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 fibrotic oral mucosa were reduced, and the collagen in the connective tissue area was significantly increased and denser (Fig. 1a-c). Immunohistochemical staining found that the number of alpha smooth muscle actin (α-SMA)-positive myofibroblasts 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.22–24 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 light25,26 and the change of collagen absorption peak,27 type I and type III collagen levels in the fibrotic 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 myofibroblasts in OSF tissue increases and the cells secrete more collagen, which accumulates in the tissue, resulting in significant changes in the ECM collagen content.
Scanning electron microscopy (SEM) was used to further study the structural changes of the fibrotic 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 fibroblast marker, increased in the epithelial cells of OSF tissue (Fig. 2e-g), which is consistent with the characteristics of EMT.34 The above findings 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 significantly 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 influence 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 significantly higher than that of the 5mg/ml collagen construct (soft constructs), and the porosity of the stiff construct was decreased significantly (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 significantly higher than that on the soft constructs (Fig. 5a). The proliferation activities of epithelial cells were further evaluated by immunofluorescent 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 fibroblasts.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.42–44 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 confirmed 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 activator Yoda1 ((2-[5-[[(2,6-Dichlorophenyl)methyl]thio]-1,3,4-thiadiazol-2-yl]-pyrazine) to treat the cells growing on the stiff matrix. Activation of Piezo1 increased the mRNA and protein expression levels of YAP and vimentin, enhanced the nuclear translocation of YAP, and further decreased mRNA and protein expression levels of E-cadherin (Fig. 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 Verteporfin. In response, the mRNA and protein expression levels of Piezo1 did not change significantly; 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 significant 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. Specifically, 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 fibrotic matrix might help to prevent the malignant transformation of epithelial cells in OSF.47–49