Fibrotic matrix induces mesenchymal transformation of epithelial cells in oral submucous brosis

Kai Jiao (  kjiao1@163.com ) School of Stomatology, The Fourth Military Medical University, Xi'an, Shaanxi 710032, China. Hao-qing Xu 1 Northwest University 2 The Fourth Military Medical University Zhen-xing Guo The Fourth Military Medical University Jia-lu Gao The Fourth Military Medical University Shu-yan Wang The Fourth Military Medical University Jian-fei Yan The Fourth Military Medical University Xiao-xiao Han 1 Northwest University 2 The Fourth Military Medical University Wen-pin Qin The Fourth Military Medical University Weicheng Lu The Third A liated Hospital of Air Force Medical University Chang-he Gao The Third A liated Hospital of Xinxiang Medical University Li-na Niu The Fourth Military Medical University https://orcid.org/0000-0002-6653-0819


Introduction
Oral submucous brosis (OSF) is de ned as a chronic progressive oral mucosal disease that is closely related to betel nut chewing. 1,2 OSF can occur in multiple parts of the patient's mouth, including the buccal mucosa, labial mucosa, molar pad, soft palate, and the bottom of the mouth, resulting in loss of ber elasticity and increased mucosal stiffness. 3 The main clinical symptom of OSF is progressive mouth opening limitation, which affects chewing, swallowing, pronunciation, and other functions, which signi cant affects the quality of a patients' life. 4 As early as 1978, the World Health Organization (WHO) classi ed OSF as a precancerous state of the oral mucosa, which is closely related to the occurrence and development of oral squamous cell carcinoma. 3,5 Many epidemiological studies have shown that the canceration rate of OSF has reached 7.6-13%. 4,6 However, the speci c mechanism of the development of cancer from OSF remains poorly understood and needs further exploration. Fibrosis can occur in many different organs of the body and is a very important risk factor for cancer occurence. [7][8][9] For example, it has been reported that the incidence of lung cancer is markedly increased in patients with pulmonary brosis, 8 and the primary alveolar epithelial cells from the brotic lung exhibited enhanced cell migration and undergo epithelial-mesenchymal transformation (EMT), a mechanism that is closely related to cancer development. [10][11][12] Moreover, when the liver disease is out of control, progressive tissue brosis will eventually lead to cirrhosis and even malignant change. The expression levels of invasion and metastasis-related genes of hepatocellular carcinoma (HCC) were signi cantly increased in a high-stiffness matrix compared with those in the controls. 13 Furthermore, high matrix stiffness is su cient to drive EMT of HCC cells, which is consistent with the results of breast cancer cells. 14 The above studies showed that increased stiffness of the extracellular matrix (ECM) could induce EMT of diseased epithelial cells; however, the mechanism by which the cells perceive the physical changes to the ECM, especially during the development of OSF, remains unclear.
Piezo-type mechanosensitive ion channel component 1 (Piezo1) is widely expressed in non-sensory cells and serves as a key sensor of mechanical stimuli. [15][16][17] The morphology and migration activities of human epidermal keratinocytes in response to mechanical stimulus are affected by Piezo1 regulation. 18 Notably, oral mucosal cells express Piezo1, which is related to the activation of the yes-associated protein (YAP) signaling pathway. 19 Moreover, recent studies have shown that Piezo1 could act upstream of YAP in the differentiation of human neural stem cells and the development of zebra sh heart. 20,21 Therefore, we hypothesized that the ECM with increased stiffness in OSF mediates the EMT process of epithelial cells through the Piezo1/YAP signaling pathway.
To verify our hypothesis, oral mucosa tissue from patients with OSF, arecoline induced rat OSF models, and their controls were collected for histopathological, microstructural, and mechanical analysis to observe the changes in the ECM and EMT in brotic lesions. Then, the effects and mechanism of matrix stiffness on EMT were explored by culturing cells on collagen matrices with different degrees of stiffness in vitro. Furthermore, implanting these collagen matrices under the oral mucosa of rats was used to further con rm whether the stiffness of the ECM triggers EMT ex vivo.

Results And Discussion
Samples of human OSF and normal oral mucosa were collected to study the pathological characteristics of OSF. After histological staining of the tissue sections, we observed that the epithelial ridges of the brotic oral mucosa were reduced, and the collagen in the connective tissue area was signi cantly increased and denser (Fig. 1a-c). Immunohistochemical staining found that the number of alpha smooth muscle actin (α-SMA)-positive myo broblasts in the connective tissue was increased, the number of CD31 (also known as PECAM1, platelet and endothelial cell adhesion molecule 1)-positive blood vessels in the tissue were reduced and became occluded ( Fig. 1d-f), which were consistent with the characteristics of OSF. [22][23][24] In addition, Sirius red staining and micro-infrared analysis were used to compare the collagen characteristics of OSF and normal mucosa tissues. According to the collagen color under polarized light 25,26 and the change of collagen absorption peak, 27 type I and type III collagen levels in the brotic mucosa tissue were increased, which is consistent with previous results reported for OSF tissues ( Fig. 1g-k). 28, 29 The above results indicated that the number of myo broblasts in OSF tissue increases and the cells secrete more collagen, which accumulates in the tissue, resulting in signi cant changes in the ECM collagen content.
Scanning electron microscopy (SEM) was used to further study the structural changes of the brotic oral mucosa. The results showed that the porosity of collagen in the connective tissue of OSF decreased, the transverse structure of some collagen disappeared, and collagen was denatured (Fig. 2a, b). Atomic force microscopy (AFM) was then used to evaluate the microscopic morphology and nano-mechanical properties of the oral mucosa connective tissue. We observed that the collagen was thick and disordered.
Young's modulus analysis showed that the stiffness of OSF tissue was increased (Fig. 2c, d), which was consistent with previous results. 30 These results showed that the OSF tissue became stiffer because of the changes in collagen components, structure, and morphology. Previous studies showed that high stiffness of the ECM was related to the EMT of tissue cells; 31-33 therefore, we further detected the protein expression of EMT markers in OSF tissue using immunohistochemical staining. The results showed that the level of E-cadherin in epithelial cells of OSF tissue was decreased compared with that of the normal tissue, while the level of vimentin, a broblast marker, increased in the epithelial cells of OSF tissue ( Fig. 2e-g), which is consistent with the characteristics of EMT. 34 The above ndings indicated that the increased stiffness of OSF tissue was accompanied by increased EMT in the OSF tissue, suggesting that the EMT changes of epithelial cells in the OSF tissue might be mediated by the changes to extracellular collagen, resulting in increased tissue stiffness.
Next, we constructed a rat model of OSF by smearing and injecting arecoline into the oral mucosa of rats for 10 weeks. 35,36 Histological staining showed decreased epithelial ridges and increased collagen contents in the connective tissue of rat OSF samples compared with those in the controls (Fig. 3a, b). Similar to the results for human OSF samples, type I and type III collagen levels increased ( Fig. 3c-g), the collagen became thicker and disordered, and tissue stiffness increased in the rat OSF samples compared with those in the controls ( Fig. 3h-k). In addition, the level of Ecadherin in epithelial cells was reduced in the rat OSF samples compared with that in the controls (Fig. 3l, m), which was consistent with the characteristics of EMT and the results for the human OSF samples. However, the levels of vimentin did not differ signi cantly between the rat OSF samples and the controls, which might be related to the different pathological process of the rat model of OSF. The present rat model of OSF exhibited changes consistent with the middle to late stages of OSF, in which the whole process of EMT may not be complete. 37,38 To further verify the in uence of the mechanical properties of the ECM on EMT, we used collagen constructs at 5 mg/ml and 35 mg/ml to simulate the soft and stiff ECM (Fig. 4a). Infrared spectrum analysis showed that the collagen in solution successfully self-assembled into collagen protein constructs with a complete structure in vitro (Fig. 4b). AFM was used to analyze the microscopic morphology and mechanical properties of the collagen constructs (Fig. 4c, d). The Young's modulus of the 35 mg/ml collagen construct (stiff construct) was signi cantly higher than that of the 5mg/ml collagen construct (soft constructs), and the porosity of the stiff construct was decreased signi cantly ( Fig. 4e, f). Subsequently, the proliferation, morphology, and EMT on soft and stiff collagen constructs were investigated.
Cell Counting Kit 8 (CCK8) assays showed that the proliferation of epithelial cells on the stiff constructs was signi cantly higher than that on the soft constructs (Fig. 5a). The proliferation activities of epithelial cells were further evaluated by immuno uorescent staining of Ki67 and quantitative real-time polymerase chain reaction analysis of Cyclin E. The results showed that the protein level of Ki67 and mRNA expression of Cyclin E both increased in stiff construct group comparing to those in the soft construct group, in accordance to the results of CCK8 (Fig. 5b, c). The cells cultured on the soft matrix were irregular polygons, while the cells cultured on the stiff matrix were spindle shaped (Fig. 5d), similar to broblasts. 39 In addition, the mRNA and protein expression levels of E-cadherin decreased and those of vimentin increased in the stiff constructs group compared with those in the soft group ( Fig. 5e-g), indicating that EMT occurred in epithelial cells cultured on the stiff collagen constructs.
However, how does the mechanical force of ECM affect the malignant transformation of cells? Previous studies have shown that Piezo1 can sense the mechanical changes of the ECM, thus opening the ion channel and mediating changes to the cell. 40,41 YAP is a key molecule of mechanical transduction, which can respond to the mechanical changes of the cell and enter the nucleus to regulate the expression of a variety of genes related to cancer. [42][43][44] Therefore, we detected these two proteins in epithelial cells, and found that their levels were increased in cells cultured on the stiff matrix, and YAP showed obvious nuclear translocation (Fig. 6a-c). These results con rmed that a stiff ECM can cause EMT in epithelial cells and that this change is related to the increased expression of Piezo1 and YAP.
To further verify the role of Piezo1 and YAP in EMT in response to a stiff matrix, we used the Piezo1  . 6d-f). This indicated that Piezo1 might mediate epithelial cell EMT through YAP. Next, epithelial cells cultured on the stiff matrix were treated with the YAP inhibitor Vertepor n. In response, the mRNA and protein expression levels of Piezo1 did not change signi cantly; however, the mRNA and protein expression levels of E-cadherin increased and those of vimentin decreased ( Fig. 6g-i). The EMT of the cells was also inhibited. This indicated that YAP plays a key role in the cell phenotypic changes caused by EMT, and Piezo1 might function upstream of YAP to sense the extracellular mechanical changes, thus mediating EMT in response to the mechanical stimulus from a stiff matrix.
Next, we implanted the soft and stiff collagen constructs into the oral mucosa of rats to verify the above results (Fig. 7a). The results showed that in the stiff collagen implantation group, the cells within the epithelium expressed increased levels of vimentin, Piezo1, and YAP, but decreased levels of E-cadherin, compared with those in the epithelial cells in the sham control and the soft collagen implantation groups (Fig. 7b, c). The levels of vimentin, E-cadherin, Piezo1, and YAP in epithelial cells did not exhibit signi cant differences between the soft collagen implantation group and sham control group (Fig. 7b, c).
In this study, we found that the stiff OSF matrix can induce EMT, in which Piezo1 and YAP play key roles for signal transduction. Speci cally, with the increased stiffness of the oral mucosa during OSF progression, Piezo1 senses the mechanical changes of the mucous matrix and induces YAP to undergo nuclear translocation, promoting the transcription and expression of EMT-related genes (e.g., SNAI1, MMP9, QSOX1, and ZEB1) 45,46 , ultimately causing the cells to undergo EMT (Fig. 8). Therefore, local injection of enzymes related to collagen degradation or cross-linking to reverse the stiff characteristics of the brotic matrix might help to prevent the malignant transformation of epithelial cells in OSF. 47-49

Conclusions
The present study demonstrated that the increased stiffness of the brotic matrix in OSF leads to increased proliferation and EMT of mucosal epithelial cells, in which the Piezo1-YAP axis plays important roles during mechanical sensing and signal transduction. Strategies to reverse the stiff brotic matrix are important to prevent EMT changes of mucosal epithelial cells in OSF.

Micro-Fourier Transform Infrared Spectroscopy (µ-FTIR)
The spotlight 400 m-FTIR imaging system (PerkinElmer, Inc., Waltham, MA, USA) was used to analyze the para n sections naturally dried after hydration. The resolution of spectral acquisition was 4 cm − 1 , ranging from 650 to 4000 cm − 1 , and 36 scans were used for each sample. Spectrum software (PerkinElmer, Inc.) was used to obtain the absorption wavelengths.

Scanning electron microscopy (SEM)
Fresh mucosa was xed with 2.5% glutaraldehyde and dehydrated using an ascending ethanol series (30%, 50%, 70%, 80%, 90%, and 100% ethanol). Then, the sample was covered with hexamethyldisilazane and air dried slowly. Finally, it was sputtered with gold and examined using a eld emission scanning electron microscope (FE-SEM, S-4800, Hitachi, Tokyo, Japan) at 5 kV. ImageJ software was used to analyze the SEM images. Arecoline (SA9640, Solarbio, Beijing, China, 10 mg/mL and 5 mg/mL) was dissolved in 0.9% normal saline, and the animal model was established by oral smearing and submucosal injection. The frequency of smearing application was once a day. The injection frequency was once every three days, and the injection volume of arecoline solution was 50 µL. After arecoline treatment, no food or water was provided for 2 hours. After 10 weeks of arecoline intervention, the rat model of OSF was established, all animals were killed, and their oral mucosa tissues were collected for subsequent analysis.

Construction of collagen matrix
A rat tail was immersed in 75% alcohol for 30 minutes to peel off the skin. The tip of the tail was clamped with tweezers to extract the collagen. After washing, the rat tail was immersed in Tris-HCl and NaCl mixture at 4 ℃ overnight. The collagen was taken out and dissolved in 0.3 M acetic acid solution. After centrifugation, the collagen solution was placed in a dialysis bag (MD25 (8000-14000 Da)) and dialyzed in phosphate-buffered saline (PBS) until the pH of the PBS remained at 7. At this time, the solid collagen block was taken and stored at 4 ℃ in 75% alcohol. Before use, the collagen block was frozen and cut into 2-4 mm thick pieces.

Cell culture
A human oral epithelial cell (HOK, HUM-iCELL-m004, China) was used. The cut collagen pieces were placed in the wells of a 24-well plates, washed PBS many times, and then the cells were inoculated onto the collagen matrix. All cells were cultured in Dulbecco's modi ed Eagle's medium high glucose medium at 37°C and 5% CO 2 .

Cell proliferation test
Cells were inoculated into 96-well plates (5000 cells/well). After 24, 48, and 72 hours of culture, cell proliferation was evaluated using Cell Count Kit-8 analysis (CCK-8, Dojindo, Japan) according to the manufacturer's instructions. A microplate reader (BioTek, Winooski, VT, USA) was used to measure the absorbance value at 450 nm.

Signal activation and inhibition
The Piezo1 activator Yoda1 (HY-18723, Med Chem Express, America) and the YAP inhibitor Vertepor n (HY-B0146, Med Chem Express, America) were respectively dissolved in DMSO according to the manufacture instructions, and added into the cell culture medium. The working concentration of Yoda1 and Vertepor n were 300nM and 3µM according to previous studies. 50,51

Immuno uorescence
The culture medium was discarded, the cells were washed using PBS three times, 4% paraformaldehyde was added to x the cells for 10 minutes, the cells were washed using PBS three times, 0.1% triton was added to treat the cells for 10 minutes, the cells were washed again, serum was incubated with the cells to block them for 30 minutes, and then the cells were exposed to the primary antibodies at 4°C overnight. The antibodies used were Piezo1 (1:300, DF12083, A nity), YAP (1:300, 66900-1-lg, Proteintech), Ecadherin (1:300, AF0131, A nity), Vimentin (1:300, 10366-1-AP, Proteintech) and Ki67 (1:300, AF0198, A nity). After PBS washing, the secondary antibody was added and incubated for 1 h in the dark. The processed cells were mounted in Antifade Mountant with 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen, Waltham, MA, USA) for confocal scanning laser microscopy (CLSM) (Nikon A1R, Nikon Corporation, Minato-ku, Tokyo, Japan) according to the manufacturer's instructions. The integrated uorescence intensity was calculated using the ImageJ software.

Quantitative reverse transcription-polymerase chain reaction (qRT-PCR)
Total RNA was extracted from the human oral epithelial cell using the Trizol reagent (Invitrogen). The concentration and purity of the extracted RNA were determined by measuring the absorbance at 260 and 280 nm (BioTek). cDNA was synthesized using a PrimeScript RT reagent kit (Takara Bio Inc., Shiga, Japan) and quantitative real-time PCR (7500 Real-time PCR System; Applied Biosystems, Carlsbad, CA, USA) was performed. GAPDH (encoding glyceraldehyde-3-phosphate dehydrogenase) was used as the housekeeping gene. Fold changes relative to the control group were estimated using the 2 −ΔΔCt method. 52 The primer sequences are presented in Supplementary Table 1.

Subcutaneous collagen implantation
Incisions were made in the oral mucosa of the rats, the collagen block was placed under the mucosa, and then the tissue incisions were sutured. Seven days later, the rats were killed to take samples.

Statistical analyses
All data are expressed as the mean ± standard deviation. The Shapiro-Wilk test was used to test the normal distribution (95% con dence interval). Levene's test was used to evaluate the homogeneity of variance. A T test was used to compare the differences between the two groups. Univariate and bivariate analysis of variance (ANOVA) were used to assess the differences between groups. The GraphPad Prism    analysis data represent the mean ± standard deviation determined using one-way analysis of variance (ANOVA) (p < 0.05).

Figure 8
Diagram of the mechanism by which matrix stiffness induces mesenchymal transformation of epithelial cells. Cells sense the mechanical changes of the extracellular matrix (ECM) through piezo type mechanosensitive ion channel component 1 (Piezo1). In the presence of exogenous transforming growth factor beta (TGF-β), when the stiffness of ECM increases, the expression of Piezo1 increases and the ion channel opens, its downstream yes-associated protein (YAP) will undergo nuclear translocation, which will affect the expression of EMT-related proteins E-cadherin (E-cad) and vimentin in cells, thus causing cells to undergo epithelial mesenchyme transition (EMT).

Supplementary Files
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