Oral Submucous Fibrosis Tissues Research Articles

Osthole mitigates the myofibroblast properties in oral submucous fibrosis by suppressing the TGF-β/smad2 signaling pathway and NCK-AS1 expression

Background/purposeNatural products are gaining increasing recognition as an alternative source for alleviating fibrosis as they can regulate various mediators or pathways against fibrosis by targeting non-coding RNAs. In the current study, we aimed to investigate the therapeutic effects of osthole in oral submucous fibrosis (OSF), a precancerous condition of the oral cavity. Materials and methodsThe cytotoxicity of osthole to normal and fibrotic buccal mucosal fibroblasts (fBMFs) derived from OSF tissues was assessed using MTT assay. Collagen gel contraction and transwell migration assays were conducted to examine the myofibroblast activities. Besides, the expression of TGF-β/Smad2 signaling as well as α-SMA and type I collagen were measured. Additionally, RNA sequencing was used to identify a potential target involved in the anti-fibrotic effect of osthole. ResultsOsthole exhibited a higher cytotoxic effect on fBMFs compared to normal BMFs and dose-dependently reduced several myofibroblast activities, including collagen gel contractility and transwell migration ability. In addition, the expression of the TGF-β/Smad2 pathway was inhibited along with a lower expression of α-SMA and type I collagen in osthole-receiving cells. Moreover, the administration of osthole downregulated the expression of NCK1-AS1 in fBMFs, which was proven to mediate the anti-fibrosis property of osthole. ConclusionOur results indicate that osthole may be a promising compound to inhibit the progression of OSF.

CircMTO1/miR-30c-5p/SOCS3 axis alleviates oral submucous fibrosis through inhibiting fibroblast-myofibroblast transition.

circRNAs have been shown to participate in diverse diseases; however, their role in oral submucous fibrosis (OSF), a potentially malignant disorder, remains obscure. Our preliminary experiments detected the expression of circRNA mitochondrial translation optimization 1 homologue (circMTO1) in OSF tissues (n = 20) and normal mucosa tissues (n = 20) collected from Hunan Xiangya Stomatological Hospital, and a significant decrease of circMTO1 expression was showed in OSF tissues. Therefore, we further explored circMTO1 expression in OSF. Target molecule expression was detected using RT-qPCR and western blotting. The migration and invasion of buccal mucosal fibroblasts (BMFs) were assessed using wound healing and Transwell assays. The interaction between miR-30c-5p, circMTO1, and SOCS3 was evaluated using dual luciferase, RNA immunoprecipitation (RIP), and RNA pull-down assays. The colocalisation of circMTO1 and miR-30c-5p was observed using fluorescence in situ hybridisation (FISH). circMTO1 and SOCS3 expression decreased, whereas miR-30c-5p expression increased in patients with OSF and arecoline-stimulated BMFs. Overexpression of circMTO1 effectively restrained the fibroblast-myofibroblast transition (FMT), as evidenced by the increase in expression of Coll I, α-SMA, Vimentin, and the weakened migration and invasion functions in BMFs. Mechanistic studies have shown that circMTO1 suppresses FMT by enhancing SOCS3 expression by sponging miR-30c-5p and subsequently inactivating the FAK/PI3K/AKT pathway. FMT induced by SOCS3 silencing was reversed by the FAK inhibitor TAE226 or the PI3K inhibitor LY294002. circMTO1/miR-30c-5p/SOCS3 axis regulates FMT in arecoline-treated BMFs via the FAK/PI3K/AKT pathway. Expanding the sample size and in vivo validation could further elucidate their potential as therapeutic targets for OSF.

Expression of IGF-1R in oral submucous fibrosis and oral squamous cell carcinoma – An RT-PCR study

ObjectivesWith the rising number of cases of oral submucous fibrosis (OSMF) undergoing malignant transformation into oral squamous cell carcinoma (OSCC), it is needed to detect the changes at the earliest. Several molecular markers have been used for this purpose, and insulin-like growth factor receptor 1 (IGF-1R) is one of the markers in vogue for several human malignancies. Thus, we compared the expression of IGF-1R mRNA in normal oral mucosa (NOM), OSMF, and OSCC. Materials and methodsThirty biopsy tissue samples for each of the three groups, NOM, OSMF, and OSCC, were taken and analyzed histopathologically and then subjected to RT-PCR for evaluation of the expression of IGF-1R mRNA in each of them. ResultsThere was an upregulation of IGF-1R mRNA in OSMF tissue samples as compared to NOM, but a downregulation as compared to OSCC. There was a 3.24-fold upregulation of IGF-1R mRNA expression in OSMF as compared to NOM and a 1.31-fold downregulation as compared to OSCC. Likewise, there was a 4.2-fold upregulation of IGF-1R mRNA expression in OSCC as compared to NOM and 1.31-fold upregulation as compared to OSMF. Moreover, the expression was more in OSMF patients with interincisal opening (IIO) < 27 mm as compared to those with IIO >27 mm and also was found to increase with the grade of OSCC. ConclusionThe study reflected a potential role of IGF-1R in the pathogenesis of OSMF and OSCC, thus implying its possible exploitation as a diagnostic and prognostic marker and also as a therapeutic target in future.

Inhibition of Oral Fibrinogenesis Through Transforming Growth Factor-Beta/SMAD2/3 Signalling Inhibition Using Mangosteen Pericarp Extract.

Background Chewing areca nuts can result in an oral disorder known as oral submucous fibrosis (OSF), which has the potential to be cancerous. Although it is only beginning to spread to European and the North American continents, it is highly prevalent in Southeast Asia. The probability of malignant transformation from OSF is raised by chewing tobacco use. In the current research, our objective was to assess the potential anti-fibrosis effects and the ability to prevent malignant transformation through the application of mangosteen pericarp extract. Methodology The Ethical Approval-IHEC/SDC/OMED-2101/23/085 from the institution was obtained to conduct this ex vivo study. The cytotoxicity effect of mangosteen pericarp extract on both normal and fibrotic buccal mucosal fibroblasts originating from OSF tissues was tested. Cell proliferationand cell migration by scratch wound healing assay was examined. Dual staining was done to determine the mode of cell death. Additionally, real-time PCR was utilized to measure the expression of TGF-β/Smad2/3 signalling, α-SMA, and type I collagen gene expression. Results Mangosteen extract exerted higher cytotoxicity of fibrotic buccal mucosal fibroblasts compared to normal cells. Furthermore, mangosteen-receiving cells exhibited downregulation in the expression of the TGF-β/Smad2 pathway, as well as reduced expression of α-SMA and type I collagen. Conclusion Findings from this study suggest that mangosteen could serve as a promising agent for averting the progression of oral fibrogenesis and halting the malignancy of the oral epithelium in patients with OSF.

Isolation of Cells with Morphological and Spatial Information from Oral Submucous Fibrosis Samples by Laser Capture Microdissection.

Oral submucous fibrosis (OSF) is a common type of potentially malignant disorder in the oral cavity. The atrophy of epithelium and fibrosis of the lamina propria and the submucosa are often found on histopathological slides. Epithelial dysplasia, epithelial atrophy, and senescent fibroblasts have been proposed to be associated with the malignant transformation of OSF. However, because of the heterogeneity of potentially malignant oral disorders and oral squamous cell carcinoma, it is difficult to identify the specific molecular mechanisms of malignant transformation in OSF. Here, we present a method to obtain a small number of epithelial or mesenchymal cells carrying morphological data and spatial information by laser capture microdissection on formalin-fixed paraffin-embedded tissue slides. Using a microscope, we can precisely capture microscale (~500 cells) dysplastic or atrophic epithelial tissue and fibrotic subepithelial tissue. The extracted cells can be evaluated by genome or transcriptome sequencing to acquire genomic and transcriptomic data with morphological and spatial information. This approach removes the heterogeneity of bulk OSF tissue sequencing and the interference caused by cells in non-lesioned areas, allowing for precise spatial-omics analysis of OSF tissue.

Knockdown of Notch Suppresses Epithelial-mesenchymal Transition and Induces Angiogenesis in Oral Submucous Fibrosis by Regulating TGF-β1.

Oral submucous fibrosis (OSF) is a chronic disorder with a high malignant transformation rate. Epithelial-mesenchymal transition (EMT) and angiogenesis are key events in OSF. The Notch signaling plays an essential role in the pathogenesis of various fibrotic diseases, including OSF. Our study aimed to explore the effects of Notch on the EMT and angiogenesis processes during the development of OSF. The expression of Notch in OSF tissues versus normal buccal mucosa samples was compared. Arecoline was used to induce myofibroblast transdifferentiation of buccal mucosal fibroblasts (BMFs). Short hairpin RNA technique was used to knockdown Notch in BMFs. Pirfenidone and SRI-011381 were used to inhibit and activate the TGF-β1 signaling pathway in BMFs, respectively. The expression of Notch was markedly upregulated in OSF tissues and fibrotic BMFs. Knockdown of Notch significantly decreased the viability and promoted apoptosis in BMFs subjected to arecoline stimulation. Downregulation of Notch also significantly suppressed the EMT process, as shown by the reduction of N-cadherin and vimentin with concomitant upregulation of E-cadherin. In addition, knockdown of Notch upregulated VEGF and enhanced the angiogenic activity of fBMFs. Moreover, inhibition of TGF-β1 suppressed viability and EMT, promoted apoptosis, and induced angiogenesis of fBMFs, while activation of TGF-β1 significantly diminished the effects of Notch knockdown on fBMFs. Knockdown of Notch suppressed EMT and induced angiogenesis in OSF by regulating TGF-β1, suggesting that the Notch-TGF-β1 pathway may serve as a therapeutic intervention target for OSF.

Fibroblast activating protein promotes the proliferation, migration, and activation of fibroblasts in oral submucous fibrosis.

Fibroblast activating protein (FAP) is associated with various organ fibrosis. However, the expression and molecular function of FAP in oral submucous fibrosis (OSF) is still unclear. The high-performance liquid chromatography was used to detect the presence of alkaloids in areca nut extract (ANE). Real-time qPCR, Western blot, and Immunohistochemistry assay were used to analyze the expression of FAP mRNA or protein in OSF and normal oral tissue. A chi-squared test analyzed the relationship between FAP protein expression and clinicopathological data of OSF patients. CCK-8, Wound-healing, and Transwell migration assay were employed to assess the effect of the proliferation and migration ability of hOMF cells with FAP overexpression or knockdown. The expression level of a-SMA, FSP1, and P13K-Akt signaling pathways-related protein in hOMF cells transfected with FAP overexpression or knockdown plasmid was verified by western blot assay. The four specific areca alkaloids (Arecoline, Guvacine, Arecaidine, and Guvacoline) were successfully detected in the ANE. The viability of hOMF cells was significantly improved in the 50 μg/mL ANE group and was inhibited in the 5 and 50 mg/mL ANE groups. The expression of FAP was upregulated in OSF tissues, and hOMF cells treated with 50 μg/mL ANE and was related to pathology grade, clinical stage, and history of chewing betel nut. Additionally, FAP may promote the proliferation, migration, and activation of hOMF cells through the P13K-Akt signaling pathway. This study found that ANE had a bidirectional effect on the viability of hOMF cells, and the FAP gene was a potential therapeutic target in OSF.

Expression and correlation of the Pi3k/Akt pathway and VEGF in oral submucous fibrosis.

Oral submucous fibrosis (OSF) has a high incidence in Asia countries, but its underlying molecular mechanism was not exploited completely. In this research, we investigated the expression of the phosphatidyl inositol 3-kinase (Pi3k)/protein kinase B (Akt) pathway and vascular endothelial growth factor (VEGF) in oral submucosal fibrosis, explore the correlation between the Pi3k/Akt pathway and VEGF, and identify the mechanisms involved in OSF. The pathological changes and fibrosis stages of OSF tissues (n = 30, 10 each of early, moderate and advanced OSF) were determined using Haematoxylin-eosin staining (HE) and Masson staining, respectively. Collagen type I (Col-I), Pi3k, Akt, VEGF, TGF-β and p-Akt expression was detected using immunohistochemistry, qPCR and WB. The correlation between Pi3k, Akt and VEGF was analysed. Col-I expression increased as OSF progressed. However, their expression was downregulated in normal and moderate to advanced OSF tissues. VEGF expression positively correlated with Pi3k and Akt expression. VEGF expression correlated positively and negatively with the Pi3k inhibitor, LY294002 below and above a concentration of 10 μM, respectively. VEGF expression correlated positively with the Pi3k/Ak activator, IGF-1. Due to the synergistic effect between Pi3k/Akt pathway and VEGF on OSF lesions and fibrosis process, targeted Pi3k/Akt pathway regulation can induce VEGF expression and improve ischemia, ultimately treating OSF.

Senescent epithelial cells remodel the microenvironment for the progression of oral submucous fibrosis through secreting TGF-β1.

Cellular senescence is strongly associated with fibrosis and tumorigenesis. However, whether the epithelium of oral submucous fibrosis (OSF) undergoes premature senescence remains unclear. This study investigates the roles of senescent epithelial cells in OSF. The immunohistochemistry and Sudan black B staining were performed to identify epithelium senescence in OSF tissues. Arecoline was used to induce human oral keratinocytes (HOKs) senescence. The cell morphology, senescence-associated β galactosidase activity, cell counting Kit 8, immunofluorescence, quantitative real-time PCR, and western blot assay were used to identification of senescent HOKs. The enzyme-linked immunosorbent assay was exerted to evaluate the levels of transforming growth factor β1 (TGF-β1) in the supernatants of HOKs treated with or without arecoline. The senescence-associated markers, p16 and p21, were overexpressed in OSF epithelium. These expressions were correlated with alpha-smooth actin (α-SMA) positively and proliferating cell nuclear antigen (PCNA) negatively. Moreover, Sudan black staining showed that there was more lipofuscin in OSF epithelium. In vitro, HOKs treated with arecoline showed senescence-associated characteristics including enlarged and flattened morphology, senescence-associated β galactosidase staining, cell growth arrest, γH2A.X foci, upregulation of p53, p21, and TGF-β1 protein levels. Moreover, senescent HOKs secreted more TGF-β1. Senescent epithelial cells are involved in OSF progression and may become a promising target for OSF treatment.

MiR-424/TGIF2-Mediated Pro-Fibrogenic Responses in Oral Submucous Fibrosis

Oral submucous fibrosis (OSF) has been recognized as a potentially malignant disorder and is characterized by inflammation and the deposition of collagen. Among various regulators of fibrogenesis, microRNAs (miR) have received great attention but the detailed mechanisms underlying the miR-mediated modulations remain largely unknown. Here, we showed that miR-424 was aberrantly overexpressed in OSF tissues, and then we assessed its functional role in the maintenance of myofibroblast characteristics. Our results demonstrated that the suppression of miR-424 markedly reduced various myofibroblast activities (such as collagen contractility and migration ability) and downregulated the expression of fibrosis markers. Moreover, we showed that miR-424 exerted this pro-fibrosis property via direct binding to TGIF2, an endogenous repressor of the TGF-β signaling. In addition, our findings indicated that overexpression of miR-424 activated the TGF-β/Smad pathway, leading to enhanced myofibroblast activities. Altogether, our data revealed how miR-424 contributed to myofibroblast transdifferentiation, and targeting the miR-424/TGIF2 axis may be a viable direction for achieving satisfactory results from OSF treatment.

XIST/let-7i/HMGA1 axis maintains myofibroblasts activities in oral submucous fibrosis

Long non-coding RNA XIST promotes the development of various types of head and neck cancers, but its role in the progression of precancerous oral submucous fibrosis (OSF) has not been determined yet. As such, we aimed to examine whether XIST implicates in the regulation of myofibroblast activation. Our results showed that the expression of XIST was upregulated in OSF tissues and fibrotic buccal mucosal fibroblasts (fBMFs), and the silencing of XIST downregulated several myofibroblasts features. We demonstrated that elevation of let-7i after inhibition of XIST may lead to reduced myofibroblast activation. On the contrary, overexpression of high mobility group AT-Hook 1 (HMGA1) following the suppression of let-7i may result in enhanced myofibroblast activities. Moreover, we showed that the suppressive effect of silencing of XIST on myofibroblasts hallmarks was reversed by let-7i inhibition or HMGA1 overexpression, suggesting the pro-fibrotic property of XIST was mediated by downregulation of let-7i and upregulation of HMGA1. These findings revealed that myofibroblast activation of fBMFs may attribute to the alteration of the XIST/let-7i/HMGA1 axis. Therapeutic approaches to target this axis may serve as a promising direction to ameliorate the malignant progression of OSF.

Downregulation of histone deacetylase 8 (HDAC8) alleviated the progression of oral submucous fibrosis

Oral submucous fibrosis (OSF) is a premalignant disorder that is associated with betel nut chewing. The purpose of the study was to establish the role of histone deacetylase (HDAC) 8, one of histone deacetylases, in the regulation of fibrotic conditions to provide a therapeutic potential for OSF. First, we examined the expression of HDAC8 in fibrotic buccal mucosal fibroblasts (fBMFs) and OSF tissues. Markers of myofibroblasts and TGF-β signaling were conducted in fBMFs with HDAC8 knockdown were examined. Furthermore, epithelial-mesenchymal transition (EMT) markers, collagen gel contraction and migration ability were also examined in fBMFs transfected with sh-HDAC8. HDAC8 inhibitor was used to analyze the collagen gel contraction and wound healing ability in fBMFs. We observed the mRNA expression of HDAC8 was significantly increased in fBMFs. Compared to normal tissues, the protein level of HDAC8 was upregulated in OSF. Next, mRNA and protein expression of HDAC8 was significantly decreased, accompanying downregulation of α-SMA and COL1A1 in fBMFs infected with sh-HDAC8. To determine the critical role of HDAC8 in OSF fibrogenesis, results revealed that TGF-β secretion and the expression of EMT transcription factor SNAIL and p-Smad were significantly decreased in HDAC8-knockdown fBMFs. We further demonstrated that collagen gel contraction and migration ability were significantly decreased in fBMFs transfected with sh-HDAC8. Last, results revealed that significantly reduced collagen gel contraction and wound healing ability in fBMFs with HDAC8 inhibitor treatment. We concluded that downregulation of HDAC8 alleviated the activities of myofibroblasts and TGF-β/Smad signaling pathway in OSF.

Identification of a BRAF/PA28γ/MEK1 signaling axis and its role in epithelial-mesenchymal transition in oral submucous fibrosis

Oral submucous fibrosis (OSF) is a chronic and insidious oral potentially malignant disorder associated with a 4–17% risk of oral squamous cell carcinoma (OSCC). Our previous study found that proteasomal activator 28 gamma (PA28γ) is frequently overexpressed in oral squamous cell carcinoma and negatively correlated with poor patient prognosis. However, the role of PA28γ in the occurrence and development of OSF remains unclear. Here, we screened PA28γ-related genes and investigated their function in OSF. We demonstrated that the expression of PA28γ was positively associated with MEK1 and gradually elevated from normal to progressive stages of OSF tissue. Arecoline, a pathogenic component of OSF, could upregulate the protein levels of PA28γ and phosphorylated MEK1 and contribute to epithelial to mesenchymal transition (EMT) in epithelial cells. Notably, PA28γ could interact with MEK1 and upregulate its phosphorylation level. Furthermore, arecoline upregulated BRAF, which can interact with PA28γ and upregulate its protein level. Additionally, BRAF, PA28γ, and MEK1 could form protein complexes and then enhance the MEK1/ERK signaling pathways. The concrete mechanism of the protein stability of PA28γ is that BRAF mediates its degradation by inhibiting its ubiquitination. These findings underscore the instrumental role of PA28γ in the BRAF/MEK1 pathway and enhanced EMT through MEK1/ERK activation in OSF.

Fucoidan-Mediated Inhibition of Fibrotic Properties in Oral Submucous Fibrosis via the MEG3/miR-181a/Egr1 Axis.

Oral submucous fibrosis (OSF) is a chronic fibrotic remodeling disease that can progress to oral cancer. However, efficient clinical diagnosis and treatment methods for OSF are still lacking. This study investigated the anti-fibrotic effect of fucoidan on oral fibrosis. To evaluate the fibrotic ability (myofibroblast activities), we performed wound-healing, Transwell migration, and collagen contraction assays by using patient-derived normal and fibrotic buccal submucous fibroblasts (BMFs and fBMFs, respectively). RNA-sequencing and dual-luciferase reporter and RNA immunoprecipitation chip assays were performed to identify the clinical significance and molecular mechanism of non-coding RNAs. Fucoidan suppressed the myofibroblast activities and inhibited the MEG3 in fBMFs. MEG3 was overexpressed in the OSF tissue and was positively associated with myofibroblast markers. Knockdown of MEG3 markedly inhibited myofibroblast activities, which were restored by inhibiting miR-181a and overexpressing Egr1. The results from luciferase reporter and RIP assays confirmed that MEG3 functioned as a competing endogenous RNA (ceRNA) and could directly target miR-181a, thereby preventing the miR-181a-mediated translational repression of Egr1. This study demonstrated that MEG3 exerts a profibrotic effect on OSF by targeting miR-181a/Egr1. Therefore, the administration of fucoidan may serve as a potential therapeutic strategy for OSF by targeting the overexpression of MEG3.

Down-regulation of miR-29c promotes the progression of oral submucous fibrosis through targeting tropomyosin-1

Various microRNAs (miRs) have been found to be associated with the development of the precancerous condition of the oral cavity, oral submucous fibrosis (OSF). The expression of miR-29c is dysregulated in oral cancer, but its role in OSF has not been investigated. The purpose of the study is to investigate the functional role of miR-29c and its target in OSF. The expression levels of miR-29c in OSF tissues and fibrotic buccal mucosal fibroblasts (fBMFs) were assessed using next-generation sequencing and real-time Polymerase Chain Reaction (PCR) analysis. MiR-29c mimic and inhibitors were employed to examine its functional role of myofibroblast transdifferentiation. In addition, several myofibroblast phenotypes, such as collagen gel contraction and migration were tested, and a luciferase reporter assay was conducted to confirm the relationship between miR-29c and its predicted target, tropomyosin-1 (TPM1). We observed that miR-29c expression was downregulated in fBMFs. fBMFs transfected with miR-29c mimics exhibited reduced migration ability and collagen gel contractility, whereas inhibition of miR-29c in normal BMFs induced the myofibroblast phenotypes. Results from the luciferase reporter assay showed that TPM1 was a direct target of miR-29c and the expression of TPM1 was suppressed in the fBMFs transfected with miR-29c mimics. Besides, we confirmed that the expression of miR-29c was indeed downregulated in OSF specimens. MiR-29c seems to exert an inhibitory effect on myofibroblast activation, such as collagen gel contractility and migration ability, via suppressing TPM1. These results suggested that approaches to upregulate miR-29c may be able to ameliorate the progression of OSF.

Paeonol inhibits profibrotic signaling and HOTAIR expression in fibrotic buccal mucosal fibroblasts

Betel nut chewing is the major risk factor of oral submucous fibrosis (OSF). Various studies have sought to discover alternative strategies to alleviate oral fibrogenesis. In the present study, we aimed to evaluate the anti-fibrosis effect of paeonol, a phenolic component derived from Paeonia Suffruticosa. The cytotoxicity of paeonol was tested using normal and fibrotic buccal mucosal fibroblasts (fBMFs) derived from OSF tissues. Collagen gel contraction, Transwell migration, invasion, and wound healing capacities were examined. Besides, the activation of TGF-β/Smad2 signaling and expression levels of type I collagen, α-SMA, and long non-coding RNA HOTAIR were measured as well. Paeonol exerted a higher cytotoxic effect on fBMFs compared to normal BMFs. The arecoline-induced myofibroblast activities, including collagen gel contractility, cell motility, and wound healing ability were all suppressed by paeonol treatment. In addition, the activation of the TGF-β/Smad2 pathway was inhibited along with a lower expression of α-SMA and type I collagen in paeonol-treated cells. Also, the administration of paeonol decreased the mRNA expression of HOTAIR in fBMFs. Our results indicate that paeonol may be a promising compound to attenuate the progression of oral fibrogenesis in OSF patients.

Depletion of miR-155 hinders the myofibroblast activities and reactive oxygen species generation in oral submucous fibrosis

Emerging evidence suggests the significance of microRNA-155 (miR-155) in fibrogenesis and oxidative stress accumulation, but its function in oral submucous fibrosis (OSF) has not been investigated. In this study, we assessed the expression of miR-155 and its effects on the maintenance of myofibroblast activation.qRT-PCR was conducted to assess the expression of miR-155 in OSF tissues and fibrotic buccal mucosal fibroblasts (fBMFs) derived from OSF samples. Collagen gel contraction, migration, and invasion capabilities were examined in fBMFs. DCFDA ROS assay was used to examine ROS generation. A luciferase reporter assay was carried out to verify the relationship between miR-155 and its potential target RPTOR.We showed that the expression of miR-155 was aberrantly upregulated in OSF specimens and fBMFs. Inhibition of miR-155 ameliorated various myofibroblast activities, including collagen gel contraction, migration, and invasion abilities as well as ROS production. Results from the luciferase reporter assay demonstrated that miR-155 directly interacted with its target RPTOR.Taken together, these findings indicate that miR-155 is implicated in the pathogenesis of oxidative stress-associated OSF, possibly through the regulation of RPTOR.

Association of NQO1 C609T (Pro187Ser) with Risk of Oral Submucous Fibrosis in Eastern Indian Population

Objective: Oral submucous fibrosis (OSF) is a debilitating disease mainly attributed to chewing areca nut with a 7.4-13% malignant transformation rate. The present study explores the role of NADPH quinone oxidoreductase 1 (NQO1) C609T (Pro187Ser) polymorphism in susceptibility to OSF among habitual areca nut chewers in an eastern Indian population. Material and Methods: In this hospital-based study, 152 controls and 179 OSF cases were genotyped at NQO1 C609T polymorphic site by PCR-RFLP and its effect on NQO1 expression in OSF tissue was studied to determine the risk of the disease. Results: Overall, about 18% of the total OSF cases were detected carrying minor TT alleles (Ser/Ser) p=0.026. When categorized by age, both CT (Pro/Ser) and TT (Ser/Ser) alleles were significantly higher (p= 0.003 &amp; 0.004 respectively) in cases above 40years of age. NQO1 protein was 42% reduced in buccal tissues of heterozygous (Pro/Ser) carriers, whereas a 70% reduction was observed in TT (Ser/Ser) OSF cases. Conclusion: Our study suggests that the NQO1 C609T polymorphism confers increased risk for OSF in habitual chewers.

Honokiol inhibits arecoline-induced oral fibrogenesis through transforming growth factor-β/Smad2/3 signaling inhibition

The habit of areca nut chewing has been regarded as an etiological factor of precancerous oral submucous fibrosis (OSF). In the present study, we aimed to evaluate the anti-fibrosis effect of honokiol, a polyphenolic component derived from Magnolia officinalis. The cytotoxicity of honokiol was tested using normal and fibrotic buccal mucosal fibroblasts (fBMFs) derived from OSF tissues. Collagen gel contraction, Transwell migration, invasion, and wound healing capacities were examined. Besides, the expression of TGF-β/Smad2 signaling as well as α-SMA and type I collagen were measured as well. Honokiol exerted higher cytotoxicity of fBMFs compared to normal cells. The arecoline-induced myofibroblast activities, including collagen gel contractility, cell motility and wound healing capacities were all suppressed by honokiol treatment. In addition, the expression of the TGF-β/Smad2 pathway was downregulated along with a lower expression of α-SMA and type I collagen in honokiol-receiving cells. Our data suggest that honokiol may be a promising compound to alleviate the progression of oral fibrogenesis and prevent the transformation of OSF oral epithelium into cancer.

Silencing periostin inhibits myofibroblast transdifferentiation of fibrotic buccal mucosal fibroblasts

Oral submucous fibrosis (OSF) a well-recognized oral premalignant disorder. Several studies have demonstrated that periostin, a matricellular protein, is involved in the development and pathogenesis of fibrosis diseases. Nevertheless, the contribution of periostin in OSF remains to be uncovered. The purpose of the study was to illustrate the functional role of periostin involved in OSF pathogenesis. RNA-sequencing was employed to screen for differentially expressed genes in normal and OSF tissues. Validation of the upregulation of periostin in OSF specimens and fibrotic buccal mucosal fibroblasts (fBMFs) was conducted by qRT-PCR. The correlation of the gene expression of periostin and various fibrosis markers was analyzed. In addition, the functional role of periostin in myofibroblast features was tested using collagen gel contraction and transwell migration assays. We observed overexpression of periostin in OSF specimens using RNA-sequencing and confirmed its upregulation in OSF tissues and patient-derived fBMFs. Besides, there was a positive relationship between the expression of periostin and several fibrosis-associated markers, including ACTA2 (α-smooth muscle actin; α-SMA), COL1A1 (type 1 collagen α1 chain), TGFB1 (TGF-β1), and FN1 (fibronectin). Furthermore, we examined the effect of silencing periostin on the maintenance of myofibroblast characteristics and showed that knockdown of periostin suppressed the expression of α-SMA. Also, inhibition of periostin markedly downregulated the myofibroblast activities (collagen gel contraction and migration capacities). Our results indicate the aberrant expression of periostin in OSF tissues and myofibroblasts. Moreover, the expression of periostin is positively associated with fibrosis markers, and repression of periostin may be a promising direction to alleviate the progression of OSF.