GAD1-mediated GABA elicits aggressive characteristics of human oral cancer cells.

Studies suggest that the expression of glutamate decarboxylase 1 (GAD1), γ-aminobutyric acid (GABA), and GABA receptors are involved in tumor progression. However, the underlying mechanisms of high expression and potential functions of GAD1 and GABA in oral squamous cell carcinoma (OSCC) are not known. In this study, we found that the expressions of GAD1 and GABA were considerably increased in OSCC samples, which were closely associated with clinical stage and lymph node metastasis. The knockdown of GAD1 expression significantly inhibited the proliferation, migration and invasion abilities of OSCC cells by reducing the expression of GABA-mediated GABAB receptors, which could be reversed by exogenous GABA, but did not cause excessive OSCC cell proliferation. And GABA secreted by OSCC cells promoted M2 macrophage polarization for inhibiting anti-tumor immunity by activating GABBR1/ERK/Ca2+. In addition, GABA/GABABR promoted the proliferation and progression of OSCC xenograft tumor. Altogether, our results showed that GAD1 synthetized GABA to promote the malignant progression of OSCC and limits the anti-tumor immunity of macrophages, thereby targeting GABA can be a novel strategy for treating OSCC.

CiRS-7 functions as a ceRNA of RAF-1/PIK3CD to promote metastatic progression of oral squamous cell carcinoma via MAPK/AKT signaling pathways.

PURPOSE: Recent findings have shown that circRNA dysregulation was involved in the development of many types of cancer. However, our knowledge of circRNA in oral squamous cell carcinoma (OSCC) remains elusive. METHODS: Here, we explored whether ciRS-7 could function as a ceRNA in promoting metastasis of OSCC via regulating miR-7 activity. The expression levels of ciRS-7 and miR-7 were examined in clinical samples and cell lines by qRT-PCR, and the effects of ectopic expression of ciRS-7 and miR-7 on cell proliferation, migration and invasion were assessed in vitro and in vivo. The effects of ciRS-7 on miR-7 activity were investigated by means of luciferase reporter assay, qRT-PCR and Western blot. In addition, the effects of miR-7 mediated ciRS-7 on the levels of MAPK/AKT signaling proteins were evaluated by Western blot. RESULTS: We found that ciRS-7 was highly expressed in OSCC tissues and cell lines compared with normal counterparts. Ectopic expression of ciRS-7 significantly promoted OSCC cell proliferation, migration and invasion through in vitro and in vivo. Based on bioinformatics analysis, qRT-PCR, Western blot and luciferase reporter assays, we determined that ciRS-7 functioned as a sponge for miR-7, resulting in attenuation of miR-7 targets RAF-1 and PIK3CD, which are core components of the MAPK/AKT signaling pathways. Moreover, miR-7 correlated with perineural and lymphovascular invasion in OSCC patients. Further experiments demonstrated that ciRS-7 overexpression could attenuate the anti-tumor effects of miR-7 on OSCC cells. CONCLUSIONS: Our results suggested that ciRS-7 can interact directly with miR-7, resulting in upregulation of RAF-1/PIK3CD expression and enhancing metastatic progression of OSCC.

Suppression of OSCC malignancy by oral glands derived-PIP identified by iTRAQ combined with 2D LC-MS/MS.

Oral squamous cell carcinoma (OSCC) is the most common malignancy in head and neck cancer and a global cause of cancer-related death. Due to the poor survival rates associated with OSCC, there is a growing need to develop novel technologies and predictive biomarkers to improve disease diagnosis. The identification of new cellular targets in OSCC tumors will benefit such developments. In this study, isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomics analysis combined with 2-dimensional liquid chromatography and tandem mass spectrometry (2D LC-MS/MS) were used to identify differentially expressed proteins (DEPs) between tumor and normal tissues. Of the DEPs detected, the most significantly downregulated protein in OSCC tissue was prolactin-inducible protein (PIP). Clonogenic and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) experiments showed that the proliferation capacity of OSCC cells overexpressing PIP decreased due to cell cycle arrest at the G0/G1 checkpoint. Wound-healing and transwell assay further showed that PIP overexpression also reduced the migration and invasion of OSCC cells. Immunohistochemistry (IHC) was used to analyze the expression in OSCC, indicating that PIP may be secreted by glandular cells and have an inhibitory effect on OSCC cells to produce. In western blot analysis, silencing studies confirmed that PIP mediates these effects through the AKT/mitogen-activated protein kinase (MAPK) signaling axis in OSCC cells. Taken together, this study reveals PIP as a key mediator of OSCC cell growth, migration, and invasion through its effect on AKT/MAPK signaling.

circ-PKD2 inhibits carcinogenesis via the miR-204-3p/APC2 axis in oral squamous cell carcinoma.

Recent findings have shown that dysregulation of circular RNAs (circRNAs) is implicated in various cancers. However, the contribution of circRNAs in oral squamous cell carcinoma (OSCC) remains largely unexplored. We screened circRNA expression profiles using a circRNA microarray in paired OSCC and normal tissues and explored the clinical significance of a downregulated circRNA, circ-PKD2. Moreover, the biological function of circ-PKD2 in OSCC was investigated both in vitro and in vivo. We found that downregulation of circ-PKD2 in OSCC correlated significantly with aggressive characteristics. Further analysis revealed that overexpression of circ-PKD2 inhibited OSCC cell proliferation, migration and invasion, induced apoptosis and cell cycle arrest, which were promoted by knockdown of circ-PKD2. In addition, circ-PKD2 was identified as a sponge for miR-204-3p and upregulated the expression of adenomatous polyposis coli 2 (APC2), which was the functional target of miR-204-3p. Moreover, circ-PKD2 attenuated the oncogenic effects of miR-204-3p-mediated APC2 on OSCC progression via multiple signaling pathways. These results demonstrate that the circ-PKD2/miR-204-3p/APC2 axis represents a novel pathway involved in the pathogenesis of OSCC and may serve as a novel therapeutic target of OSCC.

Decreased expression of hsa_circ_0072387 as a valuable predictor for oral squamous cell carcinoma.

OBJECTIVE: Increasing evidence points toward the key function of circular RNAs (circRNAs) in various carcinomas. This study aimed to identify aberrant expression of hsa_circ_0072387 in oral squamous cell carcinoma and probe its clinical significance. MATERIALS AND METHODS: Real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to assess hsa_circ_0072387 expression levels in 63 paired OSCC tissues and three OSCC cell lines. The area under the receiver operator characteristic (ROC) curve was plotted to assess any potential clinical significance. RESULTS: Our data showed that hsa_circ_0072387 expression in OSCC was significantly downregulated compared with adjacent normal tissues (p < 0.001). Compared to human normal oral keratinocyte cell, the levels of hsa_circ_0072387 were lower in three OSCC cell lines (SCC25, SCC15, CAL27). More significantly, hsa_circ_0072387 expression was associated with the TNM stage in OSCC (p = 0.050). The area under the ROC curve reached up to 0.746. Based on bioinformatics, hsa-miR-129-3p, hsa-miR-141-3p, and hsa-miR-29-3p were predicted to be potential miRNAs binding with hsa_circ_0072387. Furthermore, hsa-miR-129-3p, hsa-miR-141-3p, and hsa-miR-29-3p were involved in multiple tumor-related signaling pathways. CONCLUSION: Our finding suggested that lower expression of has_circ_0072387 could be a key circRNA in OSCC and serve as a potential biomarker in OSCC diagnosis and therapeutic targets.

Hepatotoxicity and lipid metabolism disorders of 8:2 polyfluoroalkyl phosphate diester in zebrafish: In vivo and in silico evidence.

8:2 polyfluoroalkyl phosphate diester (8:2 diPAP) has been shown to accumulate in the liver, but whether it induces hepatotoxicity and lipid metabolism disorders remains largely unknown. In this study, zebrafish embryos were exposed to 8:2 diPAP for 7 d. Hepatocellular hypertrophy and karyolysis were noted after exposure to 0.5 ng/L 8:2 diPAP, suggesting suppressed liver development. Compared to the water control, 8:2 diPAP led to significantly higher triglyceride and total cholesterol levels, but markedly lower levels of low-density lipoprotein, implying disturbed lipid homeostasis. The levels of two peroxisome proliferator activated receptor (PPAR) subtypes (pparα and pparγ) involved in hepatotoxicity and lipid metabolism were significantly upregulated by 8:2 diPAP, consistent with their overexpression as determined by immunohistochemistry. In silico results showed that 8:2 diPAP formed hydrogen bonds with PPARα and PPARγ. Among seven machine learning models, Adaptive Boosting performed the best in predicting the binding affinities of PPARα and PPARγ on the test set. The predicted binding affinity of 8:2 diPAP to PPARα (7.12) was higher than that to PPARγ (6.97) by Adaptive Boosting, which matched well with the experimental results. Our results revealed PPAR - mediated adverse effects of 8:2 diPAP on the liver and lipid metabolism of zebrafish larvae.

Probing the bioconcentration and metabolism disruption of bisphenol A and its analogues in adult female zebrafish from integrated AutoQSAR and metabolomics studies.

Plenty of emerging bisphenol A (BPA) substitutes rise to wait for assessment of bioconcentration and metabolism disruption. Computational methods are useful to fill the data gap in chemical risk assessment, such as automated quantitative structure-activity relationship (AutoQSAR). It is not clear how AutoQSAR performs in predicting the bioconcentration factor (BCF) in adult zebrafish. Herein, AutoQSAR was used to predict the logBCFs of BPA, bisphenol AF (BPAF), bisphenol B, bisphenol F and bisphenol S (BPS). For the test set, a linear relationship was shown between the observed and predicted logBCFs with a slope of 0.97. The predicted logBCFs of these five bisphenols were quite close to their experimental data with a slope of 0.94, suggesting better performance than directed message passing neural networks and EPI Suite with a slope of 0.69 and 0.61, respectively. Thus, AutoQSAR is powerful in modeling logBCFs in fish with minimal time and expertise. To link bioconcentration with metabolic effects, female zebrafish were exposed to BPA, BPAF and BPS for metabolomics analysis. BPA caused a significant disturbance in amino acid metabolism, while BPAF and BPS significantly altered another three metabolic pathways, showing chemical-specific responses. BPAF with the highest logBCF elicited the strongest metabolomic responses reflected by the metabolic effect level index, followed by BPA and BPS. Thus, BPAF and BPS elicited higher or similar metabolism disruption compared with BPA in female zebrafish, respectively, reflecting consequences of bioconcentration.

The RBP1-CKAP4 axis activates oncogenic autophagy and promotes cancer progression in oral squamous cell carcinoma.

Retinol-binding protein 1 (RBP1) is involved in several physiological functions, including the regulation of the metabolism and retinol transport. Studies have shown that it plays an important role in the pathogenesis of several types of cancer. However, the role of RBP1 and its correlation with autophagy in oral squamous cell carcinoma (OSCC) pathogenesis remain unknown. In this study, RBP1 was identified as the most significantly upregulated DEPs with a >2-fold change in OSCC samples when compared to normal tissues through iTRAQ-based proteomics analysis coupled with 2D LC-MS/MS. RBP1 overexpression was significantly associated with malignant phenotypes (differentiation, TNM stage, and lymphatic metastasis) of OSCC. In vitro experiments demonstrated that RBP1 was significantly increased in OSCC tissues and cell lines compared with control group. RBP1 overexpression promoted cell growth, migration, and invasion of OSCC cells. Silencing of RBP1 suppressed tumor formation in xenografted mice. We further demonstrated that the RBP1-CKAP4 axis was a critical regulator of the autophagic machinery in OSCC, inactivation of autophagy rescued the RBP1-CKAP4-mediated malignant biological behaviors of OSCC cells. Overall, a mechanistic link was provided by RBP1-CKAP4 between primary oncogenic features and the induction of autophagy, which may provide a potential therapeutic target that warrants further investigation for treatment of OSCC.

CircCDR1as upregulates autophagy under hypoxia to promote tumor cell survival via AKT/ERK½/mTOR signaling pathways in oral squamous cell carcinomas.

Autophagy, as an important non-selective degradation mechanism, could promote tumor initiation and progression by maintaining cellular homeostasis and the cell metabolism as well as cell viability. CircCDR1as has been shown to function as an oncogene in cancer progression, however, it remains largely unknown as to how autophagy is regulated by circCDR1as in oral squamous cell carcinoma (OSCC). In this study, we validated the functional roles of circCDR1as in regulation of autophagy in OSCC cells and further investigated how circCDR1as contributed to cell survival via up-regulating autophagy under a hypoxic microenvironment by using combination of human tissue model, in vitro cell experiments and in vivo mice model. We found that hypoxia promoted the expression level of circCDR1as in OSCC cells and elevated autophagy. In addition, circCDR1as further increased hypoxia-mediated autophagy by targeting multiple key regulators of autophagy. We revealed that circCDR1as enhanced autophagy in OSCC cells via inhibition of rapamycin (mTOR) activity and upregulation of AKT and ERK½ pathways. Overexpression of circCDR1as enhanced OSCC cells viability, endoplasmic reticulum (ER) stress, and inhibited cell apoptosis under a hypoxic microenvironment. Moreover, circCDR1as promoted autophagy in OSCC cells by sponging miR-671-5p. Collectively, these results revealed that high expression of circCDR1as enhanced the viability of OSCC cells under a hypoxic microenvironment by promoting autophagy, suggesting a novel treatment strategy involving circCDR1as and the inhibition of autophagy in OSCC cells.

Cyclosporine A Suppresses the Malignant Progression of Oral Squamous Cell Carcinoma in vitro.

BACKGROUND: The Oral Squamous Cell Carcinoma (OSCC) is one of the most frequent cancer types. Failure of treatment of OSCC is potentially lethal because of local recurrence, regional lymph node metastasis, and distant metastasis. Chemotherapy plays a vital role through suppression of tumorigenesis. Cyclosporine A (CsA), an immunosuppressant drug, has been efficiently used in allograft organ transplant recipients to prevent rejection, and also has been used in a subset of patients with autoimmunity related disorders. The present study aims to investigate novel and effective chemotherapeutic drugs to overcome drug-resistance in the treatment of OSCC. METHODS: Cells were incubated in the standard way. Cell viability was assayed using the MTT assay. Cell proliferation was determined using colony formation assay. The cell cycle assay was performed using flow cytometry. Apoptosis was assessed using fluorescence-activated cell sorting after stained by the Annexin V-fluorescein isothiocyanate (FITC). Cell migration and invasion were analyzed using wound healing assay and tranwell. The effect of COX-2, c-Myc, MMP-9, MMP-2, and NFATc1 protein expression was determined using Western blot analysis while NFATc1 mRNA expression was determined by RT-PCR. RESULTS: In vitro studies indicated that CsA inhibited partial OSCC growth by inducing cell cycle arrest, apoptosis, and the migration and invasion of OSCC cells. We also demonstrated that CsA could inhibit the expression of NFATc1 and its downstream genes COX-2, c-Myc, MMP-9, and MMP-2 in OSCC cells. Furthermore, we analyzed the expression of NFATc1 in head and neck cancer through the Oncomine database. The data was consistent with the experimental findings. CONCLUSION: The present study initially demonstrated that CsA could inhibit the progression of OSCC cells and can mediate the signal molecules of NFATc1 signaling pathway, which has strong relationship with cancer development. That explains us CsA has potential to explore the possibilities as a novel chemotherapeutic drug for the treatment of OSCC.

CBCT-based bone quality assessment in decompression of large odontogenic cystic lesions.

OBJECTIVES: Bone quality comprises bone mineral density and trabecular microstructure. The aim of this study was to explore the effectiveness of cone-beam computed tomography (CBCT) in evaluating bone quality of large odontogenic cystic lesions after decompression using CBCT and BoneJ software, and to determine whether secondary definitive surgery can be guided using CBCT data. METHODS: Twenty-seven patients with large odontogenic cystic lesions treated by decompression were evaluated by CBCT. Medical history and perioperative details were analyzed. RESULTS: The [Formula: see text]CT values for all patients with cystic lesions decreased after decompression, with no differences for age, sex, and histology (p > 0.05). Bone volume fraction and trabecular number of new cancellous bone (0.012%, 0.17/mm3) were lower than those of normal cancellous bone (0.189%, 0.47/mm3) (p < 0.05), while new cancellous bone trabecular separation (11.344 ± 2.556 mm) was stronger than normal cancellous bone trabecular separation (4.833 ± 2.232 mm) (p < 0.05). There were no differences in trabecular thickness between new cancellous bone (3.812 ± 1.593 mm) and normal cancellous bone (4.598 ± 3.573 mm) (p = 0.746). The [Formula: see text]CT values of five patients with favorable osteogenesis were - 72, -86, - 86, -47, and - 55, those of three patients with moderate osteogenesis were - 107, -120, and - 71, and those of two patients with poor osteogenesis were - 165 and - 127 during secondary definitive surgery. CONCLUSIONS: CBCT is considered beneficial for evaluating bone quality of large odontogenic cystic lesions after decompression, while providing potentially useful information for referral to secondary definitive surgery.

Virtual Planning and 3D printing modeling for mandibular reconstruction with fibula free flap

BACKGROUND: This study was to evaluate the use of virtual planning and 3D printing modeling in mandibular reconstruction and compare the operation time and surgical outcome of this technique with conventional method. MATERIAL AND METHODS: Between 2014 and 2017, 15 patients underwent vascularized fibula flap mandibular reconstruction using virtual planning and 3D printing modeling. Titanium plates were pre-bent using the models and cutting guides were used for osteotomies. 15 patients who underwent mandibular reconstruction using fibula flap without aid of virtual planning and 3D printing models were selected as control group. The operation time was recorded and compared in two groups. Accuracy of reconstruction was measured by superimposing the preoperative image onto the postoperative image of mandible. The selected bony landmark, distance and angle were measured. RESULTS: The mean total operation time and reconstruction time were 1.60±0.37 and 5.54±0.50 hours in computer-assisted group, respectively; These were 2.58±0.45 and 6.54±0.70 hours in conventional group, respectively. Both operation time and reconstruction time were shorter in computer-assisted group. The difference between the preoperative and postoperative intercondylar distances, intergonial angle distances, anteroposterior distances and gonial angles were 2.92±1.15 and 4.48±1.41mm, 2.93±1.19 and 4.79±1.48mm, 4.31±1.24 and 5.61±1.41mm, 3.85±1.68° and 5.88±2.12° in the computer-assisted and conventional group, respectively. The differences between the preoperative and postoperative mandible is smaller in the computer-assisted group. CONCLUSIONS: Virtual planning and 3D printing modeling have the potential to increase mandibular reconstruction accuracy and reduce operation time. we believe that this technology for mandibular reconstruction in selected patients will become a used method and improve the quality of reconstruction