JARID2, a novel regulatory factor, promotes cell proliferation, migration, and invasion in oral squamous cell carcinoma.

BACKGROUND: Accurate regulation of gene expression is crucial for normal development and function of cells. The prognostic significance and potential carcinogenic mechanisms of the related gene JARID2 in OSCC are not yet clear, but existing research has indicated a significant association between the two. METHODS AND MATERIALS: The relationship between the expression of the JARID2 gene in tumor samples of OSCC patients and clinical pathological factors was analyzed using immunohistochemistry experiments and RT-qPCR analysis. Based on the clinical pathological data of patients, bioinformatics analysis was conducted using public databases to determine the function of JARID2 in OSCC. Knockdown OSCC cell lines were constructed, and the impact of JARID2 on the biological behavior of OSCC cell lines was assessed through CCK-8, wound healing assay, and transwell analysis. RESULTS: Immunohistochemistry experiments confirmed the correlation between JARID2 and the prognosis of OSCC patients, while RT-qPCR experiments demonstrated its expression levels in tissue and cells. CKK-8 experiments, wound healing assays, and Transwell experiments indicated that knocking down JARID2 had a negative impact on the proliferation, invasion, and migration of OSCC cells. Bioinformatics analysis results showed that the expression of JARID2 in OSCC is closely associated with patient gene co-expression, gene function enrichment, immune infiltration, and drug sensitivity. CONCLUSION: Our study indicates that JARID2 is a novel prognostic biomarker and potential therapeutic target for OSCC.

circMTO1/miR-30c-5p/SOCS3 axis alleviates oral submucous fibrosis through inhibiting fibroblast-myofibroblast transition.

BACKGROUND: 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. METHODS: 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). RESULTS: 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. CONCLUSION: 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.

BCAT1 promotes cell proliferation, migration, and invasion via the PI3K-Akt signaling pathway in oral squamous cell carcinoma.

OBJECTIVES: To analyze the expression, biological function of branched chain amino-acid transaminase 1 (BCAT1) in oral squamous cell carcinoma (OSCC). MATERIALS AND METHODS: Real-time PCR and immunohistochemistry were used to analyze the expression of BCAT1 protein in OSCC and normal oral tissues. Based on the clinicopathological information of patients, the relationship between the expression of BCAT1 protein and other clinicopathological factors was analyzed. Real-time PCR and western blot assays were used to analyze the expression of BCAT1 gene and protein in normal human oral keratinocytes (HOK) and human OSCC cells, respectively. After BCAT1 overexpression or knockdown, the proliferation, cell cycle, migration, and invasion of human OSCC cells were analyzed by CCK8, flow cytometry, wound healing, and transwell invasion assays, respectively. After adding the BCAT1 inhibitor EGR240 to OSCC cells, the changes in cell proliferation, migration, and invasion ability in OSCC cells were analyzed. Based on the TCGA database, the involved signal pathway in BCAT1-related and BCAT1-binding genes was obtained for Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, verified by western blot assays. After inhibiting PI3K, the effect of BCAT1 on the expression of the downstream phosphorylated protein of the PI3K-Akt signaling pathway was analyzed by western blot assays. The relationship between the expression of BCAT1 and EMT-related protein of OSCC cells was also analyzed. RESULTS: The expression of BCAT1 gene and protein were upregulated in OSCC tissue, which positively correlated with the pathological grade of patients with OSCC. Compared with normal oral keratinocytes, BCAT1 gene and protein were upregulated in OSCC cells. BCAT1 overexpression promoted the proliferation, migration, and invasion of OSCC cells. BCAT1 knockdown or inhibition could reduce the proliferation, migration, and invasion abilities of OSCC cells. The results of bioinformatics analysis and Western bolt showed that BCAT1 could regulate the activation of PI3K-Akt signaling pathway, and promote epithelial-mesenchymal transition (EMT) of OSCC cells. CONCLUSIONS: BCAT1 could promote the proliferation, migration, and invasion of OSCC cells via PI3K-Akt signaling pathway, which is a potential therapeutic target for OSCC.

Areca nut-induced AREG promote oral epithelial cell proliferation, migration, and EMT.

OBJECTIVE: To analyze the biological effect and mechanism of areca nut extract (ANE) on human oral keratinocyte (HOK) cells. MATERIALS AND METHODS: The effect of gradient concentration of ANE on the proliferation activity of HOK cells was analyzed by cell counting kit-8 (CCK-8) assays. The differentially expressed genes between the ANE group and control group HOK cells were analyzed by second-generation transcriptome sequencing. Real-time PCR and western blot were, respectively, used to analyze the expression of AREG gene and protein in HOK cells. After AREG gene overexpression or knockdown, the proliferation, migration, and expression of proteins related to epithelial-mesenchymal transformation (EMT), MAPK signal pathway in HOK cells were, respectively, detected by CCK-8, wound healing, transwell, and western blot assays. RESULTS: ANE (500 µg/mL) promoted the proliferation and migration of HOK cells, ANE (2 mg/mL) promoted the EMT of HOK cells, and ANE (50 mg/mL) inhibited the proliferation of HOK cells. AREG knockdown inhibited ANE-induced proliferation and migration of HOK cells, while AREG overexpression promoted the proliferation and migration of HOK cells. Western blot assay showed that ANE activated MAPK signal pathway by upregulating AREG protein in HOK cells. CONCLUSIONS: ANE promoted HOK cell proliferation, migration, and EMT by mediating AREG-MAPK signaling pathway.

Interleukin-13 contributes to the occurrence of oral submucosal fibrosis.

Oral submucous fibrosis (OSF) is a chronic progressive fibrosis disease that affects in oral mucosal tissues. Interleukin (IL)-13 has been implicated in the development of fibrosis in multiple organs. Indeed, it contributes to diseases such as pulmonary fibrosis, liver cirrhosis among others. Currently, its expression in OSF and the specific mechanisms are not well understood. The aim of this study was to investigate the role of IL-13 in OSF and further explore whether IL-13 regulates-polarization of M2-macrophages in OSF. Initially, in the tissues of patients with OSF, we observed a high expression of M2-macrophages and IL-13 protein. Additionally, we found a correlation between the expression of IL-13 and the stage of OSF. Arecoline inhibited the proliferation of fibroblasts (FBs) and promoted IL-13 production in vitro. Furthermore, our observations revealed that M2-macrophages increased upon co-culturing M0-macrophages with supernatants containing the IL-13 cytokine. In conclusion, our study demonstrated that arecoline stimulates FBs leading to increased secretion of IL-13, which in turn IL-13 leads to polarization of M2-macrophages and promotes the occurrence of OSF. This suggests that IL-13 may be a potential therapeutic target of OSF.

The mechanism of IL-13 targeting IL-13Rα2 in regulating oral mucosal FBs through PI3K/AKT/mTOR.

OBJECTIVE: The objective of this investigation was to examine the presence of interleukin (IL)-13 and its receptor IL-13Rα2 in the tissues of oral submucous fibrosis (OSF), investigate their biological functions, and explore the underlying mechanisms involved in the development of OSF. MATERIALS AND METHODS: The expression of IL-13 and IL-13Rα2 in the oral mucosa of patients with OSF and normal individuals was determined through immunohistochemistry and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Primary fibroblasts (FBs) were extracted through enzymatic digestion and then cultured. Immunofluorescence was employed to identify the FB cultures and the location of IL-13Rα2. The effects of IL-13/IL-13Rα2/PI3K/AKT/mTOR on the migration, proliferation, and secretion of fiber-related proteins of FBs were explored via the wound healing assay, CCK-8 assay, EDU assay, and RT-qPCR. The impact of IL-13Rα2 silencing and PI3K/AKT inhibition on the effect of IL-13 on FBs was analyzed by RT-qPCR and Western blotting. RESULTS: IL-13 and IL-13Rα2 were highly expressed in OSF. Primary FBs were successfully extracted and cultured. IL-13Rα2 was found to be localized in myofibroblasts. IL-13 promoted the proliferation, migration, and secretion of fibril-associated proteins in FBs. The proliferation, migration, and secretion of fibril-associated proteins of FBs were decreased following IL-13Rα2 silencing and inhibition of the PI3K/AKT/mTOR pathway. CONCLUSION: IL-13 may promote the proliferation, migration, and secretion of fiber-related proteins of FBs through the PI3K/AKT/mTOR pathway by targeting IL-13Rα2.

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

OBJECTIVES: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

ADAMTS12, a novel prognostic predictor, promotes cell proliferation, migration, and invasion in head and neck squamous cell carcinoma.

OBJECTIVES: The prognostic significance and potential carcinogenic mechanism of ADAM metallopeptidase with thrombospondin type 1 motif 12 (ADAMTS12) in head and neck squamous cell carcinoma (HNSC) remain unclear. MATERIALS AND METHODS: Immunohistochemistry was used to analyze the correlation between ADAMTS12 protein expression and clinicopathological factors in tumor samples from 195 patients with HNSC. Based on clinicopathological data of patients, Cox regression and Kaplan-Meier analysis were used to identify the prognostic significance of the ADAMTS12 expression. The carcinogenicity of the ADAMTS12 in HNSC cells was analyzed by CCK-8 assay, the wound-healing assay, and transwell assays after transfection of ADAMTS12 overexpression or knock-down vector. RESULTS: The expression of ADAMTS12 was up-regulated in HNSC compared with normal tissue, related to pathology grade and lymph node metastasis of patients with HNSC, which was an independent prognostic factor. ADAMTS12 overexpression facilitated cell viability, invasion, and migration of HNSC cells, while ADAMTS12 knock-down had inverse results. Moreover, enrichment analysis, ADAMTS12 overexpression assay, and ADAMTS12 knock-down assay confirmed that ADAMTS12 mediated the activation of P13K/Akt pathway in HNSC. CONCLUSIONS: Our studies indicated that ADAMTS12 was a novel prognostic biomarker and potentially therapeutic target in HNSC.

Alternation of supragingival microbiome in patients with cirrhosis of different Child-Pugh scores.

OBJECTIVES: The aim of this study was to analyze the differences in the taxonomy and functions of oral microbiome between patients with and without cirrhosis. MATERIALS AND METHODS: In this study, V4-16S rDNA amplicon sequencing was used to compare the difference of supragingival microbiome in 42 patients and 12 healthy individuals. RESULTS: Overall, 3,223,529 clean reads were generated, with an average of 59,694 ± 1,548 clean reads per sample. A total of 30 phyla, 78 classes, 116 orders, 167 families, 228 genera, and 114 species were detected in the 54 samples. The differences were detected among groups at each taxonomical level. Functional prediction showed that patients with cirrhosis had a significant higher proportion of the genes associated with carbohydrate transport and metabolism, defense mechanisms, infectious diseases, membrane transport, etc. compared with healthy individuals (p < .05). CONCLUSIONS: In conclusion, significant differences were observed in compositions and predictive functions of the supragingival microbiome between patients with cirrhosis and that in healthy people. These findings will provide a new insight into the understanding of pathogenesis, diagnosis, prognosis, and therapy of cirrhosis.

Malignant Hyperthermia: A Killer If Ignored.

Malignant hypothermia (MH) is a potentially fatal hypermetabolic reaction of skeletal muscle. It is an autosomal dominant disorder that generally occurs in people with RYR1, CACNA1S, or STAC3 mutations. And these genetic abnormalities often cause the imperfection of calcium release channels of skeletal muscle. The incidence of MH among different racial groups across the world ranges from approximately 1:5,000-1:250,000, but there is no national statistic MH incidence in China. It is not clear whether there are racial or regional differences in the incidence, but patients under 18 years old may be more affected. MH can be triggered by anesthetics, or other stimuli, such as strenuous exercise, heat-stroke, and emotional stress. While viral infection, statins, hyperglycemia, and muscle metabolic dysfunctions might accelerate the onset of MH. The onset of MH is insidious and rapid, with the preclinical stage characterized by rigidity of the masseter muscle, a high level of end-tidal carbon dioxide, and a sharp and persistent increase in body temperature. Medical history, family history, clinical presentation, in vitro caffeine-halothane contracture testing (IVCT/CHCT) and genetic testing are commonly diagnostic methods of MH. As soon as the onset of MH is suspected, immediate cessation of exposure to stimuli, call for professional support, and access to dantrolene are the highest priorities. For symptomatic treatment, "5C principles" were summarized as an algorithm to guide clinicians.

The repertoire of tumor-infiltrating lymphocytes within the microenvironment of oral squamous cell carcinoma reveals immune dysfunction.

BACKGROUND: The role of tumor-infiltrating lymphocytes (TILs) in the immune remodeling of tumor microenvironments (TME) in oral squamous cell carcinoma (OSCC) remains controversial. In this study, we pursued a comprehensive characterization of the repertoire of TILs and then analyzed its clinical significance and potential prognostic value. METHODS: Fresh tumor tissue samples and peripheral blood from 83 OSCC patients were collected to comprehensively characterize the phenotypes and frequencies of TILs by flow cytometry. Archived paraffin-embedded tissues derived from 159 OSCC patients were analyzed by immunohistochemistry to further assess the TIL repertoire. The clinical significance of TILs and their potential prognostic value were further analyzed. RESULTS: A series of unique features of TILs were observed. IL-17 was highly expressed in betel nut chewers, and CD20 was abundantly expressed in patients who did not drink alcohol; high expression of CD138, PD-L1, and Foxp3 was associated with poor prognosis. The Th17/Treg ratio was an independent prognostic factor for patient survival with greater predictive accuracy for overall survival. CONCLUSIONS: Our results suggest an antigen-driven immune response; however, the immune dysfunction within the microenvironment in OSCC and the Th17/Treg balance may play important roles in the modulation of antitumor immunity.

Cytokines secreted by arecoline activate fibroblasts that affect the balance of TH17 and Treg.

BACKGROUND: Oral submucous fibrosis (OSF) is a chronic progressive oral disease with cancerous tendency. Arecoline plays an important role in the pathogenesis of OSF. Fibroblasts (FBs) are the primary cells involved in the pathogenesis of OSF. There is a change in CD4+ IL-17+ helper T cells (Th17) and CD4+ CD25+ Foxp3+ regulatory T cells (Treg) in OSF patients, but the molecular mechanisms of are not clearly understood. In this work, we studied the molecular mechanisms. METHODS: Enzyme digestion was used to extract primary FBs, and immunofluorescence was used to identify FBs. Cytotoxic experiment was then performed to determine the effect of arecoline on FB activity. Enzyme-linked immunosorbent assay (ELISA) was used to detect changes in the amount of cytokines. In addition, we treated human peripheral blood mononuclear cells (PBMC) with the above cytokines and detected their changes. Flow cytometry was used to detect the changes of Th17 and Treg, and quantitative-polymerase chain reaction (Q-PCR) was used to detect the changes of RORγt and Foxp3. RESULTS: We have found that the stimulation of arecoline on FBs increased interleukin-2, interleukin-6, and interleukin-21 (IL-2, IL-6, and IL-21) while decreased transforming growth cytokine-ß (TGF-ß). After the cytokine-containing supernatant was co-cultured with PBMC, the cytometry results showed that Th17 was increased, while Treg was significantly decreased and Q-PCR results showed that RORγt expression was increased and Foxp3 expression was decreased. CONCLUSION: The arecoline can affect inflammatory cytokines produced by FBs, which then act on immune cells Th17 and Treg, and make them change.

Oral submucous fibrosis in Asian countries.

Oral submucous fibrosis (OSF) is a chronic, insidious, and progressive oral mucosal disease that affects entire oral cavity and sometimes pharynx. This oral potentially malignant disorder has a high rate of malignant transformation (7%-30%) to oral squamous cell carcinoma (OSCC), posing global problems for public health. Due to enormous efforts dedicated to this disease in the past decades, there have been significant advances in identification of its etiology and pathogenesis as well as development of corresponding therapeutic approaches, in spite of several challenges. This study reviewed the existing literature concerning OSF in Asian countries, encompassing its etiology, histopathology, pathogenesis, clinical manifestations, diagnosis and differential diagnosis, and treatments. For improving treatment of OSF, the multifactorial etiology analysis, incorporation of effective molecular pathways, cytokines and cells for mechanism illustration, and integration of multidisciplinary modalities were also expounded to guide future research and clinical practice.

Long non-coding RNA DNM3OS/miR-204-5p/HIP1 axis modulates oral cancer cell viability and migration.

BACKGROUND: Non-coding RNAs play a critical role in the occurrence and development of oral cancer. The present study is aimed to identify long non-coding RNA (lncRNA) that might be novel effective targets for the treatments of oral cancer and the underlying mechanism. METHODS: The microarray profiling and RNA-sequencing analysis were performed to identify lncRNAs related to oral cancer development, and lncRNA DNM3OS was selected. DNM3OS knockdown was generated in cancer cell lines, and the specific effects of DNM3OS knockdown on cell phenotype were examined. DNM3OS targeted miRNA and miRNA targeted downstream mRNA were selected, the predicted bindings were verified, and the specific effects of miRNA on oral cancer cells were examined. Finally, the dynamic effects of DNM3OS and miRNA on target mRNA expression and oral cancer cell phenotype were examined. RESULTS: DNM3OS was upregulated in oral cancer tissues and cells. DNM3OS knockdown in CAL27 and SCC-9 cells inhibited cell viability and migration. DNM3OS targeted miR-204-5p to inhibit miR-204-5p expression. miR-204-5p overexpression suppressed oral cancer cell aggressiveness. miR-204-5p targeted HIP1 to inhibit HIP1 expression. HIP1 knockdown inhibited oral cancer cell viability and migration. The effects of DNM3OS knockdown were significantly reversed by miR-204-5p inhibition. Within oral carcinoma tissue samples, expression of DNM3OS and HIP1 was increased whereas the miR-204-5p expression was downregulated; miR-204-5p had a negative correlation with DNM3OS and HIP1, respectively, while DNM3OS and HIP1 were positively correlated with each other. CONCLUSION: Long non-coding RNA DNM3OS, miR-204-5p, and HIP1 form an axis that modulates oral cancer cell viability and migration.

Repertoire of peripheral T cells in patients with oral squamous cell carcinoma.

BACKGROUND: The establishment of adaptive immune responses to neoplasms involves not only the tumour tissue, but also the peripheral blood. We aimed to conduct a preliminary exploration to understand the immune response of T lymphocytes of peripheral blood mononuclear cells (PBMC-Ts) in oral squamous cell carcinoma (OSCC). METHODS: A total of 103 blood samples from OSCC patients and 18 blood samples from healthy donors (HD) were analysed by flow cytometry. RESULTS: Compared to those in HD, a series of unique features of PBMC-Ts were observed in OSCC patients including a significant increase in CD4+ T cells, a shift from naïve to memory/effector phenotype, an increased frequency of exhausted phenotypes (programmed death-1 [PD-1], T cell Ig and mucin protein-3 [Tim-3] and Tregs), an abundance of Th17s and Tc17s and an imbalance in Th17/Tc17 and Th17/Treg ratios. Furthermore, in OSCC patients, we also found that CD4+ T cells were significantly increased in patients with larger tumours than smaller tumours, memory/effector phenotype and exhausted phenotypes were significantly associated with advanced clinical stage and lymph node metastasis, and the Th17/Treg ratio was associated with early clinical stage and no lymph node metastasis. CONCLUSION: PBMC-Ts may be involved in the development and progression of OSCC, which suggested to be a manifestation of an immune response between host and tumour neoantigens.

Proliferative ability and accumulation of cancer stem cells in oral submucous fibrosis epithelium.

OBJECTIVES: The driving force of the malignant transformation of epithelial cells during oral submucous fibrosis (OSF) is an unsettled debate. We hypothesized that the expression and accumulation of cancer stem cells (CSCs) are accompanied by epithelial atrophy in OSF. MATERIALS AND METHODS: The expression levels of Ki67 (proliferation marker), SOX2, and Bmi1 (CSC marker) in the epithelium during the early, middle, and late stages of OSF were measured by immunohistochemistry. At the same time, we focused on the expression of three proteins in OSF patients with benign hyperkeratosis and epithelial dysplasia. RESULTS: The clinical cohort study showed upregulated expression of the proliferation-associated protein Ki67 in atrophic epithelium in patients with OSF. The expression levels of SOX2 and Bmi1 showed an increasing trend in the progression of OSF. Ki67, SOX2, and Bmi1 were highly expressed in OSF tissues with dysplasia. Moreover, the three proteins were located at the epithelial and mesenchymal junctions, and their expression showed a positive correlation with each other. CONCLUSION: The results suggest that CSC accumulation could be accompanied by epithelial atrophy during OSF, which may be responsible for the driving forces for OSF carcinogenesis.

Arecoline suppresses epithelial cell viability by upregulating tropomyosin-1 through the transforming growth factor-ß/Smad pathway.

CONTEXT: Oral submucous fibrosis (OSF) is a chronic and progressive disease. Arecoline, present in betel nuts, has been proposed as a vital aetiological factor. However, the underlying mechanism remains unclear. OBJECTIVES: This research elucidates the expression of tropomyosin-1 (TPM1) and its regulation mechanism in HaCaT cells treated with arecoline. MATERIALS AND METHODS: HaCaT cells were assigned into three groups: (1) Control; (2) Treated with arecoline (0.16 mM) for 48 h (3) Treated with arecoline (0.16 mM) and transfected with small interfering RNA (siRNA) for TPM1 (50 nM) for 48 h. CCK8, cell cycle, and apoptosis phenotypic analyses were performed. PCR and western blot analyses were performed to detect the expression level of TPM1 and examine the related signalling pathway. RESULTS: The IC50 of arecoline was approximately 50 µg/mL (0.21 mM). The arecoline dose (0.16 mM) and time (48 h) markedly increased TPM1 expression at the mRNA and protein levels in HaCaT cells. Arecoline suppressed the cell growth, caused cell cycle arrest at the G1 phase, and induced cell apoptosis in HaCaT cells. siRNA-mediated knockdown of TPM1 attenuated the effect of arecoline on cell proliferation, apoptosis, and cell cycle arrest at the G1 phase. Furthermore, blocking of the transforming growth factor (TGF)-ß receptor using SB431542 significantly suppressed TPM1 expression in the cells treated with arecoline. DISCUSSION AND CONCLUSIONS: Arecoline suppresses HaCaT cell viability by upregulating TPM1 through the TGF-ß/Smad signalling pathway. This research provides a scientific basis for further study of arecoline and TPM1 in OSF and can be generalised to broader pharmacological studies. TPM1 may be a promising molecular target for treating OSF.

The role of extracellular vesicles from different origin in the microenvironment of head and neck cancers.

The proliferation and metastasis ability of tumors are mediate by the "mutual dialogue" between cells in the tumor microenvironment (TME). Extracellular vesicles (EVs), mainly exosomes and microvesicles, play an important role in achieving intercellular substance transport and information transfer in the TME. Initially considered "garbage dumpsters" and later referred to as "signal boxes", EVs carry "cargo" (proteins, lipids, or nucleic acids) that can redirect the function of a recipient cell. Currently, the molecular mechanisms and clinical applications of EVs in head and neck cancers (HNCs) are still at an early stage and need to be further investigate. In this review, we provide insight into the TME of HNCs, classifying and summarizing EVs derived from different cell types and illuminating their complex signaling networks involved in mediating tumor proliferation, invasion and metastasis, vascular angiogenesis and cancer drug resistance. In addition, we highlight the application of EVs in HNCs, underlining the special pathological and physiological environment of HNCs. The application of tumor heterogeneous EVs in saliva and circulating blood diagnostics will provide a new perspective for the early screening, real-time monitoring and prognostic risk assessment of HNCs. Given the concept of precise and individual therapy, nanostructured EVs are equipped with superior characteristics of biocompatibility, low immunogenicity, loadability and modification ability, making these molecules one of the new strategies for HNCs treatment.

Anatomic study of the position of the mandibular canal and corresponding mandibular third molar on cone-beam computed tomography images.

PURPOSE: The positional relationship between the mandibular canal and corresponding third molars is a key anatomic factor of inferior alveolar nerve (IAN) injury. The aim of the present study is to classify the anatomic three-dimensional relationship between the mandibular third molar and the mandibular canal on cone-beam computed tomography (CBCT) images. METHODS: This study used CBCT images to classify the positional relationship between the mandibular canal and corresponding third molars. CBCT images of 749 patients (1296 mandibular third molars) were analyzed to draw up a classification. RESULTS: On a total of 1296 third molars, the mandibular canal relative to the roots of the mandibular third molar was on the apical side (88.1%), followed by the buccal side (7.9%), the lingual side (3.5%), and then between the roots (0.5%). Ninety-five (7.1%) third molars had a close relation with the mandibular canal, while 1201 (92.7%) third molars had no direct contact. The percentage of the mandibular canal contacts with the mandibular third molar was higher when the mandibular canal was lingually positioned. CONCLUSIONS: The anatomic structures of the mandibular third molar and the mandibular canal may be helpful to make adequate surgical planning to avoid or reduce nerve involvement.

An adaptive immune response driven by mature, antigen-experienced T and B cells within the microenvironment of oral squamous cell carcinoma.

Lymphocyte infiltrates have been observed in the microenvironment of oral cancer; however, little is known about whether the immune response of the lymphocyte infiltrate affects tumor biology. For a deeper understanding of the role of the infiltrating-lymphocytes in oral squamous cell carcinoma (OSCC), we characterized the lymphocyte infiltrate repertoires and defined their features. Immunohistochemistry revealed considerable T and B cell infiltrates and lymphoid follicles with germinal center-like structures within the tumor microenvironment. Flow cytometry demonstrated that populations of antigen-experienced CD4+ and CD8+ cells were present, as well as an enrichment of regulatory T cells; and T cells expressing programmed death-1 (PD-1) and T cell Ig and mucin protein-3 (Tim-3), indicative of exhaustion, within the tumor microenvironment. Characterization of tumor-infiltrating B cells revealed clear evidence of antigen exposure, in that the cardinal features of an antigen-driven B cell response were present, including somatic mutation, clonal expansion, intraclonal variation and isotype switching. Collectively, our results point to an adaptive immune response occurring within the OSCC microenvironment, which may be sustained by the expression of specific antigens in the tumor.