Identification of autophagy-related biomarker and analysis of immune infiltrates in oral carcinoma.

BACKGROUND: Autophagy plays a vital role in the progression of the tumor. We aimed to investigate the expression, prognostic value, and immune infiltration of autophagy-related genes in oral carcinoma via bioinformatics analysis. METHODS: The microarray datasets (GSE146483 and GSE23558) of oral carcinoma were downloaded from Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) between normal and diseased groups were identified by the Limma package. The screened autophagy-related gene was further validated by the human protein atlas (HPA) database, TCGA database, and GSE78060 dataset. RESULTS: A total of 18 upregulated (top 10: EGFR, TNF, FADD, AURKA, E2F1, CHEK1, BRCA1, BIRC5, EIF2AK2, and CSF2) and 31 downregulated (top 10: MAP1LC3A, PARK2, AGT, IGF1, TP53INP1, CXCL12, IKBKB, SESN1, ULK2, and RRAGD) autophagy-related (DEGs) were identified, and FADD was found to be related to the prognosis of oral cancer patients. Gene set enrichment analysis indicated that FADD-associated genes were significantly enriched in immune-related pathways. Moreover, correlation analysis revealed that FADD expression was associated with immune infiltrates. Upregulation of FADD is associated with poor survival and immune infiltrates in oral cancer. CONCLUSION: We speculated that FADD is involved in the immune regulation of oral cancer, as well as autophagy.

Association of Decreased Expression of Serum miR-9 with Poor Prognosis of Oral Squamous Cell Carcinoma Patients.

BACKGROUND: Dysregulation of miR-9 is a common feature of many types of cancers, including oral squamous cell carcinoma (OSCC). However, whether the expression level of serum miR-9 is changed in patients with OSCC remains unknown. MATERIAL/METHODS: Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to examine the expression level of serum miR-9 in OSCC patients, oral leukoplakia (OLK) patients, and healthy volunteers, then we evaluated the association between serum miR-9 expression level and clinical outcome of OSCC patients. RESULTS: The expression level of serum miR-9 was significantly downregulated in patients with OSCC or OLK in comparison with healthy controls (P<0.01). Serum miR-9 expression level was associated with various clinicopathological parameters, including T stage (P=0.013), lymph node metastasis (P=0.002), and TNM stage (P=0.007). In addition, the OSCC patients in the low serum miR-9 expression group had poorer overall survival rate (P=0.022) and disease-free survival rate (P=0.004) compared with those in the high serum miR-9 expression group. Multivariate analysis showed that serum miR-9 was an independent prognostic factor for OSCC. CONCLUSIONS: Serum miR-9 was downregulated in patients with OSCC and patients with OLK. In addition, low serum miR-9 was correlated with poor prognosis of OSCC, indicating miR-9 might play a tumor suppressive role in OSCC and can serve as a promising biomarker for this deadly disease.

p16 and p53 can Serve as Prognostic Markers for Head and Neck Squamous Cell Carcinoma.

OBJECTIVE: The present study aimed to explore the expression and clinical significance of human papilloma virus-related pathogenic factors (p16, cyclin D1, p53) in patients with head and neck squamous cell carcinoma (HNSCC) and construct a predictive model. METHODS: The Cancer Genome Atlas was used to obtain clinical data for 112 patients with HNSCC. Expression of p16, p53, and cyclin D1 was quantified. We used the survival package of the R program to set the cut-off value. Values above the cut-off were considered positive, while values below the cut-off were negative. Kaplan-Meier analysis and univariate and multivariate Cox regression analyses were performed to investigate prognostic clinicopathological indicators and the expression of p16, p53, and cyclin D1. A predictive model was constructed based on the results of multifactor Cox regression analysis, and the accuracy of the predictive model was verified through final calibration analysis. Follow-up of patients with HNSCC at the Affiliated Hospital of Binzhou Medical University was conducted from 2015 to 2017, and reliability of the predictive model was validated based on follow-up data and molecular expression levels. RESULTS: According to the results, expression of p16 and p53 was significantly associated with prognosis (P < .05). The predictive model constructed based on the expression levels of p16 and p53 was useful for evaluating the prognosis of patients with HNSCC. The predictive model was validated using follow-up data obtained from the hospital, and the trend of the follow-up results was consistent with the predictive model. CONCLUSION: p16 and p53 can be used as key indicators to predict the prognosis of HNSCC patients and as critical immunohistochemical indicators in clinical practice. The survival model constructed based on p16 and p53 expression levels reliably predicts patient prognosis.

Proliferation-associated 2G4 P48 is stabilized by malignant T-cell amplified sequence 1 and promotes the proliferation of head and neck squamous cell carcinoma.

Background/purpose: Proliferation-associated protein 2G4 (PA2G4) has alternative transcriptional and translational initiation. One dominant transcript ENST00000303305 could be translated into two protein isoforms (PA2G4-P42 and PA2G4-P48). In this study, we aimed to explore the effects of PA2G4-P42 and PA2G4-P48 on the proliferation of head and neck squamous cell carcinoma (HNSCC) and the mechanisms regulating PA2G4-P48 stability. Materials and methods: HNSCC cell lines HSC2 and SCC25 with relatively low PA2G4 expression were used for in-vitro cell studies. PA2G4-P42 and PA2G4-P48 overexpression lentiviruses were generated. In vitro cell proliferation was assessed by CCK-8 and colony formation. In vivo tumor cell proliferation was assessed by HSC2 cell-derived xenograft tumors. Liquid chromatography-mass spectrometry (LC-MS)/MS and co-immunoprecipitation (co-IP) assays were applied to check PA2G4-P48 interacting partners. Cycloheximide (CHX) chase and ubiquitin-based co-IP assays were also performed. Results: PA2G4-P48 was the dominant isoform, with substantially higher expression than PA2G4-P42 in HNSCC. PA2G4-P48 overexpression enhanced HNSCC cell proliferation, but PA2G4-P42 overexpression slowed the proliferation. MCTS1 interacted with PA2G4-P48, but not PA2G4-P42. PA2G4 protein but not its mRNA expression was decreased in cells with MCTS1 knockdown. MG132 treatment abrogated this alteration. MCTS1 overexpression significantly elevated the half-life of PA2G4-P48, while its knockdown drastically reduced the half-life compared with the control cells. In addition, MCTS1 overexpression significantly decreased the polyubiquitination of exogenous flag-tagged PA2G4-P48. MCTS1 overexpression-induced cell proliferation was hampered by knocking down of PA2G4-P48. Conclusion: PA2G4-P42 and PA2G4-P48 exert growth-suppressive and growth-promoting effects in HNSCC, respectively. MCTS1 can interact with PA2G4-P48 and prolong its half-life by reducing its poly-ubiquitination.

SEC61 translocon subunit gamma enhances low-dose cisplatin-induced cancer-stem cell properties of head and neck squamous cell carcinoma via enhancing Ca2+-mediated autophagy.

Background/purpose: High SEC61 translocon subunit gamma (SEC61G) expression is associated with an unfavorable prognosis in patients with head and neck squamous cell carcinoma (HNSCC), but the underlying mechanisms remain poorly understood. Materials and methods: HNSCC representative cell lines SCC15 and CAL27 were used to explore the regulation of SEC61G on Ca2+ leak from the endoplasmic reticulum (ER). Ca2+-activated autophagy was monitored by fluorescent labeling of autophagosomes and western blotting assays. CSC marker expression, sphere formation, colony formation, and transwell of invasion were detected to investigate the role of SEC61G in regulating cancer-stem cell (CSC) properties. Results: Among the SEC61 complex genes, only SEC61G upregulation is consistently associated with unfavorable progression-free interval and disease-specific survival in patients with HNSCC. Low-dose cisplatin (CDDP) treatment induced SEC61G upregulation in SCC15 and CAL27 cells. SEC61G knockdown significantly impaired CDDP-induced Ca2+ from the ER and the phosphorylation of ERK1/2 and AMPK. CDDP-induced autophagy in HNSCC cells were hampered by SEC61G shRNA, in terms of impaired autophagosome formation, lowered LC3-II/GAPDH ratio and restored p62 expression. CDDP-induced CSC properties, including CSC marker expression, sphere formation, colony formation, and invasive capabilities could be suppressed by shSEC61G and chloroquine, a specific autophagy inhibitor. Conclusion: Findings of this study revealed the contribution of SEC61G in promoting cisplatin-induced CSC properties of HNSCC cells via promoting Ca2+-mediated autophagy.

[Effects of miR-31-5p on HIF-1α/BNIP3 signaling pathway and the expression of osteoblast-related factors of dental pulp stem cells].

PURPOSE: To investigate the effects of microRNA-31-5p (miR-31-5p) on the signal pathway of hypoxia inducible factor-1α (HIF-1α)/Bcl-2/adenovirus E1B 19-kDa-interacting protein 3(BNIP3) and the expression of osteoblast-related factors of dental pulp stem cells(DPSCs). METHODS: Human dental pulp stem cells (DPSCs) were cultured in vitro and divided into the control group (no transfection), mimic NC group (transfected with negative control-miR-31-5p), miR-31-5p mimic group (transfected with hsa-miR-31-5p mimic), siRNA NC group (transfected with nonsense siRNA) and miR-31-5p siRNA group (transfected with miR-31-5p siRNA).The expressions of miR-31-5p, HIF-1α, BNIP3, alkaline phosphatase(ALP) and Runt-related transcription factor-2(Runx2) mRNA in DPSCs were detected by real-time fluorescence quantitative PCR; the proliferation of DPSCs was detected by MTT; ALP activity of DPSCs was detected by ALP activity test kit; and the protein expressions of HIF-1α, BNIP3 and Runx2 in DPSCs were detected by Western blot. Statistical analysis was carried out with SPSS 24.0 software package. RESULTS: Compared with the control group and mimic NC group, the A value, ALP mRNA expression level and activity, Runx2 mRNA and protein expression levels of DPSCs in miR-31-5p mimic group were significantly lower (P＜0.05), ALP staining decreased significantly, and the expression levels of miR-31-5p mRNA, HIF-1α, BNIP3 mRNA and HIF-1α, BNIP3, Beclin1 protein were significantly higher (P＜0.05). Compared with the control group and siRNA NC group, the A value, ALP mRNA expression level and activity, Runx2 mRNA and protein expression levels of DPSCs in miR-31-5p siRNA group were significantly higher (P＜0.05), ALP staining enhanced significantly, and the expression levels of miR-31-5p mRNA, HIF-1α, BNIP3 mRNA and HIF-1α, BNIP3, Beclin1 protein were significantly lower(P＜0.05). CONCLUSIONS: MiR-31-5p may inhibit the expression of osteoblast-related factors of DPSCs, and activating HIF-1α/BNIP3 signaling pathway.

CircRNA HIPK3 promotes the progression of oral squamous cell carcinoma through upregulation of the NUPR1/PI3K/AKT pathway by sponging miR-637.

BACKGROUND: To investigate the expression, function, and related mechanisms of circHIPK3 in oral squamous cell carcinoma (OSCC). METHODS: CircHIPK3 expression was determined by quantitative reverse transcription polymerized chain reaction (QRT-PCR) in OSCC and adjacent tissues, and the correlation between the circHIPK3 level and clinicopathological indexes of OSCC was analyzed. CircHIPK3 expressions in different OSCC cell lines were detected, cell counting kit-8 (CCK-8) and 5-bromodeoxyuridine (BrdU) assays were utilized to monitor cell proliferation and activity. Flow cytometry was adopted to detect apoptosis and transwell assay was used to detect cell invasion. The expressions of nuclear protein 1 (NUPR1), phosphatidylinositol 3 kinase (PI3K)/protein kinase B (AKT) (PI3K/AKT) pathway proteins, and E-cadherin, Vimentin, and N-cadherin markers of epithelial-mesenchymal transformation (EMT) were detected by Western blot or Quantitative Real-time PCR (QRT-PCR). RESULTS: Upregulated circHIPK3 was noted in OSCC tissues (compared with adjacent tissues), and its overexpression was related to OSCC size and histopathological grade. Functionally, overexpressed circHIPK3 can significantly promote EMT, proliferation, and invasion of OSCC cells and can inhibit cell apoptosis in vivo and in vitro. In addition, CircHIPK3 upregulated the activation of NUPR1 and PI3K/AKT. Bioinformatics analyses showed that miR-637 was the common target of circHIPK3 and NUPR1, while a dual luciferase reporting assay and RIP assay further demonstrated that circHIPK3 targeted miR-637 and bound to 3' UTR of NUPR1. CONCLUSIONS: CircHIPK3 demonstrates potential as a prognostic marker of OSCC and mediates OSCC progression via the miR-637-mediated NUPR1/PI3K/AKT axis.

VCAM1-targeted RNA interference inhibits the proliferation of human oral squamous carcinoma HN12 cells.

In the present study, RNA interference (RNAi) was used to investigate the effect of vascular cell adhesion molecule 1 (VCAM1) silencing on the proliferation of human oral squamous carcinoma HN12 cells. HN12 cells were divided into three groups: The untreated blank control cell group (CK), the negative control group transfected with non-homologous vector (NC) and the positive group transfected with the target sequence VCAM1 small hairpin RNA (KD). Reverse-transcription polymerase chain reaction and western blot analysis were used to examine the effects of VCAM1-knockdown on the mRNA expression of VCAM1 and subsequent protein expression. Furthermore, the HN12 cell growth inhibition rate was detected using the cell counting kit-8 method. The VCAM1-targeted lentiviral vector RNAi significantly inhibited VCAM1 mRNA, and subsequent protein, expression. Compared with the NC group, the VCAM1 gene knockdown efficiency was ~85%, while the expression level of VCAM1 protein was reduced by ~74% in KD group cells. In addition, cell growth was significantly inhibited in the KD group, with a growth inhibition rate of ~34%. Therefore, this targeted lentiviral vector RNAi effectively inhibited VCAM1 gene, and subsequent protein, expression, as well as the proliferation of oral squamous carcinoma cells. These results may provide an experimental reference for the diagnosis and treatment of oral squamous cell carcinoma.

[Change of zygomatic and temporal soft tissue after coronal incision].

OBJECTIVE: To investigate the change of zygomatic and temporal soft tissue after coronal incision. METHODS: A retrospective analysis was performed in 33 patients who received firm fixation for unilateral zygomatic comminuted fracture through semi-coronal incision. All the patients were followed up for more than one year. Craniofacial anthropometric measurement through 3D-CT reconstruction and facial profile was performed. The difference between the operated side and healthy side was analyzed. RESULTS: At the temporal concave point, the soft tissue thickness at healthy side was (1.60 +/- 0. 97) mm more than that at operated side, showing a significant difference between them (P < 0.01). While the soft tissue thickness was not statistically different between two sides at zygion, malar prominence, zygomaxillare, and temporal convex point (P > 0.05). CONCLUSIONS: The soft tissue atrophy may happen at temporal fat pad after semi-coronal incision, but not at zygomatic area. Intraoperative precise dissection and less stretch of soft tissue may be helpful to avoid the postoperative facial asymmetry.

[Expression and significance of vascular cell adhesion molecule-1 in human oral squamous cell carcinoma].

OBJECTIVE: To study the expression and the location of vascular cell adhesion molecule-1 (VCAM-1) gene and its clinical significance in human oral squamous cell carcinoma (OSCC). METHODS: In situ hybridization, PV-9000 polymer detection system for immunohistochemical staining was used to detect the expression and the location of VCAM-1 mRNA and VCAM-1 protein in 48 cases of OSCC and 10 cases of normal controls. Statistical analysis was performed using chi-square test in SPSS 13.0. RESULTS: VCAM-1 protein was mainly expressed in tumor cell cytoplasm and membrane, VCAM-1 mRNA was mainly expressed in tumor cell cytoplasm. The expression rate of VCAM-1 mRNA and VCAM-1 protein was significantly higher in OSCC than that in normal oral mucosa (P<0.01). The expression of VCAM-1 mRNA was positively correlated with that of VCAM-1 protein (P<0.01). In the clinicopathologic factors, lymph node metastasis and depth of infiltration were closely correlated with VCAM-1 expression (P<0.01). The expression of VCAM-1 was significantly higher in tumor with lymph node metastasis than in tumor without lymph node metastasis (P<0.01). CONCLUSION: Overexpression of VCAM-1 gene in OSCC may play a potential role in the development of OSCC. The overexpression of VCAM-1 gene in OSCC may be related to the tumor infiltration and metastasis.

[Correlations between the expression of vascular cell adhesion molecule-1 gene and clinical pathological characteristics and angiogenesis in oral squamous cell carcinoma].

PURPOSE: To investigate the correlations between the expression of vascular cell adhesion molecule-1 (VCAM-1) gene and clinicopathologic characteristics and microvessel density (MVD) in oral squamous cell carcinoma (OSCC). METHODS: Expression and location of VCAM-1mRNA and protein in 48 OSCCs and 10 normal controls were detected by in situ hybridization and immunohistochemical stainingìMVD was also assessed. Statistical analysis was performed using SPSS13.0 software package for Student's t test and Chi-square test. RESULTS: VCAM-1mRNA was mainly detected in cytoplasm of OSCC.VCAM-1 protein was mainly detected in membrane and cytoplasm of OSCC. The expression of VCAM-1mRNA and protein were significantly higher in OSCC tissues than normal oral tissues (P<0.01). In the clinicopathologic factors, lymph node metastasis and depth of infiltration were closely correlated with VCAM-1 expression (P<0.01), but there was no significant correlation between the positively expression and patients' sex, age and tumor' differentiated degree (P>0.05). CONCLUSIONS: Overexpression of VCAM-1 gene in OSCC may play a potential role in the development of OSCC.It is closely associated with lymph node metastasis and the angiogenesis.

[Influence of prostaglandin E2 on proliferation of melanocytes in full-thickness skin graft].

OBJECTIVE: To investigate the influence of the prostaglandin E2 on the proliferation of the melanocytes in the full-thickness skin graft. METHODS: Sixty-eight guinea-pigs were divided into experimental-1 group (skin graft), experimental-2 group (skin graft + diclofenac), and control groups. After the full-thickness skin graft, the dynamic changes of the prostaglandin E2 were measured and the proliferation of the melanocyte with its density was also evaluated by using histochemical and autoradiographic methods. RESULTS: In the experimental-1 group, the content of PGE2 was increasing in seven days after the operation, continued to the one month, and then returned to the base level. The labelling indices of 3H-MC-TdR of the group was also increasing postoperatively between the second day and the fourteenth day, and reach a second peak after one month, then came to the normal level. The density of the melanocytes was decreasing rapidly 3 days after the surgery, then began to increase and exceeded over the normal level 21 days after the operation. However, in the experimental-2 group, the content of PGE2 decreased in two days after the surgery, and then showed the inclination similar to the experimental-1 group with the different points in narrower range. The number of melanocytes labelled by 3H-TdR began to increase at the first day after the surgery, which appeared earlier than the experimental-1 group and was similar in the changing tendency with a less extent. The density of MC showed the similar tendency to the experimental-1 group in a narrower changing range with both of increasing and decreasing. The density of the MC was much lower in 21 after the operation than the experimental-1 group and normal control group. CONCLUSION: The increased PGE2 in the earlier stage of the skin grafting could enhance the inflammatory reaction to the tissue, as well as the melanocytes. It may stimulate the proliferation of the MC with the result of increasing their density. The use of the diclofenac might reduce the inflammation and suppress the proliferation of melanocytes, and result in the skin with light color due to decreasing the number of MC in the epidermis of the graft.