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. 2026 Jan 7;16:4384. doi: 10.1038/s41598-025-34390-9

Mechanism study of circCCDC134 regulating the biological behaviors of PDGF-BB-induced oral mucosal fibroblast

Yutong Zhou 1,2, Ni Jian 1, Qi Wang 1, Canhua Jiang 3, Haodong Guo 1, Jie Wang 1,✉
PMCID: PMC12864962  PMID: 41501162

Abstract

The transition of fibroblasts into myofibroblasts is a key factor in oral submucous fibrosis (OSF) pathogenesis. Our previous studies demonstrated that platelet-derived growth factor-BB (PDGF-BB) promotes the proliferation, migration, and transition of human oral mucosal fibroblasts (HOMF) by downregulating microRNA-503 (miR-503). However, the mechanism underlying PDGF-BB-mediated miR-503 downregulation remained unclear. We identified eight exon-derived circRNA with m6A modification sites, moderate length, and strong predicted binding affinity for miR-503. Among these candidates, circCCDC134 exhibited the most significant upregulation in PDGF-BB-stimulated HOMF. Our functional studies revealed that circCCDC134 overexpression enhanced proliferation, migration, and transition of PDGF-BB-induced HOMF, whereas circCCDC134 silencing attenuated these effects. Mechanistically, PDGF-BB upregulates circCCDC134 expression in HOMF. Subsequently, circCCDC134 acts as a molecular sponge for miR-503, sequestering miR-503 and thereby relieving its repression of RAF mRNA. This leads to increased RAF protein expression, activation of the RAF/MEK/ERK signaling pathway, and ultimately promotes HOMF proliferation, migration, and transition. These findings establish a theoretical foundation for identifying potential biomarkers and therapeutic targets for the targeted treatment of OSF.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-34390-9.

Keywords: Oral submucous fibrosis, Platelet-derived growth factor-BB, CircCCDC134, MiR-503, RAF/MEK/ERK signaling pathway

Subject terms: Cancer, Cell biology, Molecular biology

Introduction

Oral submucous fibrosis (OSF) is a chronic, progressive precancerous lesion with a secondary chronic inflammation induced by betel nut and tobacco1. Pathogenetically, OSF is an aberrant tissue repair response, characterized by three sequential processes: (1) the initiation of inflammation, (2) the activation and transition of human oral mucosal fibroblasts (HOMF), and (3) extracellular matrix (ECM) Homeostasis Dysregulation, forming a self-perpetuating vicious cycle2. Chronic inflammation is a primary driver of fibrosis3. Following tissue injury, cellular debris and damage-associated molecular patterns (DAMPs) synergistically initiate the inflammatory cascade, recruiting and activating immune cells4. Damaged epithelial cells and activated immune cells (e.g., macrophages, T cells) release inflammatory cytokines, including transforming growth factor-beta (TGF-β), platelet-derived growth factor-BB (PDGF-BB), and chemokines, to recruit and activate fibroblasts (FBs)5–9. Activated FBs migrate to inflammation loci, secrete ECM components to repair damaged tissue, and maintain ECM homeostasis. Consequently, ECM deposition enhances mechanical tension. To withstand this heightened tension, activated FBs undergo fibroblast-to-myofibroblast transition (FMT), characterized by robust synthesis of collagen-I (COL-I) and α-smooth muscle actin (α-SMA)10. PDGF-BB promotes FB proliferation, migration, and autophagy11–13. Previous studies have shown that PDGF-BB is significantly upregulated in the tumor microenvironment of oral squamous cell carcinoma (OSCC). It can promote ECM accumulation, stromal remodeling, and tumor infiltration and progression by activating FBs14,15. Mechanistically, PDGF-BB activates downstream cascades, including the MAPK and PI3K/AKT pathways, by binding PDGFRs(Fig. 1)16. These findings suggest that PDGF-BB-driven FB activation and ECM deposition are important components of the OSCC microenvironment. Our previous studies elucidated that PDGF-BB enhances HOMF proliferation, migration, FMT, and ECM synthesis17,18. The upstream regulatory network of PDGF-BB on HOMF remains incompletely understood. This study aimed to explore post-transcriptional regulation (e.g., circRNA) exists beyond this canonical framework to specifically modulate certain pathways and influence the biological behavior of HOMF.

Fig. 1.

Fig. 1

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PDGF-BB signaling and the novel regulatory axis identified in this study.

In recent years, breakthrough research on non-coding RNAs (ncRNAs) in various diseases has provided new perspectives for the OSF. ncRNAs include microRNA (miRNA), circular RNA (circRNA), long non-coding RNA (lncRNA), and so on. First discovered by Nobel laureates Victor Ambros and Gary Ruvkun in 1993, miRNAs are endogenous small ncRNAs, typically 19–25 nucleotides in length19,20. miRNAs mediate post-transcriptional gene silencing by base-pairing with complementary sequences in the 3′-untranslated region (3′UTR) of target messenger RNAs (mRNAs)21,22. Our previous studies demonstrated that PDGF-BB downregulates miR-503 to attenuate the silencing effect on rapidly accelerated fibrosarcoma (RAF), thereby indirectly enhancing RAF/MEK/ERK signaling pathway activity to promote HOMF proliferation, migration, and transition23. Both circRNA and lncRNA contain miRNA binding sites to sponge miRNAs, alleviating miRNA-mediated repression of target mRNAs and forming a Competitive Endogenous RNA (ceRNA) network24–26. Therefore, we hypothesize that circRNA or lncRNA may compete for miR-503 binding, downregulate miR-503, and indirectly activate the RAF/MEK/ERK signaling pathway to modulate the biological behavior of PDGF-BB-induced HOMF. Due to covalently closed circular structure, circRNA are resistant to degradation by exonucleases and can remain highly stable in various samples such as saliva, blood, and exosomes27. circRNA exhibits significant potential as a biomarker and therapeutic target28. Consequently, our research focuses on circRNA. circRNA are closed-loop non-coding RNAs, lacking a 5′ cap and 3′ poly(A) tail, and are widely implicated in various organs’ fibrosis, such as the heart, liver, kidneys, and lungs, through the ceRNA mechanism29–32. Therefore, we screened for circRNA targeting miR-503, integrating database analysis with experimental data. hsa_circ_0008806 (circCCDC134) is significantly elevated in OSF tissues and PDGF-BB-induced HOMF. Furthermore, under PDGF-BB stimulation, circCCDC134 overexpression downregulated miR-503 expression, suggesting its potential role as a miR-503 sponge (Fig. 1). However, the specific mechanism of circCCDC134 in modulating PDGF-BB-induced HOMF biological behavior and its precise regulatory interplay with miR-503 required further validation.

Methods

Human biospecimen acquisition

This study received ethical approval from the Ethics Committee of Xiangya Hospital, and informed consent was obtained from all participating patients. Human normal oral mucosal tissue was obtained from the gingival tissue during tooth extraction procedures performed at Xiangya Hospital. The tissue was sourced from periodontally healthy individuals aged 16–40 years. The tissue was immediately stored in DMEM high-glucose medium containing 1% antibiotic and 10% FBS after removal. Primary HOMF extraction was initiated within 30 min of tissue collection. Human OSF tissue was obtained from patients undergoing surgery in the Department of Oral and Maxillofacial Surgery, Xiangya Hospital. All specimens were pathologically confirmed OSF lesions. The tissue was immediately frozen in liquid nitrogen and stored for subsequent RNA extraction.

Primary HOMF isolation, identification, and transfection

Primary HOMF were isolated using the tissue explant method. Briefly, oral mucosal tissue was obtained under sterile conditions, minced into 1–2 mm³ fragments, and adhered to culture flasks. Cells were cultured in DMEM high-glucose medium supplemented with 10% fetal bovine serum (Cellbox, Beijing, China) and 1% antibiotic-antimycotic solution (Servicelbio, Wuhan, China). HOMF were subsequently purified through serial passaging. To exclude epithelial cell contamination, indirect immunofluorescence was performed to detect the vimentin (HOMF marker) and E-cadherin (epithelial marker). circCCDC134 overexpression plasmid (ov-circCCDC134) and its corresponding empty vector control (ov-NC) were constructed (Syngenbio, Beijing, China). miR-503 mimic and its negative control (miR-503 NC) were purchased from Ribobio (Guangzhou, China; miR10002874 and miR1N0000001-1–5). Two siRNA sequences targeting circCCDC134 (si-circCCDC134-1 and si-circCCDC134-2) and a scrambled negative control siRNA (si-NC) were synthesized by Genepharma (Shanghai, China). All transfections were performed using Lipo2000.

RT-qPCR

Total RNA was isolated from tissues and cells using the TransZol Up reagent (TransGen, Beijing, China). cDNA synthesis was performed using a reverse transcription kit (Novogene, Beijing, China) for subsequent qPCR (Novogene, Beijing, China). The gene level normalised to GAPDH or U6 was determined using the 2−ΔΔCt method.

Validation and quantification of circCCDC134

Primer design and specificity validation

To specifically detect the circular structrue of circCCDC134, a pair of divergent primers were designed to span the predicted back-spliced junction, ensuring amplification only from cDNA derived from the circular transcript. In parallel, convergent primers targeting the linear mRNA of the host gene CCDC134 were designed. The primer sequences are as follows: circCCDC134 (divergent, F: 5′-AATCGACCGCACAGAGCTCA-3′, R: 5′-GGTCCAGGGAGGTCCTCAAG-3′), CCDC134 mRNA (convergent, F: 5′-CGACATCCACCAGCAGTACAAG-3′, R: 5′-CGGCGTTTCTCTTCTTCTCTCAG-3′). The specificity of PCR amplification was confirmed by agarose gel electrophoresis.

Verification of back-spliced junction

The PCR product amplified by the divergent primers was subjected to Sanger sequencing. The obtained sequences were aligned with the genomic reference to definitively confirm the presence of the unique back-spliced junction.

RNA stability assay

To assess circCCDC134 stability conferred by the circular structure, HOMF were treated with 5 µg/mL Actinomycin D (Act D). Cells were harvested at indicated time points (0, 8, 16, and 24 h post-treatment)and analyzed by RT-qPCR to quantify expression levels of circCCDC134 and its linear host gene, CCDC134 mRNA.

Cell counting kit-8 (CCK8)

CCK8 assay quantifies viable cells to indirectly assess cell proliferation (MeilunBio, Liaoning, China). HOMF cell suspensions were seeded in 96-well plates at 5,000 cells/well with 3–6 replicate wells per group. Cells were cultured in medium containing 20 ng/mL PDGF-BB for 0, 12, 24, 36, 48, and 72 h. Absorbance at 450 nm was measured using a microplate reader.

Scratch assay

Before cell seeding, 3–5 evenly spaced reference lines were inscribed on the exterior bottom of 6-well plates to standardize imaging regions. Cells were maintained in conditioned medium containing 40 ng/mL PDGF-BB and 1% FBS (serum-starved conditions). Migration was documented at designated timepoints (e.g., 0, 6, 12, and 24 h or 0, 18, and 24 h). Scratch widths were quantified using ImageJ software.

Western blot (WB)

Total protein was extracted from HOMF (RIPA: PMSF/phosphatase inhibitor cocktail = 100:1). Protein concentration was determined by BCA assay (Bioss, Beijing, China). Lysates were mixed with 5× loading buffer, denatured at 100 °C for 5 min, and separated by SDS-PAGE. Proteins were transferred to PVDF membranes, blocked with 5% non-fat milk (Beyotime, Shanghai, China) in TBST, and probed overnight at 4 °C with primary antibodies. After secondary antibody incubation, immunoreactive bands were visualized by ECL (Sevenbio, Beijing, China) and quantified by ImageJ. Membranes were stripped with stripping buffer (Sevenbio, Beijing, China).

Dual-luciferase reporter assay

To validate putative miR-503 binding sites within circCCDC134, wild-type (WT) and mutant (MUT) circCCDC134 luciferase reporter plasmids were constructed (Ribobio, Guangzhou, China). Dual-luciferase reporter assays were performed according to manufacturer protocols (TransGen, Beijing, China). Relative luciferase activity was calculated as: Renilla luciferase activity/Firefly luciferase activity. Data were normalized to empty vector controls and expressed as fold-change relative to the circCCDC134-WT + miR-503 NC group.

Databases and related software

The starBase database(https://rnasysu.com/encori/) was utilized to identify putative mRNA targets downstream of miR-50333. Predicted circRNA interactions with miR-503 were screened via the circBank database (https://www.circbank.cn/#/home)34. For circCCDC134, we designed specific primers using the circPrimer2.0 software35.

Statistical analysis

All data are expressed as mean ± standard deviation (X̅±S). Statistical analyses and data visualization were used by GraphPad Prism 9.0. Comparisons between two groups for normally distributed data were assessed using a paired t-test. Comparisons among multiple groups (n ≥ 3) were analyzed by one-way analysis of variance (ANOVA). Statistical significance was defined as P < 0.05 (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

Results

Isolation and identification of HOMF

During the repair response, HOMF is activated to acquire enhanced proliferation, migration, and transition capabilities. Primary HOMF were isolated from normal human oral mucosal tissue. After approximately one week in culture, spindle-shaped cells migrated out from the tissue explants (Fig. 2a). These cells were large, with clear contours and an elongated spindle shape. High-power microscopy revealed oval nuclei and a network-like growth pattern (Fig. 2b, c). Immunofluorescence staining demonstrated positive vimentin staining and was negative for E-cadherin. These results confirm the successful isolation and culture of primary HOMF without epithelial cell contamination (Fig. 2d).

Fig. 2.

Fig. 2

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Observation and identification of primary HOMF under inverted microscopy and fluorescence microscopy. (a) Long spindle-shaped cells migrate out of the tissue block. (a) ×50. (b, c) Growth status of cells after passage culture. (b) ×100, (c) ×200. (d) Immunofluorescence identification of primary HOMF.

circCCDC134 was significantly elevated in PDGF-BB-stimulated HOMF with a time- and dose-dependent effect

The circBank database identified 6,071 circRNAs predicted to interact with miR-503. Given our prior observation that PDGF-BB stimulation upregulates YTHDC1 mRNA in HOMF, YTHDC1 is an m6A reader protein that regulates the generation of circRNA (including their circularization, nuclear export and stability) by binding to m6A sites36,37. Therefore, we hypothesized that the regulation of circRNA by PDGF-BB may involve m6A reader, which led us refined the candidate list. Among the initial 6,071 circRNA, only 78 contained m6A modification sites. Considering factors critical for loop formation efficiency, we imposed a length constraint of 400–1000 bp. Additionally, we selected only exon-derived circRNA with established reverse splicing circularization mechanisms. Based on their calculated free binding energy, we selected eight circRNAs with binding energies < −25 (including circKCNK12, circRBM33, circNOTCH3, circERBB2, circSMO, circPIF1, and circELF2). RT-qPCR analysis revealed that circCCDC134 exhibited the most significant upregulation in HOMF stimulated with PDGF-BB (Fig. 3a). circCCDC134 expression in HOMF increased in a time- and dose-dependent manner in response to PDGF-BB stimulation, with maximal induction observed at 60 ng/mL for 48 h (Fig. 3b, c). Importantly, PDGF-BB stimulation did not influence the parental gene CCDC134 (P > 0.05) (Fig. 3d). Moreover, circCCDC134 expression was significantly elevated in OSF tissues compared to normal oral mucosa (Fig. 3e), suggesting its potential involvement in OSF pathogenesis. Collectively, these results demonstrate that circCCDC134 is markedly upregulated in PDGF-BB-stimulated HOMF in a time- and dose-dependent manner and is associated with OSF.

Fig. 3.

Fig. 3

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circCCDC134 expression is elevated in PDGF-BB-stimulated HOMF and OSF tissues. (a) circRNA were selected using the circBank database and screening criteria, and RT-qPCR analysis of selected circRNA. (b, c) Changes in circCCDC134 expression levels in HOMF tissue stimulated with PDGF-BB at different concentrations at the same time point. (d) RT-qPCR analysis of CCDC134 mRNA. (e) Expression of circCCDC134 in normal oral mucosa tissue and OSF tissue. (* represents P < 0.05; ** represents P < 0.01; *** represents P < 0.001; **** represents P < 0.0001).

circCCDC134 is a reverse-spliced circular RNA in HOMF

circRNA are formed through back-splicing of pre-mRNA transcripts. A single gene locus can generate multiple distinct circRNA through alternative back-splicing38. circCCDC134 is an exon-derived circRNA originating from chromosome 22 (positions 42204878–42209826) and is formed by back-splicing of exons 2–6 in the CCDC134 gene (Fig. 4a). Based on the circCCDC134 sequence, we designed convergent and divergent primers; the specificity of the divergent primer was confirmed (Fig. 4b). Sanger sequencing revealed the back-splice junction sequence GAGCTC (Fig. 4c), confirming that circCCDC134 arises from the exons 2–6 of CCDC134. Crucially, amplification with divergent primers produced a distinct band exclusively in the cDNA sample, with absence in the gDNA (Fig. 4d). Due to their circular structure, circRNA generally exhibit greater stability than linear genes. Following Act D treatment, CCDC134 mRNA levels declined progressively. In contrast, circCCDC134 levels remained relatively stable. At 16 and 24 h post-treatment, circCCDC134 expression was significantly higher than that of CCDC134 mRNA (P < 0.05) (Fig. 4e).

Fig. 4.

Fig. 4

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circCCDC134 is a reverse-spliced circular RNA in HOMF. (a) The circBank database indicates that circCCDC134 is formed by reverse splicing of exons 2–6 of CCDC134. (b) The circPrimer database shows the position of the designed divergent primers for circCCDC134. Agarose gel electrophoresis demonstrates that the divergent primers can specifically amplify circCCDC134. (c) Sanger sequencing detects the splicing site of circCCDC134. d: Amplification of divergent and convergent primers for circCCDC134 and GAPDH in cDNA and gDNA. (e) Changes in the expression levels of circCCDC134 and CCDC134 mRNA after Act D treatment of HOMF at different time points. (* represents P < 0.05; *** represents P < 0.001).

Overexpression of circCCDC134 promotes proliferation, migration, and transition of PDGF-BB-induced HOMF

RT-qPCR analysis revealed circCCDC134 expression was significantly elevated in the ov-circCCDC134 group compared to the ov-NC group (Fig. 5a). CCK-8 results demonstrated a significantly increased proliferation rate in the PDGF-BB group compared to the control group at 48 h and 72 h. Furthermore, proliferation was significantly enhanced in the PDGF-BB + ov-circCCDC134 group compared to the PDGF-BB + ov-NC group at 36 h, 48 h, and 72 h (Fig. 5b). Similarly, the scratch assay showed a significantly increased migration rate in the PDGF-BB + ov-circCCDC134 group compared to the PDGF-BB + ov-NC group at 12 h and 24 h (Fig. 5c). Both α-SMA and COL-1 protein levels were significantly upregulated in the PDGF-BB group relative to control. Moreover, their expression was further significantly increased in the PDGF-BB + ov-circCCDC134 group compared to PDGF-BB + ov-NC (Fig. 5d).

Fig. 5.

Fig. 5

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Overexpression of circCCDC134 promotes proliferation, migration, and transition of PDGF-BB-induced HOMF. (a) RT-qPCR detection of circCCDC134 after transfection. Mock: transfection reagent group; ov-NC: empty vector group; ov-circCCDC134: circCCDC134-overexpressing plasmid group. (b) CCK8 assay assesses HOMF proliferation capacity. (Compared with the control group, # indicates P < 0.05; ## indicates P < 0.01; ### indicates P < 0.001; #### indicates P < 0.0001; compared with the PDGF-BB + ov-NC group, * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001; **** indicates P < 0.0001). (c) Scratch assay assesses HOMF migration capacity. (d) Western blot analysis of COL-1 and α-SMA protein levels. Bar charts show quantitative analysis of COL-1 and α-SMA protein band intensity (n = 3). (* indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001; **** indicates P < 0.0001).

Silencing circCCDC134 inhibits proliferation, migration, and transformation of PDGF-BB-induced HOMF

RT-qPCR analysis confirmed that both si-circCCDC134-1 and si-circCCDC134-2 significantly suppressed circCCDC134 levels (P < 0.05), with si-circCCDC134-1 exhibiting greater efficacy, without affecting the parental gene CCDC134 (Fig. 6a). Compared to the PDGF-BB + si-NC group, both PDGF-BB + si-circCCDC134-1 and PDGF-BB + si-circCCDC134-2 groups showed significantly reduced proliferation and migration rates (Fig. 6b, c). Furthermore, α-SMA and COL-1 demonstrated significantly reduced expression in both PDGF-BB + si-circCCDC134 groups (Fig. 6d), suggesting that circCCDC134 knockdown also attenuates PDGF-BB-induced HOMF transition.

Fig. 6.

Fig. 6

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Silencing circCCDC134 inhibits proliferation, migration, and transformation of PDGF-BB-induced HOMF. (a) RT-qPCR detect circCCDC134 and CCDC134 after siRNA transfection. (b) CCK8 assay assesses HOMF proliferation capacity. (c) Scratch assay assesses HOMF migration capacity. (d) Western blot analysis of COL-1 and α-SMA protein levels. Bar charts show quantitative analysis of COL-1 and α-SMA protein band intensity (n = 3). (* indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001; **** indicates P < 0.0001).

circCCDC134 downregulate miR-503 through sponge miR-503

Dual-luciferase reporter assays demonstrated that miR-503 overexpression significantly inhibited luciferase activity transfected with the circCCDC134-WT plasmid (approximately 50% reduction), while activity in the circCCDC134-MUT group remained unchanged (Fig. 7a). miR-503 levels were significantly downregulated in the PDGF-BB + ov-circCCDC134 group than in the PDGF-BB + ov-NC group (Fig. 7b). Conversely, circCCDC134 silencing partially restored miR-503 expression in PDGF-BB-stimulated HOMF. Transfection with si-circCCDC134-1 increased miR-503 expression (P = 0.1691), while si-circCCDC134-2 induced a significant upregulation (P < 0.05) (Fig. 7c).

Fig. 7.

Fig. 7

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circCCDC134 acts as a sponge of miR-503. (a) Dual luciferase reporter gene assay validates binding between circCCDC134 and miR-503. (b, c) RT-qPCR analysis of miR-503 expression in PDGF-BB stimulated in HOMF transfected with ov-circCCDC134/si-circCCDC134. (* indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001; **** indicates P < 0.0001).

circCCDC134 promotes the inductive effect of PDGF-BB on HOMF through the miR-503/RAF/MEK/ERK axis

Overexpression of circCCDC134 enhanced proliferation, while co-expression of the miR-503 mimic attenuated this proliferative effect (Fig. 8a). circCCDC134 overexpression greatly increased migration at 24 h; however, miR-503 mimic co-transfection prevented this enhanced migration (Fig. 8b). These findings demonstrate that circCCDC134 promotes PDGF-BB-induced HOMF proliferation and migration, while miR-503 overexpression counteracts these effects. RAF mRNA is significantly increased when circCCDC134 is overexpressed. Co-transfection with the miR-503 mimic reduced RAF mRNA (Fig. 8c). circCCDC134 overexpression significantly increased COL-1, α-SMA, RAF, p-RAF, p-MEK/MEK, and p-ERK/ERK compared to PDGF-BB + ov-NC + miR-503 NC. Co-expression of the miR-503 mimic reversed these increases (Fig. 8d, e). The concordant upregulation of RAF mRNA and protein, along with increased p-RAF, p-MEK/MEK, and p-ERK/ERK, indicates activation of the RAF/MEK/ERK pathway upon circCCDC134 overexpression. This activation was suppressed by miR-503 mimic co-expression. These results indicate that circCCDC134, by sponge miR-503, relieves miR-503-mediated repression of RAF mRNA. This leads to increased RAF protein synthesis to activate the RAF/MEK/ERK signaling pathway, The pro-fibrotic effect of circCCDC134 is closely associated with the activation of the RAF/MEK/ERK pathway.

Fig. 8.

Fig. 8

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circCCDC134 promotes proliferation, migration, and transition of PDGF-BB-induced HOMF through the miR-503/RAF/MEK/ERK axis. (a) CCK8 assay for HOMF proliferation capacity. (Compared with the control group, # indicates P < 0.05; ## indicates P < 0.01; ### indicates P < 0.001; #### indicates P < 0.0001; Compared with the PDGF-BB + ov-NC + miR-503 NC group, * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001; **** indicates P < 0.0001; Compared with the PDGF-BB + ov-circCCDC134 + miR-503 NC group, + indicates P < 0.05; ++ indicates P < 0.01; +++ indicates P < 0.001; ++++ indicates P < 0.0001). (b) Scratch assay detects the migration ability of HOMF. (c) Expression of RAF mRNA in HOMF co-transfected with ov-circCCDC134 and miR-503 mimic. (d) Western blot analysis of COL-1, α-SMA, p-RAF, RAF, MEK, ERK, p-MEK, and p-ERK in PDGF-BB-stimulated HOMF cells co-transfected with ov-circCCDC134 and miR-503 mimic. (e) Bar charts showing the gray-scale scanning quantification of protein bands (n = 3). (* represents P<0.05; ** represents P<0.01; *** represents P<0.001; **** represents P<0.0001).

Discussion

The clinical manifestations of oral submucous fibrosis (OSF) comprise oral leukoplakia, intraoral burning sensations, and progressive trismus39. Primary management employs pharmacologic agents (e.g., corticosteroids such as triamcinolone) to mitigate trismus and discomfort. While current treatments alleviate symptoms, they fail to address disease progression. Most patients suffer from trismus, lower quality of life, and poor treatment adherence, contributing to high recurrence rates. OSF frequently progresses to oral squamous cell carcinoma (OSCC)40, substantially elevating mortality. Elucidating OSF pathogenesis is imperative for developing targeted therapeutic strategies. Platelet-derived growth factor-BB (PDGF-BB) is a pivotal profibrotic mediator implicated in multiple fibrogenic processes, including biomechanical remodeling, cytokine dysregulation, and FB activation/transition41–46. Nintedanib—a multi-target tyrosine kinase inhibitor against PDGFR, VEGFR, and FGFR—is clinically approved for fibrotic disorders, notably idiopathic pulmonary fibrosis47,48.

Our prior research demonstrated that PDGF-BB stimulates proliferation, migration, myofibroblast transition, and autophagy in HOMF17,18. And PDGF-BB downregulates miR-503 indirectly activating the RAF/MEK/ERK pathway to drive HOMF proliferation, migration, and transition23. To elucidate how PDGF-BB suppresses miR-503, we selected eight candidate circRNAs and identified circCCDC134 (hsa_circ_0008806) as the most markedly upregulated in PDGF-BB-stimulated HOMF. circCCDC134 promotes PDGF-BB-driven HOMF proliferation, migration, and transition, while its silencing reverses these actions. Notably, circCCDC134 expression in negative control (NC) transfected cells was non-significantly lower than in mock controls (P = 0.416), possibly due to non-specific vector backbone effects or transcriptional resource competition. Crucially, the increased proliferative and migratory capacities found in the PDGF-BB + ov-circCCDC134 group compared to the PDGF-BB + ov-NC controls were primarily caused by circCCDC134 overexpression. Mechanistically, we confirmed circCCDC134 directly binds and negatively regulates miR-503, consistent with a ceRNA network. The classical ceRNA hypothesis posits that circRNA function as miRNA sponges through linear binding sites. However, emerging evidence suggests that the efficiency of miRNA sequestration may be influenced by the sequence-specific structural context of these sites49. Our study confirms a direct interaction between circCCDC134 and miR-503, fitting a ceRNA model. The precise structural determinants of this interaction warrant further investigation in future studies. Next, we confirmed that the promotion of PDGF-BB-induced HOMF biological behavior by circCCDC134 overexpression can be alleviated by miR-503 overexpression, and that circCCDC134 overexpression can indirectly promote the upregulation of RAF at the RNA and protein levels. The expression trend of p-RAF is consistent with that of RAF total protein, suggesting that RAF may be regulated by both transcriptional regulation and post-translational modifications. We confirmed whether RAF activation is transmitted downstream to activate the RAF/MEK/ERK pathway by detecting p-MEK/MEK and p-ERK/ERK. WB results showed that overexpression of circCCDC134 activates the RAF/MEK/ERK signaling pathway, while co-overexpression of miR-503 reduces the activity of the RAF/MEK/ERK pathway. This suggests that circCCDC134 negatively targets miR-503, alleviating the inhibitory effect on RAF, indirectly activating the RAF/MEK/ERK signaling pathway, and promoting the inductive effect of PDGF-BB on HOMF. Our study used a simplified exogenous PDGF-BB stimulation model to delineate the downstream signaling cascade in human oral mucosal fibroblast (HOMF). In vivo, oral tissue PDGF-BB is secreted by multiple cells: epithelial cells (carcinogenesis), activated platelets (injury), and macrophages/immune cells (chronic inflammation). We identified a core fibroblast response pathway that likely activates independently of PDGF-BB’s cellular source.

In colorectal cancer, circCCDC134 upregulation is modulated by m6A modification mediated by alkB homolog 5 (ALKBH5), which improves the stability of circCCDC134 in a YTHDF2-dependent manner50. This suggests that the function and expression regulation of circCCDC134 are likely associated with m6A modification. In preliminary experiments, we found that YTHDC1 may act as a downstream target of miR-503, and PDGF-BB stimulation can upregulate YTHDC1 mRNA expression in HOMF. Therefore, we specifically selected the exon-exon circRNA with m6A modification sites—circCCDC134. Using the circPrimer2.0 database, we predicted that m6A sites are present near the circularization site of circCCDC134. Additionally, YTHDC1 can recognize m6A sites and participate in regulating circRNA generation36. However, additional experimental confirmation is needed to determine whether YTHDC1 modulates the generation of circCCDC134 and whether PDGF-BB induction in HOMF is linked to m6A modification. This is a potential research avenue that we aim to continue exploring.

This study is the first to confirm that circCCDC134 can serve as a molecular biomarker for PDGF-BB-induced HOMF activation, promoting the inductive effect of PDGF-BB on HOMF. circCCDC134 expression correlates with OSF patients, providing experimental support for potential biopharmaceuticals based on circRNA-targeted therapy. circRNA is typically expressed in a tissue- or even cell type-specific manner, and it can be discovered in physiological fluids using liquid biopsy technologies. Many circRNAs are differentially expressed in tumors compared to surrounding non-malignant tissues and are related to disease stage51,52. Currently, there is no direct research reporting on the mechanism of circCCDC134 in OSCC or periodontal disease. However, numerous studies have shown that circRNA are associated with OSCC. circFNDC3B and circFOXO3 can promote the growth, metastasis, and invasion of OSCC53,54. Overexpressed circPVT1 promote OSCC cell proliferation55. These findings indicate that circRNA are widely involved in the pathogenesis of OSCC. Our results suggest that in the microenvironment of OSF, abnormally elevated PDGF-BB induces the expression of circCCDC134, which then continuously activates the RAF/MEK/ERK signaling pathway by sponging miR-503. This creates a positive feedback loop that enhances fibroblast proliferation, migration, and phenotypic transformation. Therefore, the circCCDC134/miR-503 axis is likely a key molecular bridge in the core pathological change of OSF—fibroblast activation. OSF is a significant precancerous lesion for OSCC, so circCCDC134 changes in OSCC worthy of attention. In a preliminary experiment, we observed a downregulation trend of circCCDC134 in some OSCC tissue samples compared to adjacent normal tissues (data not shown). Although our study demonstrates that circCCDC134 promotes PDGF-BB-activated HOMF, its expression pattern in the complex context of oral diseases may be cell-type and disease-specific. It may be upregulated as a pro-activation factor in fibrotic diseases like OSF, which are characterized by abnormal activation of HOMF, whereas it might act as a tumor suppressor in OSCC, a disease dominated by malignant epithelial cell proliferation.

Our study found that circCCDC134 is significantly upregulated in the oral mucosal tissues of OSF patients, and its expression level is positively correlated with the PDGF-BB-induced HOMF activation phenotype. This preliminarily establishes the clinical relevance of circCCDC134 in the pathological process of OSF. Regarding potential sample sources for detection, future research could further explore the feasibility of detecting circCCDC134 in saliva, gingival crevicular fluid, or circulating exosomes from OSF patients, considering the stable presence of circRNA in bodily fluids. These non-invasive or minimally invasive samples would be more suitable for clinical screening and long-term monitoring. And this study elucidates how circCCDC134 promotes PDGF-BB-induced proliferation, migration, and transition of HOMF through the miR-503/RAF/MEK/ERK axis, providing multiple target strategies for the clinical translation of OSF. Currently, clinical treatment for OSF is mostly based on local injections of glucocorticoids, although long-term use may result in side effects such as mucosal atrophy. Targeted therapy strategies offer high specificity and low off-target risks: The closed-loop structure of circRNA is more stable in body fluids, making it suitable as a therapeutic target. Precise regulation of the miR-503/RAF/MEK/ERK axis can mitigate interference with normal repair processes. Our data indicates that the activation of the RAF/MEK/ERK pathway is a key correlated event in regulation of circCCDC134. however, its necessity awaits further validation through functional rescue experiments using specific inhibitors or genetic approaches in the future. Therefore, treatment strategies could focus on whether inhibiting circCCDC134 or RAF and MEK/ERK pathway inhibitors can alleviate OSF progression, whether delivering miR-503 mimics via nanoparticles (such as liposomes, exosomes, polymer nanoparticles, etc.) can block OSF development56, or combining the above approaches for combined therapy. These strategies are our future research directions. However, this study still has limitations: The sample size in in vivo experiments is small, and a larger clinical cohort is needed for validation. Our study lacks molecular mechanism validation at the animal level, and it is necessary to establish a mouse OSF model with circCCDC134 knockdown for subsequent mechanism experiments to provide direct evidence for clinical application (Fig. 9).

Fig. 9.

Fig. 9

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Proposed working model of the circCCDC134/miR-503 axis in PDGF-BB-induced HOMF activation.

Conclusion

In summary, our research shows that PDGF-BB increases circCCDC134 expression, which then promotes PDGF-BB-driven HOMF proliferation, migration, and transformation through the miR-503/RAF/MEK/ERK pathway. These findings suggest new avenues for developing targeted OSF treatments.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary Material 1 (563.7KB, pdf)

Author contributions

Y.T.Z. wrote the main manuscript text, N.J analysi data, Q.W. and H.D.G. conducted part of the experiments, while J.W. and C.H.J. revised the paper. All authors reviewed the manuscript.

Funding

This work was funded by the Natural Science Foundation of Hunan Province, China (2022JJ30695).

Data availability

The datasets generated and/or analysed during the current study are available in the NCBI Sequence Read Archive, SRA repository, SAMN51603465.

Declarations

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1 (563.7KB, pdf)

Data Availability Statement

The datasets generated and/or analysed during the current study are available in the NCBI Sequence Read Archive, SRA repository, SAMN51603465.

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