CCDC71L as a novel prognostic marker and immunotherapy target via lipid metabolism in head and neck squamous cell carcinoma.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is the sixth most widespread cancer globally with high rate and poor prognosis. Coiled-coil domain containing 71 like (CCDC71L) exerts an important role in cellular lipid metabolic process. However, its function in HNSCC remains unclear. To this end, we examined the CCDC71L implications for prognosis and tumor microenvironment in HNSCC. MATERIALS AND METHODS: First, CCDC71L expression was explored through The Cancer Genome Atlas (TCGA), Human Protein Atlas (HPA), the Gene Expression Profiling Interactive Analysis 2 (GEPIA2), Clinical Proteomic Tumor Analysis Consortium (CPTAC) and the Gene Expression Omnibus (GEO) databases. The clinicpathological information were obtained from the dataset of TCGA. The Kaplan-Meier Plotter databases and Cox model were performed for the determination of prognostic values of CCDC71L, including the overall survival (OS), progress free interval (PFI), recurrence-free survival (RFS) and disease specific survival (DSS). Then, the potential mechanism of CCDC71L in HNSCC development was elucidated by means of Gene set enrichment analysis (GSEA) and Metascape databases. Furthermore, the relevance of CCDC71L to immune cells infiltration and immune checkpoints was assessed. The correlations among CCDC71L expression, mutational landscape and genome heterogeneity [mutant-allele tumor heterogeneity (MATH) and tumor purity] were detected by the data in TCGA. RESULTS: CCDC71L expression was significantly upregulated in HNSCC, and positively associated with age, gender and N stage. Higher CCDC71L expression resulted in poor OS, RFS, DSS and PFI. Multivariate Cox regression analysis showed CCDC71L would be an independent prognostic marker in patients with HNSCC. Moreover, CCDC71L and the level of macrophage and neutrophil cells infiltration were significantly correlated in HNSCC. High expression of CCDC71L was related to immune checkpoint genes, oncogene mutations and genome heterogeneity markers. CONCLUSION: These results implied that CCDC71L plays vital roles in HNSCC progression, which could be used as a underlying biomarker for the diagnosis and prognosis of HNSCC. Meanwhile, CCDC71L participates in immune regulation, which has a potential value for the immunotherapy of HNSCC patients.

circYap inhibits oral squamous cell carcinoma by arresting cell cycle.

OBJECTIVE: Circular RNAs (circRNAs) involve in the development and progression of tumour. The mechanism of circRNAs in oral squamous cell carcinoma (OSCC) has remained unclear. This study aimed to investigate the role of circular Yes-associated protein (circYap) in OSCC. METHODS: Quantification reverse transcription-polymerase chain reaction (qRT-PCR) was applied to measure circYap expression in patients with OSCC tissues and cells. Flow cytometry was performed to evaluate cell cycle. circYap interaction with CDK4 was detected by RNA immunoprecipitation (RIP) and RNA pull-down. The interaction of Cyclin D1 and CDK4 was determined using co-immunoprecipitation (co-IP). RESULTS: We showed that circYap expression was downregulated in OSCC tissues. Using small interfering circular (Si-circYap) and overexpression plasmid, we found that circYap overexpression inhibited proliferation and arrested cell cycle in OSCC cells, while, circYap knockdown yielded the opposite result. Cyclin D1/CDK4 complexes and nuclear translocation is essential for cell cycle progression. We found that CDK4 interacted with circYap was increased when circYap overexpression, meanwhile, Cyclin D1/CDK4 complexes and of nuclear distribution were decreased. CONCLUSIONS: Our findings suggest that circYap impedes progression of OSCC. Overexpression of circYap suppresses proliferation and cell cycle through binding to CDK4 to block formation and nuclear translocation of Cyclin D1/CDK4 complexes. Thus, circYap may serves as a valuable therapeutic target for OSCC.

Knockdown of microRNA-584 promotes dental pulp stem cells proliferation by targeting TAZ.

Proliferation of dental pulp stem cells (DPSCs) is crucial in tooth development and damage repairing, also includes its therapy application for tissue engineering. MicroRNAs (miRNAs) are key players in biological processes of DPSCs, and transcriptional co-activator with PDZ-binding motif (TAZ) also plays important roles in cell proliferation and differentiation, however, the roles of miR-584 and TAZ in DPSCs are not known. We found up-regulated miR-584 expression and down-regulated TAZ expression levels in aging dental pulp tissue compare to those in young dental pulp tissue. In proliferating DPSCs we demonstrated the decreased miR-584 expression and increased TAZ expression. miR-584 mimics suppressed DPSCs proliferation and migration, and significantly reduced TAZ production, whereas miR-584 inhibition exerted the converse effects. Knocking down of the TAZ in DPSCs had a similar effect as overexpression of miR-584. Furthermore, luciferase reporter assay demonstrated that miR-584 could directly bind to the TAZ mRNA 3'UTR to repress its translation. Overexpression of TAZ can partly rescue miR-584 mimic-mediated the inhibition of proliferation. Additionally, miR-584 inhibited cell proliferation and downregulated expression of cell cycle proteins by AKT signaling pathway. Together, we identified that miR-584 may be a key regulator in the proliferation of DPSCs by regulating TAZ expression via AKT signaling pathway. It would be a promising biomarker and therapeutic target for pulp disease.

PECAM1 Combines With CXCR4 to Trigger Inflammatory Cell Infiltration and Pulpitis Progression Through Activating the NF-κB Signaling Pathway.

Pulpitis is a frequent bacterially driven inflammation featured with the local accumulation of inflammatory products in human dental pulps. A GEO dataset GSE16134 comprising data of inflamed dental pulp tissues was used for bioinformatics analyses. A protein-protein interaction (PPI) analysis suggested that chemokine receptor 4 (CXCR4) owned a high correlation with platelet endothelial cell adhesion molecule-1 (PECAM1). A rat model with pulpitis was established, and lipopolysaccharide (LPS)-induced human dental pulp fibroblasts (HDPFs) were used for in vitro experiments. Then, high expression of PECAM1 and CXCR4 was validated in the inflamed dental pulp tissues in rats and in LPS-induced HDPFs. Either downregulation of PECAM1 or CXCR4 suppressed inflammatory cell infiltration in inflamed tissues as well as the inflammation and apoptosis of HDPFs. A transcription factor myocyte-enhancer factor 2 (MEF2C) was predicted and validated as a positive regulator of either PECAM1 or CXCR4, which activated the NF-κB signaling pathway and promoted pulpitis progression. To sum up, this study suggested that MEF2C transcriptionally activates PECAM1 and CXCR4 to activate the B-cell and NF-κB signaling pathways, leading to inflammatory cell infiltration and pulpitis progression.

Concentrated Growth Factor Promotes Dental Pulp Cells Proliferation and Mineralization and Facilitates Recovery of Dental Pulp Tissue.

BACKGROUND Dental pulp cells (DPCs) play vital roles in the recovery of dental pulp tissue. Concentrated growth factor (CGF) can promote proliferation and mineralization of various cells. However, the functions of CGF on DPCs and dental pulp tissue are unclear. The object of our study was to identify the roles of CGF in DPCs proliferation and mineralization in vitro and to assess the effects of CGF on direct pulp capping in vivo. MATERIAL AND METHODS We performed CCK-8 and Transwell assay to detect proliferation and migration activity of DPCs. Alizarin Red staining was performed to examine mineralized nodules. Alkaline phosphatase activity test was used to measure the mineralization capacity of DPCs. We assessed the odontogenic differentiation gene expression level by Western blot and qPCR. The effect of CGF on direct pulp capping in vivo were evaluated by radiography and histopathology. RESULTS CGF increased the number of proliferative and migratory DPCs. CGF enhanced DPCs mineralized nodules and improved the gene expression levels of DSPP, DMP-1, BSP, and ALP. CGF upregulated the protein levels of ALP, BMP2, SMAD5, Runx2, and p-Smad, and the effect could be partially reversed by Noggin. CGF promoted pulp recovery and kept its vitality in directly pulp capping. CONCLUSIONS CGF promotes DPCs proliferation and mineralization. It regulates the mineralization of DPCs via the BMP2/SMAD5/Runx2 signaling pathway. CGF can be used as the effective graft for direct pulp capping.

Characterization of different biodegradable scaffolds in tissue engineering.

The aim of the present study was to compare the characteristics of acellular dermal matrix (ADM), small intestinal submucosa (SIS) and Bio­Gide scaffolds with acellular vascular matrix (ACVM)­0.25% human­like collagen I (HLC­I) scaffold in tissue engineering blood vessels. The ACVM­0.25% HLC­I scaffold was prepared and compared with ADM, SIS and Bio­Gide scaffolds via hematoxylin and eosin (H&E) staining, Masson staining and scanning electron microscope (SEM) observations. Primary human gingival fibroblasts (HGFs) were cultured and identified. Then, the experiment was established via the seeding of HGFs on different scaffolds for 1, 4 and 7 days. The compatibility of four different scaffolds with HGFs was evaluated by H&E staining, SEM observation and Cell Counting Kit­8 assay. Then, a series of experiments were conducted to evaluate water absorption capacities, mechanical abilities, the ultra­microstructure and the cytotoxicity of the four scaffolds. The ACVM­0.25% HLC­I scaffold was revealed to exhibit the best cell proliferation and good cell architecture. ADM and Bio­Gide scaffolds exhibited good mechanical stability but cell proliferation was reduced when compared with the ACVM­0.25% HLC­I scaffold. In addition, SIS scaffolds exhibited the worst cell proliferation. The ACVM­0.25% HLC­I scaffold exhibited the best water absorption, followed by the SIS and Bio­Gide scaffolds, and then the ADM scaffold. In conclusion, the ACVM­0.25% HLC­I scaffold has good mechanical properties as a tissue engineering scaffold and the present results suggest that it has better biological characterization when compared with other scaffold types.

Cytocompatibility and biologic characteristics of synthetic scaffold materials of rabbit acellular vascular matrix combining with human-like collagen I.

Scaffold material provides a three-dimensional growing environment for seed cells in the research field of tissue engineering. In the present study, rabbit arterial blood vessel cells were chemically removed with trypsin and Triton X-100 to prepare rabbit acellular vascular matrix scaffold material. Observation by He&Masson staining revealed that no cellular components or nuclei existed in the vascular intima and media after decellularization. Human-like collagen I was combined with acellular vascular matrix by freeze-drying to prepare an acellular vascular matrix-0.25% human-like collagen I scaffold to compensate for the extracellular matrix loss during the decellularization process. We next performed a series of experiments to test the water absorbing quality, biomechanics, pressure resistance, cytotoxicity, and ultra-micro structure of the acellular vascular matrix composite material and natural rabbit artery and found that the acellular vascular matrix-0.25% human-like collagen I material behaved similarly to natural rabbit artery. In conclusion, the acellular vascular matrix-0.25% human-like collagen I composite material provides a new approach and lays the foundation for novel scaffold material research into tissue engineering of blood vessels.

Effects of TAZ on human dental pulp stem cell proliferation and migration.

Transcriptional coactivator with PDZ­binding motif (TAZ) acts as the key downstream regulatory target in the Hippo signaling pathway. TAZ overexpression has been reported to promote cellular proliferation and induce epithelial­mesenchymal transition in human mammary epithelial cells. However, the effects of TAZ in the regulation of human dental pulp stem cell (hDPSC) proliferation and migration, as well as the molecular mechanisms underlying its actions, remain to be elucidated. The present study demonstrated that TAZ was expressed in hDPSCs. TAZ silencing, following hDPSC transfection with TAZ­specific small interfering (si)RNA (siTAZ), inhibited cellular proliferation and migration in vitro. These effects appeared to be associated with the downregulation of connecting tissue growth factor (CTGF) and cysteine­rich angiogenic inducer (Cyr) 61 expression. Further investigation of the mechanisms underlying the actions of TAZ in hDPSCs revealed that TAZ silencing suppressed CTGF and Cyr61 expression by interfering with transforming growth factor (TGF)­ß signaling pathways. The present results suggested that TAZ may be implicated in the proliferation and migration of hDPSCs, through the modulation of CTGF and Cyr61 expression via a TGF­ß­dependent signaling pathway.

Hypermethylation of IFN-γ in oral cancer tissues.

OBJECTIVES: The study aimed to evaluate the methylation pattern of the interferon-gamma (IFN-γ) gene in oral cancer tissues compared with normal and benign oral disease tissues. MATERIALS AND METHODS: The oral tissues were gained from the patients of 85 cases of oral squamous cell carcinoma (OSCC), 47 cases of oral dysplastic lesions, and 53 normal biopsies. IFN -γ methylation in oral tissues was verified through methylation-specific polymerase chain reaction (PCR) and DNA sequencing analyses, and the expression levels of IFN-γ messenger RNA (mRNA) and protein were detected using real-time reverse transcription (RT)-PCR and enzyme-linked immunosorbent assays, respectively. IFN-γ was localized in macrophages from oral tissues and detected via immunostaining. RESULTS: IFN-γ mRNA and protein expression levels were evidently decreased in oral cancer tissues, whereas the IFN-γ methylation rate was significantly higher in malignant tumors than in benign and normal tissues (normal, 22.6%; benign, 38.3%; and cancer, 55.3%; P < 0.05). Furthermore, the expression of IFN-γ mRNA was significantly downregulated in oral tumors with methylation compared with tumors without methylation, as determined by real-time RT-PCR (4.76-fold difference; P < 0.05). Likewise, mRNA expression was downregulated by 6.79-fold in oral epithelial dysplasia tissues with methylation compared with those without methylation (P < 0.01). Co-immunostaining to detect MAC2 and IFN-γ demonstrated that macrophages comprised the main source of IFN-γ in oral tissues. IFN-γ methylation demonstrated a significant association with the clinical stage, histopathology grade, and primary tumor. CONCLUSIONS: Aberrant IFN-γ promoter methylation may be involved in the process of tumorigenesis of oral cancer. CLINICAL RELEVANCE: IFN-γ hypermethylation during the process of oral carcinogenesis could be useful for the clinical diagnosis and treatment for OSCC.