Carvacrol inhibits the progression of oral submucous fibrosis via downregulation of PVT1/miR-20a-5p-mediated pyroptosis

Abstract

Oral submucous fibrosis (OSF) is a precancerous condition in the oral cavity, which is closely related to the myofibroblast conversion of buccal mucosal fibroblasts (BMFs) after chronic consumption of areca nut. Emerging evidence suggests pyroptosis, a form of programmed cell death that is mediated by inflammasome, is implicated in persistent myofibroblast activation and fibrosis. Besides, numerous studies have demonstrated the effects of non-coding RNAs on pyroptosis and myofibroblast activities. Herein, we aimed to target key long non-coding RNA PVT1 with natural compound, carvacrol, to alleviate pyroptosis and myofibroblast activation in OSF. We first identified PVT1 was downregulated in the carvacrol-treated fBMFs and then demonstrated that myofibroblast features and expression of pyroptosis makers were all reduced in response to carvacrol treatment. Subsequently, we analysed the expression of PVT1 and found that PVT1 was aberrantly upregulated in OSF specimens and positively correlated with several fibrosis markers. After revealing the suppressive effects of carvacrol on myofibroblast characterisitcs and pyroptosis were mediated by repression of PVT1, we then explored the potential mechanisms. Our data showed that PVT1 may serve as a sponge of microRNA(miR)-20a to mitigate the myofibroblast activation and pyroptosis. Altogether, these findings indicated that the anti-fibrosis effects of carvacrol merit consideration and may be due to the attenuation of pyroptosis and myofibroblast activation by targeting the PVT1/miR-20a axis.

Keywords: PVT1; carvacrol; microRNA‐20a; myofibroblast; oral submucous fibrosis.

FIGURE 1

Carvacrol suppresses the cell proliferation rate and PVT1 expression of fBMFs. (A) The cytotoxicity effect of carvacrol in normal buccal mucosal fibroblasts (BMFs) and fibrotic BMFs (fBMFs). Cell survival/viability of BMFs (left panel) and fBMFs (right panel) were determined by MTT assay. IC50 values were calculated by GraFit software. (B) Hierarchical cluster heat map of the differentially expressed lncRNAs in RNA‐seq analysis of three paired fBMFs treated with or without 10 μM carvacrol. Red represented the upregulation of differentially expressed genes, and blue represented the downregulation of differentially expressed genes. (C) The relative expression of PVT1 in fBMFs treated with indicated concentrations of carvacrol. Results are means ± SD. *p < 0.05 compared to no treatment control group.

FIGURE 2

Carvacrol treatment decreases the myofibroblast activities and expression levels of fibrosis and pyroptosis of fBMFs. (A) The contractile capacity of fBMFs was determined by collagen gel contraction assay (three replicates for each concentration). Images of gels were captured and gel areas (dotted circles) were calculated by ImageJ software. (B) fBMFs were treated with the indicated concentration of carvacrol followed by transwell migration assay. The experiments were repeated for three times and data from a representative experiment were presented. Results are means ± SD. *p < 0.05 compared to control group. The expression of firosis (C) and pyroptosis (D) markers were evaluated by Western blot.

FIGURE 3

PVT1 is upregulated in OSF specimens and positively correlated with various fibrosis markers. (A) A heatmap showing PVT1 was highly expressed in two OSF tissues. (B) Gene expression of PVT1 in OSF samples and normal buccal mucosal tissues (n = 25). (C) Gene expression of PVT1 in BMFs and fibrotic BMFs derived from OSF tissues. *p < 0.05 compared to control. **p < 0.01. PVT11 was positively associated with numerous fibrosis‐related markers, including alpha smooth muscle actin (α‐SMA; D), type I collagen alpha 1 (COL1A1; E), and fibronectin (FN1; F).

FIGURE 4

Silencing of PVT1 downregulates myofibroblast features and the expression of myo‐fibroblast and pyroptosis markers. Inhibition of PVT1 in fBMFs (A) diminished the myofibroblast activities, such as collagen gel contraction (B) and transwell migration (C). The protein expression of α‐SMA, NLRP3, cleaved‐GSDMD and cleaved‐IL‐1β in fBMFs silence for PVT1 (D).

FIGURE 5

Ectopic expression of PVT1 reverses the effects of carvacrol on myofibroblast activation and cleaved‐GSDMD expression. Administration of carvacrol suppressed the transwell migration (A), collagen gel contraction (B) and protein expression of α‐SMA and cleaved‐GSDMD (C), whereas forced expression of PVT1 abolished these effects. *p < 0.05 compared to control. #p < 0.05 compared to carvacrol‐only group.

FIGURE 6

PVT1 regulates fBMFs activation and pyroptosis by direct interaction with miR‐20a. (A) The subcellular localization of PVT1 in fBMFs analysed by qRT‐PCR. *p < 0.05 compared to the nucleus group. (B) Schematic representation of the alignment of the PVT1 base pairing with miR‐20a. Wild‐type (Wt) and mutated (Mut) PVT1 reporter plasmids were co‐transfected with miR‐20a or empty vectors (miR‐Scr.). (C) The relative luciferase activity of each combination in two fBMFs is assessed and the activity was only reduced in fBMFs co‐transfected with wt‐PVT1 and miR‐20a. (D) A negative correlation between PVT1 and miR‐20a was observed. Inhibition of miR‐20a in fBMFs reverted the myofibroblast activities, including collagen gel contraction (E) and migration ability (F) in fBMFs following the silencing of PVT1. (G) The expression of myofibro‐blast marker (e.g. α‐SMA) and pyroptosis markers (e.g. NLRP3, ASC, cleaved‐GSDMD and cleaved‐IL‐1β) were all suppressed in fBMFs with knockdown of PVT1, which was prevented in the presence of miR‐20a inhibitor.

FIGURE 7

Schematic representation of the therapeutic potential of carvacrol in OSF. Carvacrol may lower pyroptosis and the susequent myofibroblast activation via targeting the PVT1/ miR‐20a axis, leading to downregulation of NLRP3, ASC, cleaved‐GSDMD and cleaved‐IL‐1β as well as myofibroblast activities.