ABSTRACT Abnormal accumulation of collagen fibrils is a hallmark feature of oral submucous fibrosis (OSF). However, the precise characteristics and underlying mechanisms remain unclear,

impeding the advancement of potential therapeutic approaches. Here, we observed that collagen I, the main component of the extracellular matrix, first accumulated in the lamina propria and

subsequently in the submucosa of OSF specimens as the disease progressed. Using RNA-seq and Immunofluorescence in OSF specimens, we screened the cartilage oligomeric matrix protein (COMP)

responsible for the abnormal collagen accumulation. Genetic COMP deficiency reduced arecoline-stimulated collagen I deposition significantly in vivo. In comparison, both COMP and collagen I

COMP-positive fibroblasts secrete more collagen I. Since COMP is a molecular bridge with Fibril-associated collagens with Interrupted Triple helices (FACIT) in the collagen network, we

further screened and identified collagen XIV, a FACIT member, co-localizing with both COMP and collagen I. Collagen XIV expression increased under arecoline stimulation in wild-type mice,

whereas it was hardly expressed in the _Comp__-/-_ mice, even with under stimulation. In summary, we found that COMP may mediates abnormal collagen I deposition by functions with collagen

XIV during the progression of OSF, suggesting its potential to be targeted in treating OSF. SIMILAR CONTENT BEING VIEWED BY OTHERS EXOSOMAL MIR-17-5P DERIVED FROM EPITHELIAL CELLS IS

INVOLVED IN ABERRANT EPITHELIUM-FIBROBLAST CROSSTALK AND INDUCES THE DEVELOPMENT OF ORAL SUBMUCOSAL FIBROSIS Article Open access 20 June 2024 PDGF-BB PROMOTES ORAL SUBMUCOSAL FIBROSIS BY

DRIVING PHENOTYPIC TRANSFORMATION AND AUTOPHAGY IN ORAL MUCOSAL FIBROBLASTS THROUGH DOWNREGULATION OF CIRCHIPK3 Article Open access 02 May 2025 STROMAL THROMBOSPONDIN 1 SUPPRESSES

ANGIOGENESIS IN ORAL SUBMUCOUS FIBROSIS Article Open access 26 February 2024 INTRODUCTION Oral submucous fibrosis (OSF) is a chronic, insidious oral potentially malignant disorder (OMPD),1

with a malignant transformation rate of 7%–13%.1,2,3 The clinical symptoms of OSF, such as pale mucosa, burning sensations, and restricted mouth opening, significantly impair patients’

quality of life.4,5 The main pathological characteristic of OSF is abnormal collagen deposition, which impacts both its malignant transformation potential and clinical symptoms.6,7,8,9,10

Therefore, understanding the underlying mechanisms of abnormal collagen accumulation during OSF progression is crucial for advancing the development of therapeutic approaches. In oral

mucosa, collagen I fibrils are the most abundant component of all types of collagen.11,12 Collagen I fibrils exhibit a hierarchical structure, requiring a multi-stage assembly process.13

Procollagen triple helix is secreted into the extracellular space to form collagen, collagen further assemble into microfibrils and finally merge into mature fibrils through longitudinal and

axial growth.13 The axial growth process of collagen fibrils plays a vital role in forming type I collagen fibrils. During this stage, Fibril-associated collagen with Interrupted Triple

helices (FACIT) can integrate into the surface of collagen fibrils that would regulate the axial growth and limit fibril diameter.14,15,16 Collagen XII and collagen XIV, both members of the

FACIT family, have been identified as being associated with collagen I fibrils contributing to collagen fibrils architecture.17,18,19,20 However, whether and how FACIT contributes to the

formation of abnormal collagen structures in OSF has not yet been investigated. COMP is a pentameric multidomain protein, which consists of an N-terminal domain, four type 2 epidermal growth

factor (EGF)-like repeats, seven type 3 calcium-binding repeats, and a C-terminal globular domain.21 Through its C-terminal domain, COMP can bind to various components of the extracellular

matrix (ECM), contributing to organ fibrosis and other pathological conditions.22 For example, In the dermis, COMP accumulates in systemic sclerosis skin and keloids, inhibiting COMP can

reduce collagen I deposited in vitro.23,24 However, the underlying mechanism of which COMP causes collagen I deposition remain unclear. A few in vitro studies reveal that COMP can act as a

molecular bridge in the collagen II network by binding to collagen IX, a FACIT that decorates the surface of collagen II fibrils,25,26 indicating its role as a molecular bridge in FACIT

decoration. However, whether and how COMP functions in the collagen I accumulation during the progression of OSF remains unknown. In this study, we examined the changes in collagen I

accumulation over time and space in OSF specimens during disease progression. We found that collagen I is abnormally accumulated in the lamina propria during the early stage, progressing

deeper into the submucosa as the disease progressed. Through RNA sequencing, we identified COMP as a key pathogenic factor in mediating this abnormal accumulation of collagen. Our results

suggest that COMP may facilitate abnormal collagen I fibrils deposition by interacting with collagen XIV during the progression of OSF. RESULTS SPATIAL-TEMPORAL ACCUMULATION OF COLLAGENS IN

THE PATHOGENESIS OF OSF The development of OSF starts with the abnormal accumulation of collagen fibers followed by hyaline degeneration (Fig. S1), indicating that characteristics of

collagen accumulation vary throughout the progression of OSF. To depict how collagen deposits over space and time, we examined the micropathological features of major collagen types,

collagen I and collagen III, using immunostaining. We observed that the micropathological characteristics of collagen I differed from those of collagen III: In the normal buccal mucosa,

collagen I was homogeneously expressed in both the lamina propria and the submucosa. In the early to middle stages, collagen I expression was upregulated in the lamina propria but not in the

submucosa. In the late stage of OSF, we observed significant deposits of collagen I in both the lamina propria and submucosa, although the collagen was much denser in the lamina propria

(Fig. 1a, b). In contrast, no significant differences were observed in the expression of collagen III in different disease stages (Fig. 1c, d). In summary, collagen I accumulation follows a

clear spatial and temporal sequence during the progression of OSF, including early accumulation in the lamina propria and gradual progression to the submucosa. COLLAGEN METABOLISM-RELATED

GENE COMP MAY BE A KEY MOLECULE MEDIATING ABNORMAL COLLAGEN I ACCUMULATION IN OSF To investigate the molecular mechanisms that mediate the abnormal accumulation of collagen I, we collected

buccal mucosal tissues from patients with early, middle, and late stage OSF and healthy controls for RNA sequencing. Differential mRNA expression profiles in OSF pathogenesis were analyzed

(Fig. S2). As we expected, collagen remodeling and metabolism-related genes were consistently upregulated during OSF progression (Supplementary Tables 1, 2). We further intersected

differential expressed genes (DEGs) in the early, middle, and late stages of OSF revealing that six genes, including _COMP_, _CCL18_, _KRT76_, _BCORP1_, _PLTP_, and _CDYL2_ were consistently

upregulated (Fig. 2a). _COMP_ enriched in most collagen remodeling-associated GO terms, indicating its crucial role in regulating collagen accumulation during OSF progression (Figs. 2b,

S3). In addition, we paid attention to the upregulated DEGs during OSF progression (Figs. S4, S5), and found that COMP may be involved in the ECM-receptor interaction pathway in the middle

stage of OSF and the PI3K-Akt signaling pathway in the late stage of OSF (Fig. S4). Therefore, we performed immunostaining of COMP in our OSF-specimen cohort. We showed that the distribution

of COMP proteins presents a low level in the lamina propria and submucosa regions of normal samples (Fig. 2c–e). However, the expression of COMP proteins was significantly upregulated in

the lamina propria and submucosa during OSF progression (Fig. 2c–e), indicating critical functional involvement of abnormal activation of COMP during OSF progression. Thus, we next focus on

the role of COMP in influencing collagen I accumulation during OSF progression. COMP DEFICIENCY REDUCES ARECOLINE-STIMULATED ACCUMULATION OF COLLAGEN I To validate whether COMP mediates

abnormal collagen accumulation, we constructed a _Comp__-/-_ mouse model (C57BL/6 N) using CRISPR/Cas (Fig. 3a). No COMP expression was observed in _Comp__-/-_ mice, indicating effective

COMP disruption in the _Comp__-/-_ mouse model (Fig. 3b, d). The _Comp__-/-_ mice exhibited normal growth and development (Fig. S6), and no apparent abnormalities were observed in the

histological structure of their buccal mucosa (Fig. 3c). However, Masson’s trichrome staining and collagen I immunofluorescence showed that _Comp__-/-_ mice had slightly less total collagen

and collagen I than WT mice (Fig. 3d–f). We next investigated the impact of COMP disruption during OSF progression. Chewing areca nuts is a significant risk factor for OSF.27,28 Previous

studies have revealed that injecting arecoline, a key component in areca nuts, into the buccal mucosa of mice develops OSF.29,30,31,32 Therefore, to further validate the role of COMP in the

abnormal deposition of collagen I, we injected arecoline (20 mg/kg) locally into the buccal mucosa of WT mice and _Comp__-/-_ mice daily for 12 weeks, and injected an equivalent volume of

phosphate buffered saline (PBS), the solvent used with arecoline, for the control group. Collagen deposition was evaluated using hematoxylin and eosin (H&E) and Masson’s trichrome

staining and collagen I immunofluorescence. The results showed that abnormal collagen deposition was significantly induced in the buccal mucosa of WT mice with arecoline injection compared

to that in the control group, whereas abnormal collagen deposition was not observed in _Comp__-/-_ mice injected with either arecoline or PBS (Fig. 4a–c). COMP expression was upregulated in

the WT mice injected with arecoline compared to those injected with PBS(Fig. 4a, d). These data uncover that COMP may play a crucial role in the deposition of collagen I. ARECOLINE

STIMULATES COLLAGEN I DEPOSITION IN FIBROBLASTS VIA COMP We next focused on how abnormal COMP expression is activated during OSF progression. As human buccal mucosal fibroblasts (hBMFs) are

the main cell type producing collagen in OSF,33,34,35 we estimate that hBMFs may be the source of abnormal COMP expression. To investigate whether hBMFs can be abnormally induced to

upregulate COMP thus causing collagen I deposition, we isolated normal hBMFs and treated them with arecoline (Figs. 5a, S7). Under normal conditions, very few fibroblasts expressed COMP

(Fig. 5b, c). Upon arecoline stimulation, the expression levels of both COMP and collagen I were upregulated time-dependent (Figs. 5b, d, e, S8). In addition, the number of COMP-positive

fibroblasts gradually increased under stimulation and was the highest at 24 h post arecoline stimulation (Fig. 5b, c). Interestingly, Immunofluorescence co-localization revealed that hBMFs

present heterogeneous response to arecoline treatment. COMP-positive fibroblasts exhibit elevated COMP expression and secrete higher levels of collagen I. (Fig. 5f). Due to the heterogeneous

expression level of collagen I within COMP-negative fibroblasts, we classified them into collagen I high and collagen I low fibroblasts using the lower quartile of collagen I expression

intensity as the threshold. Quantitative analysis showed that most COMP-negative fibroblasts had low collagen I levels (Fig. 5g, h). These results indicate that high COMP expression in

fibroblasts correlates with high collagen I production, suggesting the intracellular function of COMP in collagen I secretion. To explore the effect of COMP on fibroblast function, we

knocked down COMP in hBMFs and performed RNA sequencing, we observed a significant reduction in prolyl 3-hydroxylase 1(P3H1) and procollagen C-endopeptidase enhancer 2(PCOLCE2) expression.

Both of them are essential for collagen assembly (Fig. 6a). Additionally, GO enrichment analysis indicated abnormal functions associated with collagen remodeling in the extracellular matrix

following knock down COMP (Fig. 6b, c). To validate whether activation of COMP expression upregulates collagen I production, we stimulated the hBMFs with arecoline and found that collagen I

synthesis was greatly reduced following the silencing of COMP, particularly following stimulation by arecoline. Meanwhile, arecoline promoted the secretion of COMP and collagen I in control

fibroblasts (Fig. 6d–f). COLLAGEN XIV, NOT COLLAGEN XII, MODIFIES COLLAGEN I DURING COLLAGEN DEPOSITION IN OSF Previous studies indicate the role of COMP as a extracellular molecular bridge

in the collagen II network by modifying the surface of collagen IX,25,26 indicating it may have similar function in collagen I deposition. STRING protein interactions revealed that COMP may

interact with COL12A1 and COL14A1, both of which belong to the FACIT family (Fig. 7a). The correlation heatmap showed that, compared to COL12A1, COMP was more strongly correlated with

COL14A1 (Fig. 7b). Immunofluorescence showed that the expression level of collagen XIV was elevated during OSF development, and the collagen XIV protein largely co-localized with collagen I

(Fig. S9, S10). Notably, collagen XIV expression in the lamina propria was more significantly upregulated than that in the submucosa, similar to the patterns of collagen I and COMP (Fig. 1a,

b, Fig. 2c–e, Fig. S9, S10). In contrast, the expression level of collagen XII was much lower than that of collagen XIV, and co-localization with collagen I was rarely observed (Fig. S9,

S11). These results indicate that collagen XIV, not collagen XII, modifies collagen I to promote abnormal collagen deposition during OSF pathogenesis. COMP MEDIATES THE ARECOLINE-INDUCED

SECRETION OF COLLAGEN I AND COLLAGEN XIV To verify whether COMP is involved in the formation of collagen XIV and collagen I collagen structures that facilitate the progression of OSF, we

first found their local co-localization through immunofluorescence techniques(Fig. 7c). The interaction between COMP, collagen XIV and collagen I was subsequently confirmed via

co-immunoprecipitation experiment (Fig. 7d). To determine whether collagen XIV can be regulated by arecoline stimulation, we analyzed the expression levels of collagen XIV in the buccal

mucosa of WT mice with or without arecoline treatment. Collagen XIV expression was upregulated following arecoline stimulation (Fig. 8a, b). Meanwhile, we observed co-localization of COMP,

collagen XIV, and collagen I in the arecoline-stimulated buccal mucosa of WT mice (Fig. 8a). At the cellular level, collagen XIV expression was upregulated in arecoline-stimulated

fibroblasts in a time-dependent manner in vitro (Figs. 8c, S12a). We then studied whether arecoline- stimulated secretion of collagen I and collagen XIV were mediated by COMP. We found that

collagen XIV expression in the buccal mucosa of _Comp__-/-_ mice was not elevated following arecoline treatment (Fig. 8a, b). At the cellular level, following COMP knockdown, the expression

of collagen XIV was not significantly elevated, even in the presence of arecoline stimulation (Figs. 8d, S12b). DISCUSSION In this study, we observed a sequential accumulation of collagen I

in the lamina propria at an early stage and later in the submucosa as the disease progressed. Through RNA sequencing, we identified COMP as a crucial causative factor in the abnormal

deposition of collagen. By constructing _Comp__-/-_ mice, we demonstrated that deleting COMP reduced the extent of arecoline-stimulated collagen I deposition. Given that COMP usually binds

to FACITs and acts as a molecular bridge in the collagen network, we found that collagen XIV, a member of FACIT, was co-localized with COMP and collagen I. Functional experiments showed that

collagen XIV expression was upregulated in response to arecoline stimulation in WT mice, whereas it was hardly expressed in _Comp__-/-_ mice, even under stimulation. These findings suggest

that COMP may mediate abnormal collagen I deposition by interacting with Collagen XIV during the progression of OSF. Abnormal collagen accumulation is associated with an abnormal collagen

structure. The skin of patients with scleroderma exhibits a large number of thin collagen fibrils, either in bundles alone or intermingled with large-diameter fibrils.36 During the early

stages of OSF, a transition occurs from uniformly sized collagen fibril bundles to the presence of immature, fine fibrils within the interfibrous matrix. As the disease progresses, the

diameter of the fiber bundles varies, leading to a dense array of fibers.37,38 The fibrous network structure of the ECM in the oral mucosa is composed of a variety of macromolecules,

primarily collagen I and III. While collagen I levels gradually increase with the progression of OSF, previous research has not clarified the spatial and temporal dynamics of collagen I

accurately.7,9 Our study, however, found for the first time that collagen I abnormally accumulates in the lamina propria in the early stage of OSF and subsequently penetrates deeper into the

submucosa as the disease progresses. COMP is an ECM glycoprotein that is involved in fibrosis, skeletal diseases, cancer, and cardiovascular diseases.21,22 In fibrotic diseases, COMP

expression levels often correlate with disease progression across different disorders, including scleroderma,23,39,40,41 skin keloid,24,40 liver fibrosis42,43 and idiopathic pulmonary

fibrosis.44 In skin keloids, COMP is expressed at higher levels in larger lesions but at lower levels in small scars.24 Similarly, in idiopathic pulmonary fibrosis, COMP is expressed at

higher levels in areas of dense fibrosis.44 The upregulation of COMP stimulates the accumulation of collagen and other ECM proteins, exacerbating fibrosis severity.23,39,45,46 In this study,

we found that COMP expression was upregulated in both the lamina propria and submucosa of OSF samples. Further, we found that COMP mediates abnormal collagen I deposition. The observation

of the pathological changes in the abnormal structure of the collagen fibrils network prompted us to investigate the process of collagen fibrils formation. Collagen categories include

classical fibrillar collagen, FACITs, and membrane-associated collagens with interrupted triple helices (MACITs), etc. 47 Fibrillar collagen is the main component of the ECM in the

connective tissues, and collagen I is the most abundant fibril of the oral mucosa.48 The structure of FACITs is characterized by “collagenous domains” interrupted by short non-helical

domains, which play a role in modifying the surfaces of classical fibrillar collagen fibrils. For example, collagen IX, a member of FACIT, is associated with collagen II fibrils, while

collagen XII and collagen XIV modifies the surface of collagen I fibrils.18,49 FACIT also interacts with other ECM molecules, such as COMP.20,50,51,52 COMP acts as a molecular bridge in the

collagen II network by binding to collagen IX in cartilage.21,22,53 In addition, COMP binds to collagen XII and XIV, which associate with collagen I fibrils in the ECM of the skin.20 In our

study, we found that collagen XIV, a member of the FACIT family, correlated with collagen I and co-localized with collagen I and COMP in the oral mucosa. These results suggest that COMP

mediates abnormal collagen I deposition by serving as a molecular bridge interacting with collagen XIV. COMP may play different roles during the progress of OSF. COMP’s interaction with

collagen I and XIV was significantly increased in OSF tissues, helping to explain the observed collagen deposition. Interestingly, by visualizing the co-localization of COMP and collagen I

in arecoline-stimulated fibroblasts, we found that COMP-positive fibroblasts had relatively high collagen I expression, which indicates COMP promotes collagen synthesis intracellularly.

These results indicate the dual intracellular and extracellular roles of COMP, although its specific mechanism remains to be further studied. As OSF advances to the late stage, the cellular

population diminishes significantly, and COMP may primarily function as an organizer of collagen fibers. TGF-beta signaling is critical for oral submucosal fibrosis. TGF-beta was associated

with fibrotic disease by induces COMP expression. In skin fibrosis and lung fibrosis, COMP regulate the TGF-beta signaling pathway, and both COMP and TGF-beta form a positive feedback

loop.44,54 Furthermore, COMP promotes the progression of fibrosis by interacting with CD36 and activating the MEK/ERK and PI3K/AKT signaling pathways.46,55,56,57 COMP has intracellular

functions that facilitate collagen secretion of dermal fibroblasts.45 In COMP-deficient fibroblasts, ER stress is caused by collagen retention in the ER.45 In this study, although we

revealed the possible intracellular and extracellular roles of COMP, the detailed regulation mechanisms of COMP mediated collagen synthesis or deposition needs further studies. This study

has some limitations. First, the quantification of COMP expression level is needed in a larger cohort of OSF patients. Second, given that COMP expression was only in part of hBMFs, future

studies based on single cell sequencing from controls and OSF patients at different stages will help us to identified the characterized fibroblast subset expressing COMP. Third, this study

did not provide detailed quantitative data on collagen I expression levels across different OSF stages in vivo in mice, which would help assess the progression’s severity and translate the

results to humans. Last, this study did not investigate additional potential factors like TGF-beta or compensatory mechanisms that may influence collagen deposition other than COMP. This

study focused on the abnormal assembly of collagen structures during OSF progression and revealed that COMP functions as an important mediator of this process. Given that COMP expression can

be upregulated by arecoline, it suggests that COMP-mediated collagen deposition may be a key pathological mechanism underlying OSF. Therefore, COMP may function as a significant biomarker

of OSF. Furthermore, targeting COMP is a promising treatment for OSF, and future studies should focus on investigating potential small molecules that could inhibit the function of COMP.

MATERIALS AND METHODS PATIENT TISSUE SAMPLES The study protocol was conducted in accordance with the principles of the Declaration of Helsinki and approved by the Research Ethics Board of

the Scientific and Ethical Committee of Xiangya Hospital (Approval numbers 2019030562 and 2024020145). Mucosal tissues were obtained after written informed consent was obtained from the

patients. Normal tissues were obtained from patients with low impacted tooth extraction and buccal mucosa urethral reconstruction, and OSF samples were obtained from the biopsy samples of

the patients during their first visit, the final diagnosis was made by professional pathologists. The pathological manifestations of OSF at different stages are as follows. In the early

stage of OSF, a large number of inflammatory cells were infiltrated and collagen fiber edema was observed, but no significant fibrotic changes were observed. In the middle stage, the

connective tissues show hyalinization of collagen, with mild edema and inflammatory cell infiltration. In the late stage, collagen fibers are severely hyalinized, the number of fibroblasts

is significantly reduced, inflammatory cell infiltration is almost invisible, and blood vessels are narrowed or blocked. The clinical characteristics of the patients are shown in

Supplementary Tables 3 and 4. PATHOLOGICAL OBSERVATION Tissues were fixed in 4% paraformaldehyde overnight, dehydrated using an ethanol gradient, embedded in paraffin, and sectioned into

4-μm sections. H&E staining was performed, and the sections were embedded in neutral resin and photographed using a Leica DM750 light microscope (Leica Microsystems, Wetzlar, Germany).

Total collagen accumulation was evaluated using a Modified Masson’s trichrome staining kit (G1346, Solarbio, Beijing, CHN). Images were captured using a Leica DM750 light microscope (Leica

Microsystems, Wetzlar, Germany). Collagen fibers are shown in blue, cytoplasm and muscle tissue are shown in red, and nuclei are shown in blue-brown. The area of the collagen volume fraction

was determined using ImageJ software. IMMUNOFLUORESCENCE ASSAY Paraffin-embedded samples were sectioned into 4-µm-thick slices, dried at 60 °C for 2 h, dewaxed in xylene, and hydrated using

the gradient ethanol method. The antigen was microwave-repaired and blocked with goat serum for 30 min. Primary antibodies were added and kept overnight at 4 °C, then the tissue was

incubated with the appropriate secondary antibody at room temperature for 1 h, and the tablet was sealed with DAPI-containing anti-quenching sealing tablets. Images were captured using a

Leica DM6B Thunder fluorescence microscope (Leica Microsystems, Wetzlar, Germany) and analyzed using ImageJ software. The primary antibodies used were as follows: collagen I (CST, Danvers,

MA, USA; #72026; 1:200), collagen III (Proteintech, Wuhan, China; 22734-1-AP, 1:200), COMP (Abcam, Cambridge, MA, USA; ab300055, 1:1 000), collagen XIV (Invitrogen, Carlsbad, CA, USA;

PA5-49916, 1:150), and collagen XII (Santa Cruz Biotechnology, Santa Cruz, CA,USA; sc-166020, 1:200). A tyramide signal amplification (TSA) kit (AFIHC034; Ai Fang biological, Changsha,

China) was used for multiple immunofluorescence tests: (1) Each slide was dewaxed using xylene; (2) Antigen repair was the same as mentioned above; (3) A 3% hydrogen peroxide solution was

used to block endogenous peroxidases; (4) Goat serum was used as a non-specific target; (5) The primary antibody was incubated at 4 °C overnight; (6) The poly-horseradish peroxidase (HRP)

secondary antibody was incubated at room temperature for 50 min; (7) The TSA fluorescent dye reaction solution was administered under a fluorescence microscope; (8) Antibodies were eluted;

(9) Steps 3–8 were repeated (replaced by the second TSA fluorescent dye label); 10) Steps 3–7 were repeated (replaced by the third TSA fluorescent dye label); (11) Slides were mounted with

DAPI-containing Prolong Gold antifade reagent (P36935, Invitrogen); (12) Images were captured using a Leica DM6B Thunder fluorescence microscope (Leica Microsystems, Wetzlar, Germany).

ANIMAL STUDIES _Comp__-/-_ mice were generated using CRISPR/Cas9-mediated genome editing. _Comp_ (NCBI reference sequence: NM_016685; Ensembl: ENSMUSG00000031849) is located on mouse

chromosome 8. Nineteen exons have been identified in mouse chromosome 8, with an ATG start codon in exon 1 and a TAG stop codon in exon 19 (Transcript: ENSMUST0000000003659). Exons 2–9 were

selected as target sites. Heterozygous mutant mice were mated with homozygous mutant mice to generate heterozygous mutant mice, which were intercrossed to produce homozygous mutant mice. The

mice were identified by PCR, and the genotyping primers used are listed in Supplementary Table 5. Western blotting of the positive tissue (mouse cartilage) was used to verify the COMP

knockout. Animal experiments were approved by the Animal Ethics Committee of the Department of Laboratory Animals, Central South University, and. housed at the Central South University

Department of Laboratory Animal Science, Changsha, China. At 7 weeks of age, the mice were randomly allocated into the WT + PBS, WT+ arecoline, _Comp__-/-_+PBS, and _Comp__-/-_+arecoline

groups. Arecoline was diluted to 4 mg/mL in PBS immediately before injection. The WT+ arecoline and _Comp__-/-_+arecoline groups were treated with a regional local injection of arecoline (20

 mg/kg, daily) on the left cheek using a 30G insulin syringe (KDL, Wenzhou, China). An equal volume of PBS was administered to the control group. After 12 weeks of treatment, the mice were

euthanized and buccal tissues were collected. CELL CULTURE Primary fibroblasts were isolated from normal human buccal tissue using a nonenzymatic procedure. Tissue samples were inserted into

15-mL polypropylene tubes containing 5 mL Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Carlsbad, CA, USA) with 3% penicillin-streptomycin. The tissues were processed as quickly as

possible after surgery. Tissue samples were rinsed three times in a cell culture dish with PBS containing 3% penicillin-streptomycin. The tissue samples were finely chopped into 1 mm 2

pieces using sterile scissors. A T25 cell culture flask (Nest, China) was washed with DMEM, the medium was removed, and the tissue pieces were gently pressed into the T25 flask. The tissue

pieces were dried for 4 h in a humidified 5% CO2 incubator at 37 °C. Fibroblasts between passages 3 and 8 were cultured in DMEM containing 10% fetal bovine serum and 1%

penicillin-streptomycin in a humidified 5% CO2 incubator at 37 °C. STABLE CELL LINE GENERATION LV-COMP-RNAi recombinant lentivirus and GV112 vector lentivirus were purchased from GeneChem

(Shanghai, China). The lentivirus design is illustrated in Supplementary Table 6. Fibroblasts were infected with lentivirus (MOI = 10) according to the manufacturer’s protocol. Selective

culture medium containing puromycin (2 µg/mL) was used to select the stable expression cells. COMP expression was detected using western blotting. CELL IMMUNOCYTOCHEMISTRY The fibroblasts

were cultured on coverslips, fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 in PBS for 20 min, blocked in 3% bovine serum albumin for 1 h, and incubated with primary

antibody overnight at 4 °C. The primary antibodies used were against collagen I (CST, Danvers, MA, USA; #72026; 1:200) and COMP (Abcam, Cambridge, MA, USA; ab300055, 1:1 000). The

fibroblasts were then washed with PBS, and incubated with Alexa Fluor 488 or 594 secondary antibodies (Abcam, Cambridge, MA, USA; ab150080, ab150077,1:600) for 1 h at room temperature. Then,

the coverslips were washed and mounted with histology mounting medium with DAPI (sigma-aldrich, Louis Missouri, FLORIDA, USA; F6057). Images were acquired using a Leica DM6B fluorescence

microscope (Leica Microsystems, Wetzlar, Germany). CO-IMMUNOPRECIPITATION hBMFs was treated with arecoline (10 μg/mL) or PBS and exocytosis inhibitor EXO1 for 24 h, Cultured cells were lysed

on ice in NP40 buffer containing protease inhibitors. The whole cell lysate was transferred to 1.5 mL centrifuge tube and centrifuged at 12 000 r/min at 4 °C for 15 min. The liquid

supernatant was collected and the protein concentration was measured by BCA kit (Thermo Fisher Scientific, MA, USA; 23225). Five percent of the protein sample was taken as the input group.

Protein sample (2 mg) was taken from PBS and AREC group for follow-up experiment. The sample was divided into two parts. One part was added with magnetic beads coupled with target

antibodies, and the other part was added with magnetic beads coupled with IgG antibodies as control group. After being rotated at 4 °C overnight, the samples were washed with TBST with

magnetic beads for three times. Finally, 50 μL elution buffer was added and treated for 5 min at 95 °C. WESTERN BLOT Cultured cells and mouse cartilaginous tissues were homogenized and lysed

on ice in lysis buffer containing protease inhibitors. Protein samples were isolated using a 7.5% sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS–PAGE) gel and transferred to

polyvinylidene difluoride membranes. The samples were then blocked with 5% skim milk powder for 1 h at room temperature and incubated with collagen I (Proteintech, Wuhan, China; 22734-1-AP,

1:2 000), COMP (Abcam, Cambridge, MA, USA; ab300055, 1:1 000), collagen XIV (Invitrogen, Carlsbad, CA, USA; PA5-49916, 1:1 000), β-actin (Zenbio, Chengdu, China; 250136, 1:5 000) antibodies

overnight at 4 °C. HRP-conjugated secondary antibodies were added and incubated at room temperature for 1 h. RNA EXTRACTION, LIBRARY PREPARATION, AND SEQUENCING RNA sequencing was performed

on human buccal mucosa tissues, including the healthy control group, OSF early-stage group, OSF middle-stage group, and OSF late-stage group; each group contained three independent samples.

Total RNA was extracted using the mir Vana miRNA isolation kit (Cat#AM1561, Ambion, Waltham, Ma,USA). Total RNA was analyzed using a NanoDrop ND-2000 spectrophotometer (Thermo Fisher

Scientific, Waltham, MA, US) and an Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, US). The cDNA libraries were sequenced using an Illumina sequencing platform (Genedenovo

Biotechnology Co. Ltd., Guangzhou, China). Raw reads obtained by sequencing were filtered using Seqtk, Genome mapping of the preprocessed reads was performed using the spliced mapping

algorithm in Hisat2 version 2.0.4. Subsequently. DEGs were analyzed using edgeR software. The criteria for identifying DEGS were a false discovery rate (FDR) < 0.05 (the p-value adjusted

by the Benjamini–Hochberg method) and |log2(fold change (FC))| ≥ 1. Part of the data was analyzed using the bioinformatics analysis website sangerbox.58 BIOINFORMATICS ANALYSIS Statistical

analyses were performed using GraphPad Prism 8.0 software. Unpaired two-tailed t-tests were used to determine the significance of the differences between two groups. Analysis of variance

(ANOVA) with Tukey’s Honestly Significant Difference (Tukey’s HSD) tests were used to compare more than two groups. The Pearson correlation coefficient was used for correlation analysis. All

data are presented as mean ± standard deviation (SD). _P_ < 0.05 indicated statistical significance (*_P_ < 0.05, **_P_ < 0.01, ***_P_ < 0.001, ****_P_ < 0.000 1). REFERENCES

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supported by the National Natural Science Foundation of China grant (81974150). AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Oral and Maxillofacial Surgery, Xiangya Hospital,

Central South University, Changsha, China Yafei Xiong, Jie Zhang, Xiaoshan Wu & Feng Guo * Academician Workstation for Oral-Maxillofacial Regenerative Medicine, Central South University,

Changsha, China Yafei Xiong, Xuechun Li, Bincan Sun, Jie Zhang, Xiaoshan Wu & Feng Guo * Research Center of Oral and Maxillofacial Development and Regeneration, Xiangya Hospital,

Central South University, Changsha, China Yafei Xiong, Xuechun Li, Bincan Sun, Jie Zhang, Xiaoshan Wu & Feng Guo * National Clinical Research Center for Geriatric Diseases, Xiangya

Hospital, Central South University, Changsha, China Yafei Xiong, Xuechun Li, Bincan Sun, Jie Zhang, Xiaoshan Wu & Feng Guo Authors * Yafei Xiong View author publications You can also

search for this author inPubMed Google Scholar * Xuechun Li View author publications You can also search for this author inPubMed Google Scholar * Bincan Sun View author publications You can

also search for this author inPubMed Google Scholar * Jie Zhang View author publications You can also search for this author inPubMed Google Scholar * Xiaoshan Wu View author publications

You can also search for this author inPubMed Google Scholar * Feng Guo View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS Yafei Xiong

designed, and conducted the experiments; collected clinic samples; and wrote the manuscript. Xuechun Li contributed to data analysis and interpretation; and revised the manuscript. Bincan

Sun and Jie Zhang collected clinic samples; Feng Guo and Xiaoshan Wu contributed to design, supervise the experiments; data interpretation; and revised the manuscript. CORRESPONDING AUTHORS

Correspondence to Xiaoshan Wu or Feng Guo. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. SUPPLEMENTARY INFORMATION SUPPLEMENTARY TABLES SUPPLEMENTARY

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THIS ARTICLE CITE THIS ARTICLE Xiong, Y., Li, X., Sun, B. _et al._ Abnormal collagen deposition mediated by cartilage oligomeric matrix protein in the pathogenesis of oral submucous

fibrosis. _Int J Oral Sci_ 17, 25 (2025). https://doi.org/10.1038/s41368-025-00355-x Download citation * Received: 08 July 2024 * Revised: 05 February 2025 * Accepted: 14 February 2025 *

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