Oral submucous fibrosis (OSF) is a chronic, progressive disease characterized by fibrosis and stiffness of the oral mucosa, severely affecting the oral functions of patients and carrying a potential risk of malignancy. The etiology of OSF is complex, often associated with habits such as betel nut chewing, but the specific molecular mechanisms remain incompletely understood. Recent studies have shown that oxidative stress and myofibroblast transdifferentiation are key factors in the progression of OSF. Long non-coding RNAs (lncRNAs) play important roles in various diseases, especially fibrotic diseases. However, the specific roles and mechanisms of lncRNAs in OSF still require further investigation. MetaLnc9, a newly discovered lncRNA, has unclear roles in OSF.

Recently, Min-Te Chang's team published a research titled "Targeting MetaLnc9/miR-143/FSCN1 Axis Inhibits Oxidative Stress and Myofibroblast Transdifferentiation In Oral Submucous Fibrosis" in the Journal of Dental Sciences. The study found that MetaLnc9 is upregulated in OSF tissues, and its pro-fibrotic characteristics in fibrotic buccal mucosal fibroblasts (fBMFs) may be mediated by sequestering miR-143 and reducing the availability of miR-143 to target FSCN1. Therefore, targeting the MetaLnc9/miR-143/FSCN1 axis might be a therapeutic approach to treat OSF by inhibiting fBMF activation.

Materials and Methods

Normal and fibrotic buccal mucosa specimens were collected from healthy individuals and Oral Submucous Fibrosis (OSF) patients at Chung Shan Medical University Hospital, following Institutional Review Board-approved protocols and informed consent. Normal and fibrotic buccal mucosa fibroblasts were extracted and cultured in DMEM with 10% FBS and 1% penicillin-streptomycin. MetaLnc9 was silenced using a lentiviral-mediated knockdown system via shRNA sequences cloned into pLV-RNAi vectors. For RNA analysis, tissues were stored in liquid nitrogen at -80°C, and RNA was extracted using Trizol. cDNA synthesis and qRT-PCR were performed with specific primers. Various assays, including collagen contraction, wound healing, transwell migration, ROS production, Western blotting, and dual-luciferase reporter assays, were conducted to evaluate cellular responses.

Results

The study investigates the role of MetaLnc9 in the pathology of oral submucous fibrosis (OSF) by conducting high-throughput RNA sequencing and qRT-PCR analysis. MetaLnc9 was found to be differentially expressed in OSF tissues compared to normal mucosal samples (Fig. 1A). qRT-PCR validation indicated that MetaLnc9 was significantly upregulated in OSF specimens (n = 25) (Fig. 1B), and positively correlated with several fibrosis-associated markers including alpha-smooth muscle actin (α-SMA), alpha-1 type I collagen (COL1A1), and fibronectin 1 (FN1) (Fig. 1C).

Fig. 1 MetaLnc9 is overexpressed in oral submucous fibrosis tissues and positively correlated to several fibrosis-associated markers.

Investigations further revealed that MetaLnc9 expression was elevated in fibrotic buccal mucosal fibroblasts (fBMFs) derived from OSF tissues compared to normal buccal mucosal fibroblasts (BMFs) (Fig. 2A). Reduction of MetaLnc9 expression decreased transwell migration ability (Fig. 2B), the expression of α-SMA (Fig. 2C), and collagen gel contraction (Fig. 2D). Additionally, intracellular reactive oxygen species (ROS) production was significantly reduced in fBMFs when MetaLnc9 was inhibited (Fig. 2E).

Fig. 2 Inhibition of MetaLnc9 in fibrotic buccal mucosal fibroblasts attenuates multiple myofibroblast features.

Localization studies revealed that MetaLnc9 predominantly resides in the cytoplasm of fBMFs (Fig. 3A). Analysis extended to its interaction with miR-143, a microRNA implicated in fibrosis. Complementarity between MetaLnc9 and miR-143 was observed, and lower miR-143 expressions were found in OSF tissues compared to normal tissues (Fig. 3B, 3C). Luciferase reporter assays confirmed that MetaLnc9 interacts directly with miR-143, with activity suppressed in cells co-transfected with wild-type MetaLnc9 and miR-143, but not in those with the mutant MetaLnc9 (Fig. 3D).

Fig. 3 MetaLnc9 interacts with miR-143.

Subsequent experiments explored miR-143's impact on myofibroblast activities. Overexpression of miR-143 in fBMFs resulted in marked suppression of collagen gel contractility (Fig. 4A, 4B), transwell migration (Fig. 4C), and wound healing capacities (Fig. 4D). This suggested that miR-143 inhibits critical myofibroblast functionalities.

Fig. 4 Overexpression of miR-143 diminishes myofibroblast phenotypes.

Further examination corroborated that MetaLnc9's effect on myofibroblast activities is mediated through miR-143 modulation. There was a negative relationship between MetaLnc9 and miR-143 expression (Fig. 5A). Silencing of MetaLnc9 led to reduced transwell migration, collagen gel contractility, and the expression of fibrosis markers, but these effects were reversed by miR-143 inhibition (Fig. 5B-5D). These results indicate that MetaLnc9 modulates myofibroblast activation through direct interaction with miR-143, reducing its expression and consequently promoting fibrotic activities.

Fig. 5 MetaLnc9 regulates myofibroblast characteristics through repression of miR-143.

Furthermore, FSCN1 (fascin actin-bundling protein 1), known for its role in cell motility and fibrosis, was identified as a potential miR-143 target. Bioinformatics analysis indicated complementarity between FSCN1 and miR-143, which was validated by luciferase reporter assays showing reduced activity when co-transfected with miR-143 and wild-type FSCN1, but not the mutant FSCN1 (Fig. 6A-6B). The expression of FSCN1 was downregulated in fBMFs treated with miR-143 mimics (Fig. 6C), and FSCN1 was positively correlated with MetaLnc9 expression (Fig. 6D).

Fig. 6 FSCN1 is a putative target of miR-143 and is positively associated with MetaLnc9.

Collectively, these findings suggest that MetaLnc9 exerts its pro-fibrosis properties through the miR-143/FSCN1 axis. MetaLnc9 enhances fibrotic characteristics by modulating miR-143 expression, which in turn affects FSCN1 levels, leading to increased cell motility and fibrosis-associated activities. This study implies that targeting the MetaLnc9/miR-143/FSCN1 pathway might serve as a potential therapeutic strategy for treating fibrosis-related diseases such as OSF.

Conclusion

The study highlights the potential role of MetaLnc9 in the progression of oral submucous fibrosis (OSF), a precancerous condition often linked to betel nut chewing. MetaLnc9 is upregulated in OSF tissues and interacts with miR-143, thereby influencing fibrosis markers. While previous research indicated MetaLnc9's anti-fibrosis properties, this study shows its pro-fibrosis effects through the miR-143/FSCN1 pathway. This suggests that MetaLnc9 can have both pro- and anti-fibrotic roles depending on conditions. Areca nut exposure upregulates MetaLnc9 through transcription factors FOXC2, SP1, and STAT3, further promoting OSF. Hence, the MetaLnc9/miR-143/FSCN1 axis could be a therapeutic target for OSF, warranting future studies on betel nut chewing's impact on MetaLnc9 expression in oral tissues.

• Cell Culture
• Cells
• Cytokine
• Organoid Development