Oral submucous fibrosis (OSF) is an irreversible fibrosis disease and a potentially malignant disorder in the oral cavity. Various studies have shown that miR-21 was implicated in the fibrogenesis and carcinogenesis, but its functional role in the development of OSF has not been investigated. The expression levels of miR-21 in arecoline-stimulated normal buccal mucosal fibroblasts (BMFs) and OSF specimens were determined by qRT-PCR. Exogenous administration of TGF-β and its inhibitor (SB431542) were utilized to examine the involvement of TGF-β signaling in miR-21 alteration. Collagen gel contraction, transwell migration, and invasion assays were used to assess the myofibroblast activities. The relationship between α-SMA and miR-21 was calculated using the Pearson correlation coefficient. MiR-21 expression was induced in BMFs by arecoline treatment in a dose-dependent manner. Our results showed that this upregulation was mediated by TGF-β signaling. Subsequently, we demonstrated that the administration of the miR-21 inhibitor suppressed the arecoline-induced myofibroblast characteristics, including a higher collagen gel contractility and cell motility, in normal BMFs. Furthermore, inhibition of miR-21 was sufficient to attenuate the myofibroblast features in fibrotic BMFs. Besides, we showed that the expression of miR-21 was aberrantly upregulated in the OSF tissues and there was a positive correlation between miR-21 and myofibroblast marker, α-SMA. MiR-21 overexpression in OSF may be due to the stimulation of areca nut, which was mediated by the TGF-β pathway. Our data suggested that the repression of miR-21 was a promising direction to palliate the development and progression of OSF.

Accumulating studies have indicated that long non-coding RNAs (lncRNAs) participate in the regulation of cancer stem cells (CSCs), which are crucial in tumor initiation, metastasis, relapse, and therapy resistance. In the current study, RT-PCR analysis was employed to evaluate the expression of LINC00963 in tumor tissues and oral CSCs. Stemness phenotypes and the expression of CSCs markers in oral cancer cells transfected with sh-LINC00963 were examined. Our results showed that the expression of the lncRNA LINC00963 was up-regulated in oral cancer tissues and CSCs. We found that the downregulation of LINC00963 inhibited CSC hallmarks, such as migration, invasion and colony formation capacity. Moreover, suppression of LINC00963 reduced the activity of stemness marker ALDH1, the percentage of self-renewal, chemoresistance and the expression of multidrug-resistance transporter ABCB5. Most importantly, we demonstrated that knockdown of LINC00963 decreased self-renewal, invasion and colony formation ability via ABCB5. Analysis of TCGA (the Cancer Genome Atlas) datasets suggested that the level of LINC00963 was positively correlated with the expression of the cancer stemness markers (Sox2 and CD44) and drug resistance markers (ABCG2 and ABCB5). Altogether, our results showed that suppression of LINC00963 may be beneficial to inhibit chemoresistance and cancer relapse in oral cancer patients.

Oral submucous fibrosis (OSF) has been recognized as a precancerous disorder in the oral cavity. Great effort has been made to inhibit the malignant progression of OSF over the past decades, but the cure of this fibrosis disease has not been discovered. In the present study, we found that a long noncoding RNA, LINC00312, was upregulated in OSF tissues, and positively associated with several fibrosis factors, such as α-SMA, type I collagen, and fibronectin. As such, we sought to investigate the role of LINC00312 in OSF progression and identify its interacting factor that mediated oral fibrogenesis. Our results showed that the inhibition of LINC00312 downregulated the myofibroblast activities, including collagen gel contractility, transwell migration, and wound healing, as well as the gene expression of myofibroblast markers. We verified that YBX1 was a downstream factor of LINC00312 and revealed that the downregulation of YBX1 repressed the gene expression of α-SMA and p-Smad2 along with the reduced myofibroblast phenotypes. Most importantly, we demonstrated that the LINC00312-induced myofibroblast activities were reverted by the knockdown of YBX1, suggesting that the LINC00312-mediated myofibroblast transdifferentiation was through YBX1. Collectively, our findings revealed that the LINC00312/ YBX1 axis may serve as a target for the development of therapies against OSF.

Purpose: Although clinicians encounter patients aged ≥70 years with locally advanced oral cavity squamous cell carcinoma (LA-OCSCC), no evidence is available to facilitate decision making regarding treatment for this elderly population. Methods: We selected elderly (≥70 years) patients from the Taiwan Cancer Registry database who had received a diagnosis of LA-OCSCC. Propensity score matching was performed. Cox proportional hazards model curves were used to analyze all-cause mortality in patients in different age groups receiving different treatments. Results: The matching process yielded a final cohort of 976 patients in concurrent chemoradiotherapy (CCRT), non-treatment, radiotherapy (RT) alone, and surgery cohorts who were eligible for further analysis. After stratified analysis, the adjusted hazard ratios (aHRs) (95% confidence intervals [CIs]) derived for surgery, RT alone, and non-treatment compared with CCRT were 0.66 (0.52 to 0.83), 1.02 (0.81 to 1.28), and 1.52 (1.21 to 1.91), respectively, in patients aged 70 to 80 years. In the oldest patients (aged >80 years), multivariate analysis indicated that the results of surgery or RT alone were nonsignificant compared with those of CCRT. The aHR (95% CI) derived for the highest mortality was 1.81 (1.11 to 2.40) for non-treatment compared with CCRT. Conclusions: Surgery for elderly patients with LA-OCSCC is associated with a significant survival benefit, but the association is nonsignificant in the oldest elderly patients. No survival differences were observed between RT alone and CCRT in these elderly patients. Non-treatment should not be an option for these patients.

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.

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.