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Abstract Background Head and neck squamous carcinoma (HNSCC) is known for its high aggressiveness and susceptibility to cervical lymph node metastasis, which greatly contributes to its poor prognosis. During tumorigenesis, many types of cancer cells acquire oncogenic super-enhancers (SEs) that drive the overexpression of oncogenes, thereby maintaining malignant progression. This study aimed to identify and validate the role of oncogenic SE-associated genes in the malignant progression of HNSCC. Methods We identified HNSCC cell-specific SE-associated genes through H3K27Ac ChIP-seq and overlapped them with HNSCC-associated genes obtained from The Cancer Genome Atlas (TCGA) dataset and Gene Expression Omnibus (GEO) datasets using weighted gene coexpression network analysis (WGCNA) to identify hub genes. The expression of IGF2BP2 and KLF7 in HNSCC was detected using clinical samples. To determine the biological role of IGF2BP2, we performed CCK-8, colony formation assay, Transwell migration assay, invasion assay, and orthotopic xenograft model experiments. Furthermore, we utilized a CRISPR/Cas9 gene-editing system, small-molecule inhibitors, ChIP-qPCR, and dual-luciferase reporter assays to investigate the molecular mechanisms of IGF2BP2 and its upstream transcription factors. Results Our study identified IGF2BP2 as a hub SE-associated gene that exhibited aberrant expression in HNSCC tissues. Increased expression of IGF2BP2 was observed to be linked with malignant progression and unfavorable prognosis in HNSCC patients. Both in vitro and in vivo experiments confirmed that IGF2BP2 promotes the tumorigenicity and metastasis of HNSCC by promoting cell proliferation, migration, and invasion. Mechanistically, the IGF2BP2-SE region displayed enrichment for H3K27Ac, BRD4, and MED1, which led to the inhibition of IGF2BP2 transcription and expression through deactivation of the SE-associated transcriptional program. Additionally, KLF7 was found to induce the transcription of IGF2BP2 and directly bind to its promoter and SE regions. Moreover, the abundance of KLF7 exhibited a positive correlation with the abundance of IGF2BP2 in HNSCC. Patients with high expression of both KLF7 and IGF2BP2 showed poorer prognosis. Lastly, we demonstrated that the small molecule inhibitor JQ1, targeting BRD4, attenuated the proliferation and metastatic abilities of HNSCC cells. Conclusions Our study reveals the critical role of IGF2BP2 overexpression mediated by SE and KLF7 in promoting HNSCC progression. Targeting SE-associated transcriptional programs may represent a potential therapeutic strategy in managing HNSCC.

Conclusion: This study suggests that sensitization of small- to medium-sized Dil+ TG neurons and CGRP- and IB4-positive Dil+ TG neurons might contribute to the development of TMJOA chronic pain in rats. This will provide valuable information for more efficient control of TMJOA chronic pain.

Cleft palate only (CPO) is one of the most common craniofacial birth defects. Environmental factors can induce cleft palate by affecting epigenetic modifications such as DNA methylation, histone acetylation, and non-coding RNA. However, there are few reports focusing on the RNA modifications. In this study, all-trans retinoic acid (atRA) was used to simulate environmental factors to induce a C57BL/6J fetal mouse cleft palate model. Techniques such as dot blotting and immunofluorescence were used to find the changes in m6A modification when cleft palate occurs. RNA-seq and KEGG analysis were used to screen for significantly differentially expressed pathways downstream. Primary mouse embryonic palate mesenchymal (MEPM) cells were successfully isolated and used for in vitro experimental verification. We found that an increased m6A methylation level was correlated with suppressed cell proliferation in the palatine process mesenchyme of cleft palate mice. This change is due to the abnormally high expression of m6A methyltransferase METTL14. When using siRNAs and the m6A methyltransferase complex inhibitor SAH to interfere with the expression or function of METTL14, the teratogenic effect of atRA on primary cells was partially alleviated. In conclusion, METTL14 regulates palatal mesenchymal cell proliferation and cycle-related protein expression relies on m6A methylation modification, affecting the occurrence of cleft palate.

The role of Pleckstrin homology-like domain family B member 2 (PHLDB2) in the regulation of cell migration has been extensively studied. However, the exploration of PHLDB2 in head and neck squamous cell carcinoma (HNSCC) is still limited in terms of expression, function, and therapeutic potential. In this study, we discovered an upregulation of PHLDB2 expression in HNSCC tissues which was correlated with a negative prognosis in patients with HNSCC. Additionally, we determined that a high level of expression of PHLDB2 is crucial for maintaining cell migration through the regulation of the epithelial–mesenchymal transition (EMT). Furthermore, we demonstrated that the ablation of PHLDB2 in tumor cells inhibited tumorigenicity in a C3H syngeneic tumor-bearing mouse model. Mechanistically, PHLDB2 was found to be involved in the regulation of T cell anti-tumor immunity, primarily by enhancing the activation and infiltration of CD8+ T cells. In light of these findings, PHLDB2 emerges as a promising biomarker and therapeutic target for interventions in HNSCC.

Ferroptosis is a newly established form of regulated cell death characterized by intracellular lipid peroxidation and iron accumulation that may be a promising cancer treatment strategy. However, the function and therapeutic value of ferroptosis in oral squamous cell carcinoma (OSCC) remain inadequately understood. In the present study, we investigated the biological role of the fat mass and obesity-associated gene (FTO) in ferroptosis in the context of OSCC. We found that OSCC had greater potential for ferroptosis, and FTO is associated with ferroptosis. Furthermore, higher FTO expression sensitized OSCC cells to ferroptosis in vitro and in vivo. Mechanistically, FTO suppressed the expression of anti-ferroptotic factors, acyl-CoA synthetase long-chain family member 3 (ACSL3) and glutathione peroxidase 4 (GPX4), by demethylating the m6A modification on the mRNA of ACSL3 and GPX4 and decreasing their stability. Taken together, our findings revealed that FTO promotes ferroptosis through ACSL3 and GPX4 regulation. Thus, ferroptosis activation in OSCC with high FTO levels may serve as a potential therapeutic target.

Abstract Background Serine hydroxymethyltransferase 2 (SHMT2) is a vital metabolic enzyme in one carbon metabolism catalyzing the conversion of serine to glycine, which has been reported to play a crucial role in the progression of tumors. However, its function in tongue squamous cell carcinoma (TSCC) remains unclear. Methods SHMT2 expression was analyzed using samples in online databases, and was assessed through immunohistochemistry staining of collected clinical specimens. The correlation between SHMT2 expression and the cell cycle was predicted through bioinformatic analysis, including weighted gene co-expression network analysis (WGCNA) and gene set enrichment analysis (GSEA). After transfection with siRNA, CCK8 assay, Edu staining, flow cytometry, trans-well assay, and wound healing experiments were performed to verify the functional role of SHMT2 in vitro. A stable cell line with SHMT2 silencing was established to detect the oncogenic function of SHMT2 in vivo. Results The expression of SHMT2 was up-regulated in TSCC tissues and cell lines compared with normal groups, and highly expressed SHMT2 significantly indicated a poorer clinical outcome for TSCC patients. Bioinformatic analysis found that high expression of SHMT2 was closely related with biologic process including cell cycle and cell cycle G1/S transition. Down regulating of SHMT2 significantly suppressed the proliferation, invasive and migrative ability of TSCC cells, and induced the prolongation of the G1 phase of the cell cycle in vitro. Furthermore, western blot showed that cell cycle-related regulators such as cyclin-dependent kinase 4 (CDK4) and cyclinD1 expression levels were decreased, while the expression levels of the cyclin-dependent kinase inhibitors p21Cip1 and p27Kip1 were increased after SHMT2 knockdown. Silencing SHMT2 in the HN6 cell line using short hairpin RNA also impeded tumor growth in vivo. Conclusions Overexpression of SHMT2 in TSCC indicated low survival rates, and was associated with aggressive behaviors of TSCC. It was also found to be involved in cell cycle regulation of TSCC cells. SHMT2 may serve as a novel prognostic indicator of TSCC.

Excessive differentiation of osteoclasts contributes to the disruption of bone homeostasis in inflammatory bone diseases. Methyltransferase-like 3 (METTL3), the core methyltransferase that installs an N6-methyladenosine (m6A) modification on RNA, has been reported to participate in bone pathophysiology. However, whether METTL3-mediated m6A affects osteoclast differentiation in inflammatory conditions remains unelucidated. In this study, we observed that the total m6A content and METTL3 expression decreased during LPS-induced osteoclastogenesis. After knocking down METTL3, we found reduced levels of the number of osteoclasts, osteoclast-related gene expression and bone resorption area. A METTL3 deficiency increased osteoclast apoptosis and pro-apoptotic protein expression. RNA sequencing analysis showed that differentially expressed genes in METTL3-deficient cells were mainly associated with the mitochondrial function. The expression of the mitochondrial function-related genes, ATP production and mitochondrial membrane potential decreased after METTL3 knockdown. Moreover, the most obviously upregulated gene in RNA-Seq was Nos2, which encoded the iNOS protein to induce nitric oxide (NO) synthesis. METTL3 knockdown increased the levels of Nos2 mRNA, iNOS protein and NO content. NOS inhibitor L-NAME rescued the inhibited mitochondrial function and osteoclast formation while suppressing osteoclast apoptosis in METTL3-silenced cells. Mechanistically, a METTL3 deficiency promoted the stability and expression of Nos2 mRNA, and similar results were observed after m6A-binding protein YTHDF1 knockdown. Further in vivo evidence revealed that METTL3 knockdown attenuated the inflammatory osteolysis of the murine calvaria and suppressed osteoclast formation. In conclusion, these data suggested that METTL3 knockdown exacerbated iNOS/NO-mediated mitochondrial dysfunction by promoting a Nos2 mRNA stability in a YTHDF1-dependent manner and further inhibited osteoclast differentiation and increased osteoclast apoptosis in inflammatory conditions.

N6-methyladenosine (m6A) is the most abundant internal mRNA modification in eukaryotes and is related to stability, localization, or translation efficiency in tumorigenesis. Autophagy plays an important role in the occurrence and development of tumours. However, the relationship between m6A and autophagy remains unclear. In this study, we used a rapamycin-induced autophagy model of oral squamous cell carcinoma (OSCC) cells, and observed increased m6A RNA methylation. When autophagy was activated, the methyltransferase-like 14 (METTL14) expression was upregulated and influenced the proliferation, migration, and invasiveness of OSCC cells. Through meRIP-seq and RNA-seq analysis, we found that METTL14 directly combined with eukaryotic translation initiation factor gamma 1 (eIF4G1) mRNA and decreased its RNA stability. According to the dual-luciferase reporter and mutagenesis assay, the mutated site 1 of exon 11 of eIF4G1 is the key target of METTL14. Knockdown of the main m6A binding protein YTHDF2 may rescue the shortened half-life of eIF4G1 mRNA induced by METTL14 overexpression. Furthermore, an in vivo tumour xenograft model confirmed that a high METTL14 expression can effectively reduce OSCC growth. Additionally, using clinical samples, we found that patients with advanced or moderately/poorly differentiated tumours exhibited lower METTL14 levels. Taken together, our results revealed that METTL14 mediated eIF4G1 expression via m6A modification and regulated autophagy levels and biological functions in OSCC. Our findings not only expand our understanding of the correlation between autophagy and RNA methylation in tumorigenesis but also present an opportunity to develop new therapeutic options.

Objective: Osteoradionecrosis of the jaws (ORNJ) is among the most serious oral complications of head and neck cancer treatment with radiation therapy. This study aimed to examine the level of symptom distress and interference of ORNJ in head and neck cancer patients in China. Methods: A cross-sectional study was conducted to profile patient reported symptom severity. Ninety-five hospitalized ORNJ patients were recruited. Participants completed the MD Anderson Symptom Inventory–Head and Neck Module-Chinese version. Results: The percentage of participants who reported that they experienced at least one type of symptom was 97.9%, and 85.2% patients reported interference. The 10 most severe symptoms were as follows: limited mouth opening, problem with teeth/gums, difficulty swallowing/chewing, dry mouth, oral malodor, difficulty with voice/speech, dental ulcer, tinnitus/ear obstruction, skin pain/burning/rash, and difficulty hearing. The problem of limited mouth opening was more severe in patients with longer time to onset of ORNJ after radiotherapy. The interference of patients positively correlated with core symptoms (r = 0.612), head and neck symptoms (r = 0.709), and ORNJ symptoms (r = 0.440) (P

MicroRNAs have been proposed as novel biomarkers for the diagnosis and treatment of many types of cancer. The levels of five candidate microRNAs (miRNAs) (miR-99a-5p, miR-31-5p, miR-138-5p, miR-21-5p, and miR-375-3p) in sera from oral cancer patients and paired tumor and normal tissues were detected by real-time qPCR. The diagnostic power of these miRNAs was analyzed by receiver operating characteristic (ROC) curves. Patient-derived xenograft (PDX) models of oral cancer were established and utilized to verify the potential therapeutic effect of miR-31-5p. Candidate miRNAs were screened from our previous studies and verified in 11 paired oral cancer and adjacent normal tissues. Only serum miR-31-5p levels were significantly different between oral cancer patients and healthy controls and between pre- and postoperative patients. Based on the logistic regression model, this panel of five miRNAs distinguished oral cancer patients from healthy control, with an area under the ROC curve (AUC) of 0.776 (sensitivity = 76.8% and specificity = 73.6%). Furthermore, a miR-31-5p mimic enhanced the proliferation of normal epithelial cells, and antagomiR-31-5p inhibited the proliferation of oral cancer cells in vitro. In vivo, antagomiR-31-5p significantly inhibited tumor growth in oral cancer PDX models. Our findings suggest that circulating miR-31-5p might act as an independent biomarker for oral cancer diagnosis and could serve as a therapeutic target for oral cancer. Keywords: oral cancer, biomarker, circulating miRNA, miR-31-5p, target therapy

ObjectivesRecently long non-coding RNAs (lncRNAs) have emerged as novel gene regulators involved in tumorigenic processes, including oral squamous cell carcinoma (OSCC). Here, we identified a differentiation-related lncRNA, terminal differentiation-induced non-coding RNA (TINCR). However, its biological function and clinicopathological significance in OSCC still remain unclear.MethodsThe lncRNA expression profiles in OSCC tissues and paired adjacent non-tumor tissues (NATs) from 10 patients were detected by lncRNA microarrays. Weighted gene co-expression network analysis (WGCNA) and gene ontology (GO) enrichment were performed to identify the most significant module and module functional annotation, respectively. Potential differentiation-related lncRNAs were screened by differential expression analysis. TINCR was further confirmed in OSCC cell lines and tissues of another patient cohort by using qRT-PCR. The correlation between the TINCR expression level and clinicopathological characteristics was analyzed. The effects of TINCR on cell differentiation, migration and invasion were assessed by knockdown or knock-in in vitro and in vivo.ResultsWGCNA and GO enrichment analysis showed that one co-expression network was significantly enriched for epithelial cell differentiation, among which, TINCR was significantly downregulated. qRT-PCR analyses validated down-regulation of TINCR in tumor tissues compared with paired NATs, and its expression was closely correlated with pathological differentiation and lymph node metastasis in patients with OSCC. Patients with lower TINCR expression levels had worse survival. Cell function experiments showed that TINCR played a crucial role in epithelial differentiation. Both TINCR and epithelial differentiation-associated genes, including IVL and KRT4, were significantly upregulated during OSCC cell calcium-induced differentiation but were reduced when cell dedifferentiation occurred in tumor spheres. Overexpression of TINCR dramatically suppressed cell dedifferentiation, migration and invasion in vitro, while knockdown of TINCR had the opposite effects. Upregulation of TINCR significantly elevated the expression of terminal differentiation genes and repressed tumor growth in vivo. Moreover, TINCR significantly suppressed the activation of JAK2/STAT3 signaling in OSCC cells.ConclusionOur study suggests that TINCR functions as a tumor suppressor by inducing cell differentiation through modulating JAK2/STAT3 signaling in OSCC. TINCR may serve as a prognostic biomarker and therapeutic target for OSCC.

BackgroundAutophagy, a highly conserved self-digesting process, has been deeply involved in the development and progression of oral squamous cell carcinoma (OSCC). However, the prognostic value of autophagy-related genes (ARGs) for OSCC still remains unclear. Our study set out to develop a multigene expression signature based on ARGs for individualized prognosis assessment in OSCC patients.MethodsBased on The Cancer Genome Atlas (TCGA) database, we identified prognosis-related ARGs through univariate COX regression analysis. Then we performed the least absolute shrinkage and selection operator (LASSO) regression analysis to identify an optimal autophagy-related multigene signature with the subsequent validation in testing set, GSE41613 and GSE42743 datasets.ResultsWe identified 36 prognosis-related ARGs for OSCC. Subsequently, the multigene signature based on 13 prognostic ARGs was constructed and successfully divided OSCC patients into low and high-risk groups with significantly different overall survival in TCGA training set (p

Aerobic glycolysis is the main pathway for energy metabolism in cancer cells. It provides energy and biosynthetic substances for tumor progression and metastasis by increasing lactate production. Lactate dehydrogenase A (LDHA) promotes glycolysis process by catalyzing the conversion of pyruvate to lactate. Despite LDHA exhibiting carcinogenesis in various cancers, its role in oral squamous cell carcinoma (OSCC) remains unclear. This study demonstrated that LDHA was over-expressed in both OSCC tissues and cell lines, and was significantly associated with lower overall survival rates in patients with OSCC. Using weighted gene correlation network analysis and gene set enrichment analysis for the gene expression data of patients with OSCC (obtained from The Cancer Genome Atlas database), a close association was identified between epithelial–mesenchymal transition (EMT) and LDHA in promoting OSCC progression. The knockdown of LDHA suppressed EMT, cell proliferation, and migration and invasion of OSCC cells in vitro. Moreover, the silencing of LDHA inhibited tumor growth in vivo. Oxamate, as a competitive LDHA inhibitor, was also suppressed diverse malignant biocharacteristics of OSCC cells. Our findings reveal that LDHA acts as an oncogene to promote malignant progression of OSCC by facilitating glycolysis and EMT, and LDHA may be a potential anticancer therapeutic target.

Postoperative pneumonia (POP) is common and results in prolonged hospital stays, higher costs, increased morbidity and mortality. However, data on the incidence and risk factors of POP after oral and maxillofacial surgery are rare. This study aims to identify perioperative risk factors for POP after major oral cancer (OC) surgery.Perioperative data and patient records of 331 consecutive subjects were analyzed in the period of April 2014 to March 2016. We individually traced each OC patient for a period to discharge from the hospital or 45 days after surgery, whichever occur later.The incidence of POP after major OC surgery with free flap construction or major OC surgery was 11.6% or 4.5%, respectively. Patient-related risk factors for POP were male sex, T stage, N stage, clinical stage and preoperative serum albumin level. Among the investigated procedure-related variables, incision grade, mandibulectomy, free flap reconstruction, tracheotomy, intraoperative blood loss, and the length of the operation were shown to be associated with the development of POP. Postoperative hospital stay was also significantly related to increased incidence of POP. Using a multivariable logistic regression model, we identified male sex, preoperative serum albumin level, operation time and postoperative hospital stay as independent risk factors for POP.Several perioperative risk factors can be identified that are associated with POP. At-risk oral cancer patients should be subjected to intensified postoperative pulmonary care.

N6-methyladenosine (m6A) plays crucial roles in tumorigenesis and autophagy. However, the underlying mechanisms mediated by m6A and autophagy in the malignant progression of oral squamous cell carcinoma (OSCC) remain unclear. In the present study, we revealed that down-regulated expression of METTL14 was correlated with advanced clinicopathological characteristics and poor prognosis in OSCC. METTL14 knockdown significantly inhibited autophagy and facilitated malignant progression in vitro, and promoted tumor growth and metastasis in vivo. A cell model of rapamycin-induced autophagy was established to identify RB1CC1 as a potential target gene involved in m6A-regulated autophagy in OSCC, through RNA sequencing and methylated RNA immunoprecipitation sequencing (meRIP-seq) analysis. Mechanistically, we confirmed that METTL14 posttranscriptionally enhanced RB1CC1 expression in an m6A-IGF2BP2-dependent manner, thereby affecting autophagy and progression in OSCC, through methylated RNA immunoprecipitation qRT-PCR (meRIP-qPCR), RNA stability assays, mutagenesis assays and dual-luciferase reporter. Collectively, our findings demonstrated that METTL14 serves as an OSCC suppressor by regulating the autophagy-related gene RB1CC1 through m6A modification, which may provide a new insight for the diagnosis and therapy of OSCC. [ABSTRACT FROM AUTHOR]

Chemerin, which is encoded by retinoic acid receptor responder 2 (RARRES2), has been found to be related to malignant tumours, but its role in the development of oral squamous cell carcinoma (OSCC) is largely unexplored. In the present study, a higher serum level of chemerin was evident in patients with OSCC than in healthy individuals, and this high level of chemerin significantly decreased after tumour resection. In addition, high chemerin levels were positively associated with advanced tumour stage and lymph node metastasis. The expression levels of chemerin and Chemerin Receptor 23 (ChemR23) were positively correlated with the migration and invasion of OSCC cell lines. Recombinant chemerin (R-chemerin) enhanced the in vitro migration, invasion and proliferation of OSCC cells in a concentration-dependent manner, and short hairpin RNAs (shRNAs) targeting RARRES2 decreased chemerin expression and inhibited OSCC cell metastasis and proliferation both in vitro and in vivo. Additionally, R-chemerin activated manganese superoxide dismutase (SOD2) and increased the amount of intracellular hydrogen peroxide (H2O2), leading to a significant decrease in E-cadherin expression and dramatic increase in the expression of phosphorylated ERK1/2 (p-ERK1/2), Slug, Vimentin and N-cadherin, but shRNAs targeting RARRES2 reversed these effects. Moreover, knockdown of ChemR23 with small interfering RNAs (siRNA) significantly inhibited chemerin-induced OSCC cell migration/invasion and SOD2 activity. Our results revealed that chemerin is a novel biomarker for OSCC. Chemerin/ChemR23 promotes tumorigenesis and metastasis in OSCC and may be a new therapeutic target for OSCC. [ABSTRACT FROM AUTHOR]

Reconstruction of the tongue after glossectomy is a primary objective in the process of improving the life quality of patients suffering from tongue cancer. We developed a new method of surgical reconstruction of the tongue after hemiglossectomy, with reinnervated rectus abdominis musculoperitoneal flaps. The rectus abdominis musculoperitoneal flap consisted of the rectus muscle, posterior rectus sheath, peritoneum, the 10th, 11th and 12th intercostal nerves, and the deep inferior epigastric artery and veins. Five patients underwent immediate reconstruction of the tongue with rectus abdominis musculoperitoneal flaps after hemiglossectomy. The intercostal nerves were anastomosed to the descending branch of the hypoglossal nerve. All transplanted flaps survived. The peritoneum was replaced by squamous epithelium eight weeks after surgery. During the follow-up period, the contour and function of the reconstructed tongues was satisfactory. Our experience indicates that reconstruction of the tongue with rectus abdominis musculoperitoneal flaps after hemiglossectomy is a suitable, cosmetically acceptable method. [ABSTRACT FROM AUTHOR]