Slug mediates myofibroblastic differentiation to promote fibrogenesis in buccal mucosa

2.1 Chemicals, OSF tissue, and cell culture

Arecoline (an alkaloid from areca nut) and collagen solution from bovine skin were purchased from Sigma-Aldrich (St. Louis, MO). Ingredients in areca nut have been shown to be critical in the pathogenesis of OSF and oral cancer via regulation of inflammatory factors and oxidative stress (M. C. Chang et al., 2001, 2004; Jeng et al., 2000). Hence, we utilized arecoline to induce the myofibroblast transdifferentiation (M. C. Chang et al., 2013).

For tissue acquisition, all procedures have followed the tenets of the Declaration of Helsinki and were reviewed by the Institutional Review Committee at Chung Shan Medical University, Taichung, Taiwan. Specimens of histologically normal or fibrotic mucosa from 25 normal subjects and OSF patients were collected in Department of Dentistry, Chung Shan Medical University Hospital for quantitative real-time polymerase chain reaction (qRT-PCR), heat map, western blot analyses. Normal BMFs and fibrotic BMFs (fBMFs) were cultivated as previously described (Yu, Tsai, Hsu, & Chang, 2013). Cell cultures between the third and eighth passages were used in this study.

2.2 qRT-PCR

Trizol reagent was used for total RNA isolation and Superscript III first-strand synthesis system (Invitrogen Life Technologies, Carlsbad, CA) was used to reverse-transcribe the messenger RNAs (mRNAs) according to the manufacturer’s instruction. qRT-PCR reactions on resulting cDNAs were performed on an ABI StepOne Real-Time PCR Systems (Applied Biosystems, Foster City, CA; Y. C. Chang et al., 2015). Fifty nanograms of complimentary DNA (cDNA) sample were used in an SYBR Green-based qPCR reaction (Kapa Biosystems, Inc., Wilmington, MA); the cycling conditions were as follows: 50°C for 2 min, 95°C for 10 min, followed by 40 cycles of 95°C for 10 s and 60°C for 1 min. The end point used in the real-time quantification was calculated by the StepOne software (Applied Biosystems, Foster City, CA), and the threshold cycle number (C_t value) for each analyzed sample was calculated. The primer sequences were listed in Table 1.

2.3 Western blot analysis

Western blot analysis was carried out as previously described (Y. C. Chang et al., 2014). Briefly, cells were lysed with NP-40 lysis buffer, and protein concentration was determined by BCA protein assay reagent (Thermo Fisher Scientific Inc., Rockford, IL). A total of 25 μg of total protein was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane (Millipore, Billerica, MA). The membranes were then incubated with primary antibodies followed by corresponding secondary antibodies. The primary antibodies against Slug, α-SMA, collagen type I α1 (COL1A1), and vimentin were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Immunoreactive protein bands were detected by the ECL detection system (Amersham Biosciences Co., Piscataway, NJ).

2.4 Collagen contraction assay

fBMFs (2 × 10⁵) were suspended in 0.5 ml of a 2 mg/ml collagen solution (Sigma-Aldrich) and cell per collagen mixture was added into 24-well plate. The plate was incubated at 37°C for 2 hr, which caused polymerization of the collagen gels. After detaching from well using a 200 μl pipet tip, the gels were further incubated in 0.5 ml MEMα medium for 48 hr. Contraction of the gels was photographed and contraction index was calculated using the ImageJ software (Yu, Tsai, Hsu, et al., 2013).

2.5 Migration and invasion assays

The Transwell system with a polycarbonate filter membrane of 8-μm pore size (Corning, NY) was utilized to assess the migration and invasion capacities. For invasion assay, the membrane was coated with Matrigel (BD Pharmingen, Franklin Lakes, NJ). In the upper chamber, cell were seeded at a cell density of 1×10⁵ in serum-free medium followed by 24 hr of incubation. Serum-containing media (10% fetal bovine serum) was used as the chemoattractant in the lower chamber. Cells on the other side of the membrane were stained with 0.1% crystal violet (Sigma-Aldrich) after fixation. These cells were counted from five different visual areas of 100-fold magnification under a microscope (Chung et al., 2017).

2.6 Wound healing assay

Cells were seeded into a six-well culture plate, and then wounds were introduced to the confluent monolayer of cells with a sterile 200 µl plastic pipette tip to create a denuded area in the center. Cell movement into the wound area was photographed at 0 and 48 hr under a microscope (Yu, Tsai, Wang, et al., 2013).

2.7 Immunofluorescent image

After fixation with 4% paraformaldehyde for 10 min, cells were incubated with the primary antibody against Snail or α-SMA (Santa Cruz Biotechnology) for 3 hr followed by the corresponding secondary antibodies. Cells were also stained with 4′6-diamidino-2-phenylindole (DAPI) to indicate the nuclei (Hsin et al., 2017).

2.8 Lentiviral-mediated RNAi for silencing Slug

The pLV-RNAi vector was purchased from Biosettia Inc. (San Diego, CA). The method of cloning the double-stranded short hairpin RNA (shRNA) sequence was described in the manufacturer’s protocol. Oligonucleotide sequence of lentiviral vectors expressing shRNA that targets Slug was synthesized and cloned into pLVRNAi to generate a lentiviral expression vector. The target sequences for Slug were listed as follows: Sh-Slug-1: 5′-AAAAGGACACACATACAGTGATTTTGGATCCAAAATCACTGTATGTGTGTCC-3′; Sh-Slug-2: 5′-AAAAGCCCAGTCTAGAAAATCTTTTGGATCCAAAAGATTTTCTAGACTGGGC-3.

2.9 Overexpression of Slug

Slug cDNA was cloned into pLV-EF1a-MCS-IRES-Puro (Cat. No: cDNA-pLV01; Biosettia Inc.). Lentivirus production was performed by cotransfection of plasmid DNA mixture with lentivector plus helper plasmids (VSVG and Gag-Pol) into 293T cells (American Type Culture Collection, Manassas, VA) using Lipofectamine 2000 (LF2000; Invitrogen, Carlsbad, CA).

2.10 Luciferase reporter assays

For transactivation assays, luciferase reporter plasmid was cotransfected with Slug into the constructs. A β-galactosidase-expressing vector was included as an internal control for transfection efficiency. After 24 hr, the cells were lysed and both luciferase and β-galactosidase activities were determined with enzyme assay kits (Promega). Luminescence was quantitated using a TD-20e luminometer (Turner Design) from duplicate plates. The representative results from three independent experiments were used.

2.11 Chromatin immunoprecipitation (ChIP) analysis

A ChIP assay will be conducted using the ChIP Assay Kit according to the manufacturer’s instructions (Upstate-Millipore Corporation, Billerica, MA).

2.12 Statistical analysis

Pearson and Spearman’s correlation were used to analyze the relationship between the expression of myofibroblast-related markers and Slug. Data were presented as mean ± SD. A Student’s t test was used to compare the differences. p < 0.05 was considered statistically significant.

3.1 The expression of Slug is elevated in OSF

RNA-sequencing and heat map analysis of differential RNA expressions revealed that various fibrosis-associated factors (such as fibronectin and transforming growth factor-β [TGF-β] signaling) were upregulated in OSF tissues (Figure 1a), including Slug (>2-fold). We found that Slug was positively correlated to other myofibroblast-related markers (Figure 1b) and confirmed that the gene expression of Slug was significantly higher in OSF specimens and fBMFs (Figure 1c,d). Results from western blot also showed that the expression of Slug was elevated in OSF samples (Figure 1e).

3.2 Downregulation of Slug attenuates the hallmarks of fibrosis

To examine the significance of Slug in myofibroblasts, knockdown of Slug was used to study its functional role (Figure 2a). Given that myofibroblasts migrate to the injured cites and participate in the wound healing process, cell mobility and contractility are common features to investigate (Hinz et al., 2007). Our results showed that the behavioral phenotypes of fBMFs, including collagen gel contractility (Figure 2b), migration (Figure 2c), invasion (Figure 2d), and wound healing (Figure 2e) were all reduced following downregulation of Slug, indicating that modulation of Slug was sufficient to inhibit the myofibroblast activation. Additionally, the relative protein expression levels of myofibroblast markers, including α-SMA, type I collagen, and vimentin were inhibited in the Slug-knockdown fBMFs (Figure 2f). Together, these data suggested that Slug was critical to the existence of myofibroblasts characteristics.

3.3 Inhibition of Slug prevents the increased myofibroblast activities induced by arecoline

The main etiology of OSF has been attributed to areca nut chewing and its major component arecoline-induced myofibroblast transdifferentiation has been reported previously (Y. C. Chang et al., 2014). To understand the contribution of Slug in the pathogenesis of OSF, we evaluated the expression of Slug after arecoline treatment. As shown in Figure 3a,b, the gene and protein expression of Slug increased in a dose-dependent manner following arecoline stimulation. In consistent with these results, the immunofluorescent imaging also showed that the expression of Slug and α-SMA-positive stress fibers were dose-dependently enhanced in response to arecoline (Figure 3c). Subsequently, we knockdown Slug (Figure 3d) to investigate whether it could impede the arecoline-induced transdifferentiation of myofibroblasts. Our results demonstrated that inhibition of Slug successfully blocked the myofibroblast hallmarks including increased collagen gel contraction (Figure 3e), migration (Figure 3f), and invasion (Figure 3g). These findings suggested that Slug may be the key factor that involves in the arecoline-increased myofibroblasts activities.

3.4 Overexpression of Slug increases the features of myofibroblasts

To verify that Slug was able to induce the myofibroblast activation, ectopic expression of Slug in BMFs was carried out and we showed that increased Slug upregulated the myofibroblast marker, α-SMA, and type I collagen (Figure 4a). Moreover, the contractility of BMFs embedded into a collagen gel was increased (Figure 4b) as well as cell motility, including migration (Figure 4c) and invasion (Figure 4d).

3.5 Slug activates the expression of type I collagen by direct binding to its E-box

We analyzed the data from The Cancer Genome Atlas (TCGA) and found that Slug was positively correlated with several myofibroblast- and ECM-associated markers in oral cancer tissues, including, α-SMA, type I collagen, fibronectin, and vimentin (Figure 5a–d), suggesting that Slug may promote not only fibrosis but also tumorigenesis.

Apart from modulating the myofibroblast activities, we sought to further elucidate whether Slug contributes to the excessive ECM accumulation in OSF. We noticed that there were E-box regions in the promoter region of type I collagen (COL1A1; Figure 5e), indicating that Slug may induce COL1A1 expression by directly binding to its promoter. Hence, we performed luciferase-based reporter assay to investigate whether the COL1A1 promoter can be activated by Slug and identify the binding site for Slug within the COL1A1 promoter. This was done using reporter constructs containing wild-type, mutated, and deleted lengths of the COL1A1 promoter (Figure 5e). An increase in COL1A1 promoter activity was observed after transient transfection with Slug (Figure 5f, the “738 bp” group), while deleting a region containing the E2 box (Figure 5f, the “288 bp” group) and site-directed mutagenesis of the E2 E-box hampered COL1A1 activation. By ChIP, we found that arecoline treatment of BMFs increased the binding of Slug to the COL1A1 promoter (Figure 5g). Taken together, our data demonstrated that arecoline stimulation enhanced the direct binding of Slug to the E-box region in the COL1A1 promoter, which may increase the deposition of collagen and further exacerbate the activation of myofibroblast (Figure 5h).