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

BACKGROUND/PURPOSE: 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. METHODS: 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). RESULTS: 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. CONCLUSION: 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.

BACKGROUND/PURPOSE: 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. METHODS: 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-ß/Smad2 signaling and expression levels of type I collagen, α-SMA, and long non-coding RNA HOTAIR were measured as well. RESULTS: 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-ß/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. CONCLUSION: Our results indicate that paeonol may be a promising compound to attenuate the progression of oral fibrogenesis in OSF patients.

Ex Vivo Expanded Circulating Tumor Cells for Clinical Anti-Cancer Drug Prediction in Patients with Head and Neck Cancer.

The advanced-stage head and neck cancer (HNC) patients respond poorly to platinum-based treatments. Thus, a reliable pretreatment method for evaluating platinum treatment response would improve therapeutic efficiency and outcomes. This study describes a novel strategy to predict clinical drug responses in HNC patients by using eSelect, a lab-developed biomimetic cell culture system, which enables us to perform ex vivo expansion and drug sensitivity profiling of circulating tumor cells (CTCs). Forty liquid biopsies were collected from HNC patients, and the CTCs were expanded ex vivo using the eSelect system within four weeks. Immunofluorescence staining confirmed that the CTC-derived organoids were positive for EpCAM and negative for CD45. Two illustrative cases present the potential of this strategy for evaluating treatment response. The statistical analysis confirmed that drug sensitivity in CTC-derived organoids was associated with a clinical response. The multivariant logistic regression model predicted that the treatment accuracy of chemotherapy responses achieved 93.75%, and the area under the curves (AUCs) of prediction models was 0.8841 in the whole dataset and 0.9167 in cisplatin specific dataset. In summary, cisplatin sensitivity profiles of patient-derived CTCs expanded ex vivo correlate with a clinical response to cisplatin treatment, and this can potentially underpin predictive assays to guide HNC treatments.

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

BACKGROUND/PURPOSE: 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. METHODS: 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-ß/Smad2 signaling as well as α-SMA and type I collagen were measured as well. RESULTS: 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-ß/Smad2 pathway was downregulated along with a lower expression of α-SMA and type I collagen in honokiol-receiving cells. CONCLUSION: 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.

miR-145 mediates the anti-cancer stemness effect of photodynamic therapy with 5-aminolevulinic acid (ALA) in oral cancer cells.

5-Aminolevulinic acid-mediated photodynamic therapy (ALA-PDT) has been used in the treatment of various precancerous and malignant lesions. Our previous work has demonstrated that ALA-PDT possesses the potential to serve as an adjuvant therapy against head and neck cancer via eliminating the cancer stem cells (CSCs) property. This study aimed to further investigate the possible molecular mechanism underlying the effect of ALA-PDT. Our results revealed that ALA-PDT upregulated the expression of microRNA-145 (miR-145) in two oral cancer cell lines. Overexpression of miR-145 in oral CSCs further enhanced the treatment effect of ALA-PDT with lower self-renewal, invasion capacities and reduced CD44 expression, while inhibition of miR-145 exhibited the opposite phenomena. These findings suggest that the anti-CSCs effect of ALA-PDT is due to an elevation of miR-145.

A clinically validated prediction method for facial soft-tissue changes following double-jaw surgery.

PURPOSE: It is clinically important to accurately predict facial soft-tissue changes prior to orthognathic surgery. However, the current simulation methods are problematic, especially in anatomic regions of clinical significance, e.g., the nose, lips, and chin. We developed a new 3-stage finite element method (FEM) approach that incorporates realistic tissue sliding to improve such prediction. METHODS: In Stage One, soft-tissue change was simulated, using FEM with patient-specific mesh models generated from our previously developed eFace template. Postoperative bone movement was applied on the patient mesh model with standard FEM boundary conditions. In Stage Two, the simulation was improved by implementing sliding effects between gum tissue and teeth using a nodal force constraint scheme. In Stage Three, the result of the tissue sliding effect was further enhanced by reassigning the soft-tissue-bone mapping and boundary conditions using nodal spatial constraint. Finally, our methods have been quantitatively and qualitatively validated using 40 retrospectively evaluated patient cases by comparing it to the traditional FEM method and the FEM with sliding effect, using a nodal force constraint method. RESULTS: The results showed that our method was better than the other two methods. Using our method, the quantitative distance errors between predicted and actual patient surfaces for the entire face and any subregions thereof were below 1.5 mm. The overall soft-tissue change prediction was accurate to within 1.1 ± 0.3 mm, with the accuracy around the upper and lower lip regions of 1.2 ± 0.7 mm and 1.5 ± 0.7 mm, respectively. The results of qualitative evaluation completed by clinical experts showed an improvement of 46% in acceptance rate compared to the traditional FEM simulation. More than 80% of the result of our approach was considered acceptable in comparison with 55% and 50% following the other two methods. CONCLUSION: The FEM simulation method with improved sliding effect showed significant accuracy improvement in the whole face and the clinically significant regions (i.e., nose and lips) in comparison with the other published FEM methods, with or without sliding effect using a nodal force constraint. The qualitative validation also proved the clinical feasibility of the developed approach.

Design, development and clinical validation of computer-aided surgical simulation system for streamlined orthognathic surgical planning.

PURPOSE: There are many proven problems associated with traditional surgical planning methods for orthognathic surgery. To address these problems, we developed a computer-aided surgical simulation (CASS) system, the AnatomicAligner, to plan orthognathic surgery following our streamlined clinical protocol. METHODS: The system includes six modules: image segmentation and three-dimensional (3D) reconstruction, registration and reorientation of models to neutral head posture, 3D cephalometric analysis, virtual osteotomy, surgical simulation, and surgical splint generation. The accuracy of the system was validated in a stepwise fashion: first to evaluate the accuracy of AnatomicAligner using 30 sets of patient data, then to evaluate the fitting of splints generated by AnatomicAligner using 10 sets of patient data. The industrial gold standard system, Mimics, was used as the reference. RESULT: When comparing the results of segmentation, virtual osteotomy and transformation achieved with AnatomicAligner to the ones achieved with Mimics, the absolute deviation between the two systems was clinically insignificant. The average surface deviation between the two models after 3D model reconstruction in AnatomicAligner and Mimics was 0.3 mm with a standard deviation (SD) of 0.03 mm. All the average surface deviations between the two models after virtual osteotomy and transformations were smaller than 0.01 mm with a SD of 0.01 mm. In addition, the fitting of splints generated by AnatomicAligner was at least as good as the ones generated by Mimics. CONCLUSION: We successfully developed a CASS system, the AnatomicAligner, for planning orthognathic surgery following the streamlined planning protocol. The system has been proven accurate. AnatomicAligner will soon be available freely to the boarder clinical and research communities.