, and mRNA components.

Dual-luciferase Reporter Assay

To validate direct interactions between hsa_circ_0003472 and miR-1253, as well as between miR-1253 and ERCC1, dual-luciferase reporter assays were performed. Wild-type (WT) sequences containing putative miR-1253 binding sites and corresponding mutant (Mut) sequences with disrupted binding sites were cloned into the pmiRGLO reporter vector (Synthgene Biotech, Nanjing, China). Site-directed mutagenesis was performed to generate Mut constructs by altering the seed sequence complementary to miR-1253. Cells were co-transfected with the reporter constructs and either miR-1253 mimic or mimic-NC. At 48 h post-transfection, luciferase activity was measured using the Dual-Luciferase Reporter Assay Kit (Promega). Firefly luciferase activity was normalized to Renilla luciferase activity to account for transfection efficiency.

Immunofluorescence Staining and Quantification

Immunofluorescence staining was performed to evaluate target protein expression in PDAC tissues and cell lines. Cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100, and blocked with 5% bovine serum albumin (BSA; Sigma, Cat #A2153) for 1 h at room temperature. Primary antibodies were applied and incubated overnight at 4 °C. Subsequently, fluorophore-conjugated secondary antibodies were incubated for 1 h in the dark at room temperature, followed by nuclear counterstaining with DAPI. Images were acquired using a fluorescence microscope, and fluorescence intensity was quantified using ImageJ software. Results represent the mean of three independent experiments with consistent imaging parameters.

Xenograft Tumor Model

Six male BALB/c nude mice (8 weeks old, approximately 18 g body weight) were obtained and randomly assigned to two groups (n = 3 per group). BxPC-3 cells stably transfected with either sh-NC or sh-circ_0003472 were harvested, digested with 0.25% trypsin, centrifuged at 800 rpm for 3 min, resuspended in PBS, and adjusted to a concentration of 6 × 10⁷ cells/mL. Each mouse received a subcutaneous injection of 150 µL cell suspension into the axillary region. Tumor dimensions were measured weekly using digital calipers, and tumor volume was calculated using the formula: volume = (length × width²)/2. After 6 weeks, mice were euthanized, and tumors were carefully excised, photographed, and weighed. Tumor tissues were harvested for histological examination and molecular analysis. All animal procedures were approved by the Nanjing University of Chinese Medicine Animal Ethics Committee (approval number 202302A048).

Histological Examination

Tumor tissues were fixed in 10% neutral buffered formalin, embedded in paraffin, and sectioned at 5 μm thickness. Sections were deparaffinized, rehydrated through graded alcohols, and subjected to hematoxylin and eosin (H&E) staining for morphological assessment. Apoptosis in tissue sections was detected using the TUNEL assay with the ApopTag^® Peroxidase In Situ Oligo Ligation (ISOL) Kit (Chemicon International, Inc., Cat #S7200) according to the manufacturer’s protocol. For immunohistochemical analysis, sections were subjected to antigen retrieval and incubated with anti-Ki-67 antibody. Stained sections were digitized using a Pannoramic MIDI scanner (3DHISTECH Ltd., Budapest, Hungary) at 20× magnification, and images were analyzed using Panoramic Viewer software.

Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)

Total RNA was isolated from tissues or cells using TRIzol reagent (T9424, Sigma-Aldrich, Beijing, China) according to the manufacturer’s protocol. RNA concentration and purity were assessed using a NanoDrop spectrophotometer, with A260/A280 ratios between 1.8 and 2.0 considered acceptable. Complementary DNA (cDNA) was synthesized using a reverse transcription kit (RR037Q, Takara, Dalian, China). Quantitative PCR was performed in a 25 µL reaction volume containing 12.5 µL Maxima SYBR Green qPCR Master Mix (A46012, Invitrogen, ThermoFisher Scientific), 2.5 µL cDNA template, and 0.3 µM of each primer. Amplification was conducted using a MyiQ2 PCR thermocycler (Sigma-Aldrich, Beijing, China). Relative gene expression was calculated using the 2^−ΔΔCT^ method with GAPDH serving as the internal reference for mRNA and circRNA, while U6 small nuclear RNA served as the reference for miRNA quantification.

Western Blotting

Cells were lysed in radio-immunoprecipitation assay (RIPA) buffer (Beyotime, Beijing, China) supplemented with protease inhibitors. Total protein concentration was determined using a BCA protein assay kit (Beyotime, Beijing, China). Equal amounts of protein (60 µg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; ISEQ00010, Sigma-Aldrich, Beijing, China) and transferred onto polyvinylidene fluoride (PVDF) membranes. Membranes were blocked with 5% non-fat milk powder in Tris-buffered saline containing 0.05% Tween-20 (TBST; 37573, Thermo Scientific, Beijing, China) for 1 h at room temperature. Primary antibodies against Ki-67 (1:2000), PCNA (1:1000), Bax (1:1000), Bcl-2 (1:2000), caspase-3 (1:1000), caspase-9 (1:2000), MMP-2 (1:1000), MMP-9 (1:1000), ERCC1 (1:2000), and GAPDH (1:1000) were incubated overnight at 4 °C. Following three washes with TBST, membranes were incubated with HRP-conjugated goat anti-mouse IgG secondary antibody (A0208, 1:2000 dilution; Beyotime, Beijing, China) for 1 h at room temperature. Protein bands were visualized using enhanced chemiluminescence detection. GAPDH served as the loading control, and densitometric analysis was performed using Quantity One software (Bio-Rad, Hercules, USA).

Statistical Analysis

All experiments were conducted with a minimum of three independent biological replicates to ensure reliability and reproducibility. Statistical analyses were performed using IBM SPSS Statistics 19.0 software. Data are presented as mean ± standard deviation (SD). Comparisons between two groups were performed using two-tailed unpaired Student’s t-test, while comparisons among three or more groups were analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test for multiple comparisons. For dose-response experiments with two independent variables (gemcitabine concentration and treatment group), two-way ANOVA followed by Bonferroni post-hoc test was applied. IC50 values were calculated by nonlinear regression analysis using a four-parameter dose-response model (log[inhibitor] vs. normalized response, variable slope) in GraphPad Prism (version 9.0; GraphPad Software, San Diego, CA, USA). A p-value of less than 0.05 was considered statistically significant.

Results

Elevated Expression of hsa_circ_0003472 in PDAC Tissues and Cell Lines

To investigate the potential involvement of hsa_circ_0003472 in PDAC pathogenesis, we first examined its expression profile in clinical specimens and pancreatic cancer cell lines. Quantitative RT-PCR analysis revealed that hsa_circ_0003472 expression was significantly upregulated in PDAC tumor tissues compared with matched adjacent non-cancerous tissues (P < 0.001; Fig. 1A). Consistent with the clinical observations, comparative analysis across multiple pancreatic cancer cell lines demonstrated markedly elevated hsa_circ_0003472 levels in CFPAC-1, BxPC-3, PANC-1, and ASPC-1 cells relative to the immortalized normal pancreatic epithelial cell line HPNE (P < 0.001; Fig. 1B). These findings established a consistent pattern of hsa_circ_0003472 upregulation in PDAC, providing the rationale for subsequent functional investigations.

Fig. 1.

Expression of hsa_circ_0003472 in PDAC tissues and cell lines. A Quantitative RT-PCR analysis comparing hsa_circ_0003472 expression between PDAC tumor tissues and matched adjacent non-cancerous tissues (n = 30). B Relative expression of hsa_circ_0003472 in pancreatic cancer cell lines (CFPAC-1, BxPC-3, PANC-1, and ASPC-1) compared with the normal pancreatic epithelial cell line HPNE. Data are presented as mean ± SD from three independent experiments. ***P < 0.001

Silencing hsa_circ_0003472 Suppresses PDAC Cell Proliferation and Enhances Chemosensitivity

Given the observed upregulation of hsa_circ_0003472 in PDAC, we sought to characterize its functional significance through loss-of-function experiments. BxPC-3 and ASPC-1 cells were transfected with sh-circ_0003472 to achieve stable knockdown, and successful downregulation was confirmed by qRT-PCR (P < 0.01; Fig. 2A). Functional assessment using the CCK-8 assay demonstrated that hsa_circ_0003472 silencing resulted in a significant reduction in cell viability at 72 h post-transfection (P < 0.001; Fig. 2B). Furthermore, drug sensitivity analysis revealed that knockdown of hsa_circ_0003472 substantially enhanced the sensitivity of PDAC cells to gemcitabine treatment (Fig. 2C).

Fig. 2.

Effects of hsa_circ_0003472 knockdown on proliferation and apoptosis in BxPC-3 and ASPC-1 cells. A Confirmation of hsa_circ_0003472 knockdown efficiency by qRT-PCR following sh-circ_0003472 transfection. B Cell viability assessed by CCK-8 assay at 24, 48, and 72 h post-transfection. C Gemcitabine sensitivity evaluated by IC50 determination using CCK-8 assay. D Representative images and quantification of apoptotic cells detected by TUNEL assay. E Western blot analysis of apoptosis-related proteins (Bax, Bcl-2, caspase-3, and caspase-9) with GAPDH as loading control. Data are presented as mean ± SD from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001

hsa_circ_0003472 Knockdown Promotes Apoptosis in PDAC Cells

The impact of hsa_circ_0003472 silencing on programmed cell death was evaluated through TUNEL staining and Western blot analysis of apoptosis-associated proteins. Downregulation of hsa_circ_0003472 induced a significant increase in apoptotic cell populations as quantified by TUNEL assay (P < 0.01; Fig. 2D). At the molecular level, Western blot analysis revealed corresponding alterations in the expression of key apoptotic regulators, with notable upregulation of pro-apoptotic markers including Bax (P < 0.001), caspase-3 (P < 0.01), and caspase-9 (P < 0.001), accompanied by downregulation of the anti-apoptotic protein Bcl-2 (P < 0.05; Fig. 2E). These results collectively indicate that hsa_circ_0003472 normally functions to suppress apoptosis in PDAC cells.

hsa_circ_0003472 Silencing Inhibits Migration and Invasion of PDAC Cells

To assess the contribution of hsa_circ_0003472 to the metastatic potential of PDAC cells, Transwell assays were performed following circRNA knockdown. Silencing of hsa_circ_0003472 produced a dramatic reduction in both migratory and invasive capacities of BxPC-3 and ASPC-1 cells (P < 0.001; Fig. 3A). Western blot analysis of matrix metalloproteinases, key enzymes facilitating extracellular matrix degradation during metastasis, demonstrated significantly decreased expression of both MMP-2 and MMP-9 following hsa_circ_0003472 knockdown (P < 0.001; Fig. 3B). These findings support a role for hsa_circ_0003472 in promoting the invasive phenotype of PDAC cells.

Fig. 3.

Effects of hsa_circ_0003472 knockdown on migration and invasion in BxPC-3 and ASPC-1 cells. A Representative images and quantification of migrated and invaded cells assessed by Transwell assay. B Western blot analysis of MMP-2 and MMP-9 expression with GAPDH as loading control. Data are presented as mean ± SD from three independent experiments. ***P < 0.001

hsa_circ_0003472 Functions as a Competitive Endogenous RNA for miR-1253

Bioinformatic analysis identified miR-1253 as a potential target of hsa_circ_0003472, with predicted binding sites within the circRNA sequence analogous to miRNA-binding regions in linear mRNAs (Fig. 4A). To validate this interaction, dual-luciferase reporter assays were conducted in BxPC-3 cells. Co-transfection with miR-1253 mimic significantly reduced the luciferase activity of the wild-type hsa_circ_0003472 reporter construct (P < 0.001), while the mutant construct harboring disrupted binding sites showed no change in luciferase activity (Fig. 4B). Furthermore, in order to eliminate the non-specific effects of the overexpression operation itself, we also introduced an irrelevant miRNA (miR-23a) as a control. The results showed that regardless of whether the vector was the wild type or the mutant type, miR-23a mimic did not cause significant changes in the fluorescence activity. Importantly, under control conditions with NC-mimic, the mutation in hsa_circ_0003472 did not independently affect luciferase activity, confirming the specificity of the miR-1253-mediated effect.

Fig. 4.

Identification of miR-1253 as a target of hsa_circ_0003472. A Schematic representation of the predicted miR-1253 binding site within hsa_circ_0003472 and the corresponding mutant sequence. B Dual-luciferase reporter assay in BxPC-3 cells co-transfected with wild-type (WT) or mutant (Mut) hsa_circ_0003472 reporter constructs and miR-1253 mimic or NC-mimic. C Expression of miR-1253 in PDAC tissues compared with adjacent non-cancerous tissues (n = 30) by qRT-PCR. D Correlation analysis between the expression of hsa_circ_0003472 and miR-1253in 30 PDAC tissues. E Relative miR-1253 expression in BxPC-3 cells compared with HPNE cells. Data are presented as mean ± SD from three independent experiments. ***P < 0.001

Expression analysis in clinical specimens revealed that miR-1253 levels were significantly downregulated in PDAC tissues compared with adjacent normal tissues (P < 0.001; Fig. 4C). The correlation analysis revealed that the expression levels of hsa_circ_0003472 and miR-1253 showed a significant negative correlation (R² = 0.3173, p = 0.0012; Fig. 4D). Similarly, reduced miR-1253 expression was observed in BxPC-3 cells relative to HPNE cells (P < 0.001; Fig. 4E). The inverse expression pattern between hsa_circ_0003472 and miR-1253, combined with the luciferase reporter data, supports the function of hsa_circ_0003472 as a miR-1253 sponge in PDAC.

miR-1253 Overexpression Recapitulates the Effects of hsa_circ_0003472 Silencing

To further establish the functional relationship between hsa_circ_0003472 and miR-1253, gain-of-function experiments were performed by transfecting BxPC-3 cells with miR-1253 mimic. Successful overexpression was confirmed by qRT-PCR (P < 0.001; Fig. 5A). Consistent with the effects observed upon hsa_circ_0003472 knockdown, miR-1253 restoration led to significant reductions in cell viability (P < 0.001; Fig. 5B) and proliferative capacity as assessed by EdU incorporation (P < 0.001; Fig. 5D). Notably, combined treatment with sh-circ_0003472 and gemcitabine produced a synergistic reduction in BxPC-3 cell proliferation (P < 0.01; Fig. 5C). Furthermore, TUNEL staining demonstrated enhanced apoptosis following miR-1253 overexpression (P < 0.001; Fig. 5E), and Transwell assays revealed marked suppression of both migration and invasion capabilities (P < 0.001; Fig. 5F). These phenotypic similarities between miR-1253 overexpression and hsa_circ_0003472 silencing reinforce the regulatory axis between these molecules.

Fig. 5.

Effects of miR-1253 overexpression on BxPC-3 cell behavior. A Confirmation of miR-1253 overexpression by qRT-PCR following miR-1253 mimic transfection. B Cell viability assessed by CCK-8 assay. C Drug sensitivity analysis showing effects of combined sh-circ_0003472 and gemcitabine treatment on cell proliferation (#: GEM + sh-NC + NC inhibitor vs. NC; $: GEM + sh-NC + NC inhibitor vs. GEM + sh-circ_0003472 + miR-1253 inhibitor; *: GEM + sh-NC + NC inhibitor vs. GEM + sh-circ_0003472 + miR-1253 inhibitor). D Cell proliferation assessed by EdU incorporation assay with representative images and quantification. E Apoptosis detection by TUNEL assay with representative images and quantification. F Migration and invasion assessed by Transwell assay with representative images and quantification. Data are presented as mean ± SD from three independent experiments. *,$P < 0.05,^##P < 0.01, ***P < 0.001

ERCC1 is a Direct Target of miR-1253

Bioinformatic prediction identified ERCC1 as a putative downstream target of miR-1253, with conserved binding sites within its 3′-UTR (Fig. 6A). Dual-luciferase reporter assays confirmed this prediction, demonstrating that miR-1253 mimic transfection significantly suppressed luciferase activity of the wild-type ERCC1 3′-UTR construct (P < 0.001), while the mutant construct remained unaffected (Fig. 6B). The specificity of this interaction was further validated by the observation that mutation of the ERCC1 3′-UTR did not independently alter luciferase activity in cells transfected with NC-mimic.

Fig. 6.

Identification of ERCC1 as a direct target of miR-1253. A Schematic representation of the predicted miR-1253 binding site within the ERCC1 3′-UTR and the corresponding mutant sequence. B Dual-luciferase reporter assay in BxPC-3 cells co-transfected with wild-type (WT) or mutant (Mut) ERCC1 3′-UTR reporter constructs and miR-1253 mimic or NC-mimic. C Expression of ERCC1 in PDAC tissues compared with adjacent non-cancerous tissues (n = 30) by qRT-PCR. D Correlation between hsa_circ_0003472 and ERCC1 expression in 30 PDAC tissues. E Correlation analysis of ERCC1 and miR-1253 expression in 30 PDAC tissues. F Relative ERCC1 expression in BxPC-3 cells compared with HPNE cells by qRT-PCR. G ERCC1 mRNA expression in BxPC-3 cells following miR-1253 mimic transfection. H Western blot analysis of ERCC1 protein expression following miR-1253 mimic transfection with GAPDH as loading control. Data are presented as mean ± SD from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001

Expression profiling revealed ERCC1 upregulation in PDAC patient tissues compared with adjacent normal tissues (P < 0.001; Fig. 6C). The expression of ERCC1 was significantly positively correlated with hsa_circ_0003472 (R² = 0.6759, P < 0.0001; Fig. 6D), while the expression of ERCC1 was significantly negatively correlated with miR-1253 (R² = 0.5587, P < 0.0001; Fig. 6E); moreover, an increase in ERCC1 expression was also observed in BxPC-3 cells compared to HPNE cells (P < 0.01; Fig. 6F). Functional validation demonstrated that transfection with miR-1253 mimic resulted in significant downregulation of ERCC1 at both the mRNA level (P < 0.01; Fig. 6G) and protein level (P < 0.05; Fig. 6H), confirming ERCC1 as a bona fide target of miR-1253.

The hsa_circ_0003472/miR-1253/ERCC1 Axis Regulates PDAC Cell Behavior

To delineate the regulatory relationships among components of the proposed molecular axis, rescue experiments were performed. Knockdown of hsa_circ_0003472 with sh-circ_0003472 resulted in elevated miR-1253 expression (P < 0.001; Fig. 7A) and concomitant reduction in ERCC1 levels (P < 0.05; Fig. 7B), consistent with the proposed ceRNA mechanism.

Fig. 7.

Functional validation of the hsa_circ_0003472/miR-1253/ERCC1 axis in BxPC-3 cells. A Expression of miR-1253 following sh-circ_0003472 transfection by qRT-PCR. B Expression of ERCC1 following sh-circ_0003472 transfection by qRT-PCR. C Cell viability assessed by CCK-8 assay in cells transfected with miR-1253 inhibitor alone or in combination with sh-ERCC1. D Cell proliferation assessed by EdU assay with representative images and quantification. E Apoptosis detection by TUNEL assay with representative images and quantification. F Migration and invasion assessed by Transwell assay with representative images and quantification. Data are presented as mean ± SD from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 versus control; ^#P < 0.05, ^##P < 0.01,^###P < 0.001 versus miR-1253 inhibitor group

Functional rescue experiments revealed that inhibition of miR-1253 enhanced cell viability at 72 h (P < 0.001), an effect that was reversed upon concurrent ERCC1 silencing (P < 0.05; Fig. 7C). Similarly, EdU proliferation assays demonstrated increased proliferation with miR-1253 inhibition (P < 0.05), which was attenuated by sh-ERCC1 treatment (P < 0.05; Fig. 7D). TUNEL assays showed reduced apoptosis following miR-1253 inhibition (P < 0.001), with partial restoration of apoptotic activity upon ERCC1 knockdown (P < 0.05; Fig. 7E). Finally, the enhanced migration and invasion observed with miR-1253 inhibition (P < 0.001) were substantially suppressed by concurrent sh-ERCC1 treatment (P < 0.001; Fig. 7F). These rescue experiments establish ERCC1 as the functional effector downstream of the hsa_circ_0003472/miR-1253 regulatory axis.

hsa_circ_0003472 Knockdown Suppresses Tumor Growth in Vivo

To validate the in vitro findings in a physiologically relevant context, xenograft tumor models were established in nude mice using BxPC-3 cells stably transfected with sh-circ_0003472 or control vector. Tumors derived from hsa_circ_0003472-silenced cells exhibited significantly reduced volume and weight compared with control tumors (P < 0.001; Fig. 8A, B). Histological examination by H&E staining revealed distinct morphological alterations in the sh-circ_0003472 group, while TUNEL staining demonstrated substantially increased apoptosis within these tumors (Fig. 8C). Immunohistochemical analysis revealed reduced Ki-67 expression in the knockdown group, indicating decreased proliferative activity (Fig. 8D). Western blot analysis of tumor lysates confirmed the molecular changes observed in vitro, with decreased Bcl-2 expression and increased levels of Bax and cleaved caspase-3 (P < 0.01; Fig. 8E).

Fig. 8.

Effects of hsa_circ_0003472 knockdown on tumor growth in a xenograft mouse model. A Representative images of excised tumors from nude mice bearing BxPC-3 cells transfected with sh-NC or sh-circ_0003472 (n = 3 per group). Tumor weight quantification is shown. B Tumor volume measurements recorded weekly over the 6-week experimental period. C Representative images of H&E staining and TUNEL staining of tumor tissue sections. Scale bars are indicated. D Immunohistochemical staining for Ki-67 in tumor tissue sections with quantification. E Western blot analysis of Bcl-2, Bax, and cleaved caspase-3 expression in tumor tissues with GAPDH as loading control. F Expression of hsa_circ_0003472, miR-1253, and ERCC1 in tumor tissues by qRT-PCR. Data are presented as mean ± SD. **P < 0.01, ***P < 0.001

Molecular characterization of the tumor tissues by qRT-PCR demonstrated that sh-circ_0003472-transfected tumors exhibited reduced hsa_circ_0003472 expression (P < 0.001), elevated miR-1253 levels (P < 0.001), and decreased ERCC1 expression (P < 0.01; Fig. 8F). These in vivo findings corroborate the in vitro observations and confirm the oncogenic role of hsa_circ_0003472 in PDAC progression through regulation of the miR-1253/ERCC1 axis.

Discussion

Circular RNAs were first identified approximately four decades ago as a distinct class of RNA molecules generated through back-splicing of linear precursors to form covalent closed-loop structures [25]. Accumulating evidence has implicated circRNAs in the development and progression of numerous diseases [26], with their role in PDAC pathogenesis receiving increasing attention [27, 28]. In this study, we demonstrate that hsa_circ_0003472 is significantly upregulated in PDAC tissues and cell lines and functions as an oncogenic factor by sponging miR-1253, thereby modulating ERCC1 expression and influencing gemcitabine sensitivity.

Our findings revealed consistent elevation of hsa_circ_0003472 in both clinical PDAC specimens and pancreatic cancer cell lines compared with their respective controls. Functional characterization demonstrated that silencing hsa_circ_0003472 in BxPC-3 and ASPC-1 cells reduced proliferation, migration, and invasion while enhancing apoptosis. These observations align with the established paradigm that circRNAs can function as miRNA sponges to regulate oncogene or tumor suppressor expression [15]. The accompanying reduction in MMP-2 and MMP-9 expression following hsa_circ_0003472 knockdown suggests involvement of this circRNA in modulating the metastatic potential of PDAC cells. Furthermore, our drug sensitivity analysis revealed that hsa_circ_0003472 silencing enhanced the responsiveness of PDAC cells to gemcitabine, indicating a role in chemoresistance. These findings are consistent with previous reports demonstrating that hsa_circ_0074298 promotes PDAC growth and metastasis [29].

Mechanistically, we identified miR-1253 as a direct target of hsa_circ_0003472 through bioinformatic prediction and dual-luciferase reporter assays. The inverse expression pattern between hsa_circ_0003472 and miR-1253 in PDAC tissues, combined with the observation that hsa_circ_0003472 knockdown elevated miR-1253 levels, supports a ceRNA mechanism whereby hsa_circ_0003472 sequesters miR-1253 to prevent target gene suppression. Prior studies have established miR-1253 as a tumor suppressor that targets genes involved in DNA repair pathways, including ERCC1, which is frequently overexpressed in PDAC and associated with resistance to DNA-damaging agents such as gemcitabine [21, 30]. Our data confirm ERCC1 as a direct target of miR-1253 and demonstrate that its expression is regulated through the hsa_circ_0003472/miR-1253 axis.

The rescue experiments provided functional validation of this regulatory axis. Inhibition of miR-1253 enhanced proliferation, migration, and invasion while reducing apoptosis, effects that were reversed by concurrent ERCC1 knockdown. These findings indicate that ERCC1 functions as the downstream effector mediating the oncogenic effects of hsa_circ_0003472. By sponging miR-1253, hsa_circ_0003472 relieves the suppression of ERCC1, thereby promoting DNA repair capacity and enabling tumor cells to evade apoptosis. This mechanism may also contribute to the poor chemotherapeutic response commonly observed in PDAC patients. Similar circRNA-miRNA interactions have been reported to contribute to chemoresistance in other malignancies [31].

The in vivo xenograft experiments corroborated our in vitro observations. Tumors derived from hsa_circ_0003472-silenced cells exhibited reduced volume and weight, increased apoptosis, and altered expression of the molecular axis components. These findings strengthen the evidence for hsa_circ_0003472 as an oncogenic driver in PDAC and support its potential as a therapeutic target.

The stable circular structure of circRNAs renders them resistant to exonuclease degradation, making them attractive biomarker candidates. Their presence in body fluids and association with disease stage and prognosis further support their clinical utility [32, 33]. Our identification of hsa_circ_0003472 as a regulator of PDAC progression adds to the growing repertoire of clinically relevant circRNAs in this malignancy.

Beyond chemoresistance, hsa_circ_0003472 appears to influence multiple malignant characteristics of PDAC cells. The effects on proliferation, migration, and invasion are consistent with reports that circRNAs can modulate epithelial-mesenchymal transition (EMT), apoptosis, and cell cycle progression through regulation of miRNA targets [34]. Studies in gastric cancer have demonstrated that circRNAs can promote EMT and invasive behavior through similar mechanisms [35].

Several limitations of this study warrant consideration. First, the migration and invasion assays were conducted in serum-containing medium, which may introduce confounding effects from cell proliferation. However, the concurrent reduction in MMP-2 and MMP-9 expression supports genuine alterations in migratory and invasive capacity beyond proliferative effects. Future studies employing serum-free conditions or proliferation controls would help distinguish these phenotypes. Second, BxPC-3 and ASPC-1 cells lack the KRAS mutation present in over 90% of PDAC cases. Although BxPC-3 cells harbor a BRAF mutation that activates downstream pathways, validation in KRAS-mutant cell lines such as PANC-1 or MIA PaCa-2 would enhance the generalizability of our findings. Third, the relatively small patient cohort limits the broader applicability of our observations, and larger, multi-center studies are needed for validation. Fourth, while we focused on the miR-1253/ERCC1 axis, hsa_circ_0003472 may interact with additional miRNAs or proteins that warrant investigation. Finally, our study examined tumor cell-intrinsic mechanisms without addressing the potential influence of hsa_circ_0003472 on the tumor microenvironment, which plays a critical role in PDAC progression and therapeutic resistance.

In conclusion, our study identifies hsa_circ_0003472 as a regulator of PDAC progression through the miR-1253/ERCC1 axis. Elevated hsa_circ_0003472 expression promotes proliferation, migration, and invasion while suppressing apoptosis and reducing gemcitabine sensitivity. Targeting this circRNA may offer a strategy for disrupting multiple tumor-promoting pathways in PDAC. Further investigation is required to fully elucidate the clinical potential of hsa_circ_0003472-directed therapies.

Authors’ Contributions

Conceptualization: CB.X., Y.C., HJ.S., and H.Z. Methodology: YK.Z., DX.C., T.S., and GY.W. Software: Y.C. and XJ.Y. Validation: CB.X. and Y.C. Formal Analysis: CB.X., Y.C., and XJ.Y. Investigation: CB.X., Y.C., HJ.S., and H.Z. Resources: CB.X., Y.C., HJ.S., and H.Z. Data Curation: CB.X., Y.C., HJ.S., and H.Z. Writing – Original Draft: CB.X. Writing – Review & Editing: All authors Visualization: CB.X., Y.C., HJ.S., and H.Z. Supervision: CB.X. and XD.Y. Project Administration: CB.X. and YK.Z. Funding Acquisition: CB.X. and YK.Z. All authors have read and approved the final version of the manuscript.

Funding

This research was supported by the Natural Science Foundation of Jiangsu Province (grant number: Y2022037) and the Natural Science Foundation of Nanjing University of Chinese Medicine (grant number: XZR2023044).

Data Availability

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics Approval and Consent to Participate

This study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of the Second Affiliated Hospital of Nanjing Medical University (approval number: 2022-KY-058-01). Written informed consent was obtained from all participants prior to tissue collection. All animal experiments were performed in accordance with institutional guidelines and approved by the Nanjing University of Chinese Medicine Animal Ethics Committee (approval number: 202302A048).

Consent for Publication

Not applicable.

Competing Interests

The authors declare no competing interests.