C1QTNF6 promotes oral squamous cell carcinoma by enhancing proliferation and inhibiting apoptosis.

BACKGROUND: C1QTNF6 (CTRP6), a member of the CTRP family, has recently been implied to play a role in the tumorigenesis of for a variety of cancer types. However, the role of C1QTNF6 in oral squamous cell carcinoma (OSCC) and its potential molecular remains unclear. METHODS: C1QTNF6 expression was detected by qRT-PCR and western blot analysis. Lentiviral vectors were constructed to knockdown C1QTNF6 in CaL27 and SCC-9 human OSCC cell lines. Cell viability, cell cycle and cell apoptosis analyses were performed by MTT assay, PI/Annexin V staining, and flow cytometry. The effect of C1QTNF6 knockdown on in vivo tumorigenicity of OSCC cells in vivo was evaluated using nude mouse xenograft tumor model. Downstream signaling mechanisms were identified by microarray and Ingenuity Pathway Analysis. RESULTS: Immunohistochemistry of OSCC tissue and data from TCGA demonstrate that C1QTNF6 was overexpressed in OSCC tissues, and that cellular proliferation was significantly decreased after C1QTNF6 was knockdown in CaL27 and SCC-9 cell lines. Knockdown of C1QTNF6 also resulted in cell cycle arrest at the G2/M phase and enhanced cell apoptosis in in CaL27 and SCC-9 cell lines. Furthermore, knockdown of C1QTNF6 in Cal-27 cells inhibited tumor growth of OSCC in vivo. Microarray analysis revealed that C1QTNF6 silencing resulted in significant alterations of gene expression, with the Acute Phase Response signaling pathway significantly activated following C1QTNF6 silencing. CONCLUSIONS: These results suggest that C1QTNF6 plays an important role in promoting OSCC tumorigenesis, which indicates that C1QTNF6 may comprise a promising therapeutic target for OSCC treatment.

Carcinoembryonic antigen cell adhesion molecule 1 inhibits the antitumor effect of neutrophils in tongue squamous cell carcinoma.

Carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1), a transmembrane glycoprotein, has multiple functions. In tongue squamous cell carcinoma (TSCC), CEACAM1 overexpression is correlated with neutrophil infiltration, and both are associated with poor clinical outcomes. However, the mechanism underlying CEACAM1's effect on neutrophil function in TSCC remains unclear. We cocultured tongue carcinoma cells overexpressing CEACAM1-4L, CEACAM1-4S and differentiated HL-60 cells. This significantly upregulated the expression of MMP-9, interleukin 8, and VEGF-A in the differentiated HL-60 cells and downregulated the expression of TNF-α, relative to vector and blank control groups (P < 0.05). Additionally, CEACAM1 overexpression in tongue carcinoma cells weakened the cytotoxicity of differentiated HL-60 cells in the coculture system (P < 0.05). Thus, CEACAM1 expression in TSCC may induce an antitumor to protumor transformation of neutrophils. We performed qRT-PCR and ELISA to evaluate the underlying mechanism, and found that CEACAM1 expression in tongue carcinoma cells upregulated transforming growth factor ß1 (TGF-ß1) expression, while blocking of TGF-ß1 inhibited the neutrophils' changes in the coculture system. Immunohistochemical analysis of clinical specimens revealed strong expression of TGF-ß1 protein in TSCC. TGF-ß1 expression was positively correlated with CEACAM1 expression, lymph node metastasis, and tumor recurrence. Double immunofluorescence results revealed colocalization of CEACAM1 and TGF-ß1 protein in TSCC. A xenograft nude mouse model revealed that CEACAM1 overexpression in TSCC promoted tumor formation and growth, and was associated with more neutrophils infiltration. Our results indicate that CEACAM1 overexpression in TSCC may induce transformation of neutrophils from antitumor to protumor type via TGF-ß1, which may further promote tumor progression.

Endothelial cells modified by adenovirus vector containing nine copies hypoxia response elements and human vascular endothelial growth factor as the novel seed cells for bone tissue engineering.

Vascularization is one of the hotspots during the development of new therapeutic strategies for bone tissue engineering, which can alleviate hypoxic circumstance and prevent transplant failure. Vascular endothelial growth factor (VEGF) gene transfection using recombinant adenovirus (Ad) vector can effectively promote angiogenesis, but uncontrolled long-term continuous expression of VEGF brings safety concern. Here we constructed a recombinant Ad vector containing nine copies of HRE promoter and the hVEGF165 gene, which conserved the oxygen sensitivity of hypoxia-inducible factor-1/hypoxia response elements (HIF-1/HRE). After transfection into human umbilical vein endothelial cells (HUVEC), the hVEGF165 mRNA and protein levels were much higher in response to hypoxia, as revealed by RT-PCR and ELISA, respectively. Furthermore, Ad-9HRE-hVEGF165 vector effectively promoted proliferation, migration and tube formation of HUVEC under hypoxic conditions. Thus we believe that the Ad-9HRE-hVEGF165 vector can contribute to the regulation of vascularization, which may provide a new approach for a better control of the expression of hVEGF165 during bone tissue engineering.

In situ expression and serum level of thymic stromal lymphopoietin in oral lichen planus.

BACKGROUND: Oral lichen planus (OLP) is a chronic inflammatory disease of oral mucosa in which the CD8(+) T cell-mediated cytotoxicity is regarded as a major mechanism of pathogenesis. The main objective of this study is to investigate in situ expression and secretion of thymic stromal lymphopoietin (TSLP) in specimens and sera from patients with oral lichen planus. METHODS: Thirty-six patients with OLP and 35 donors enrolled in specimen and serum collection. Immunohistochemical method and immunofluorescence double-staining method were used to detect the expression of thymic stromal lymphopoietin and its receptor (TSLPR) together with CD8 in OLP specimens. Enzyme-linked immunosorbent assay (ELISA) was used to detect TSLP secretion. RESULTS: More TSLP- or TSLPR-positive cells showed in OLP specimens than in normal controls, and TSLP-positive cells were mainly in the epithelium, while TSLPR-positive cells mainly in the lamina propria. Furthermore, the number of TSLP-positive cells in the stratum basal was associated with the amount of mononuclear cells infiltrating in the lamina propria of OLP specimens. Among infiltrating mononuclear cells in the lamina propria, some CD8-positive cells also expressed TSLPR. The TSLP serum level of patients with OLP was significantly higher than of healthy donors, but there was no statistically difference between two clinical subtypes of OLP. CONCLUSIONS: Our findings provided the first evidence that TSLP may enroll in the pathology of OLP and the TSLP-TSLPR interaction may play an important role in it.

Ischemia preconditioning protects rat submandibular glands from ischemia/reperfusion injuries.

To investigate the effects of ischemia/reperfusion on rat submandibular glands without denervation and the possible protective effects of ischemia preconditioning on the glands that experienced ischemia/reperfusion, in-situ ischemia/reperfusion and ischemia preconditioning experimental models of submandibular glands of healthy male Wistar rats were conducted. For ischemia/reperfusion groups, the glands were subjected to 90 min of ischemia without denervation, followed by 1, 12, 24, or 72 h of reperfusion. Ischemia preconditioning was achieved by 3 min of ischemia following 3 min of reperfusion, performed three times before ischemia/reperfusion. Salivary secretion, histological changes, alterations of tight junctions, myeloperoxidase activity, cellular apoptosis, and reactive oxygen species levels were detected. In ischemia/reperfusion glands, rising acute-inflammation responses, reduced tight-junction width, and increased myeloperoxidase activity, reactive oxygen species levels, and apoptotic cell numbers were observed, along with secretory dysfunction, especially at 1 and 12 h post-reperfusion, which seemed to gradually return to normal by 72 h post-reperfusion. In contrast, ischemia preconditioning showed the potential to ameliorate the injury-stress responses caused by ischemia/reperfusion. Our study revealed that ischemia/reperfusion could cause a series of injury-stress responses and ultimately lead to hyposecretion, independently of the parasympathetic nerve supply, which might play an important role in the early-phase dysfunction of the transplanted glands. Ischemia preconditioning could protect the involved glands and improve ischemia/reperfusion-induced hyposecretion.

Galectin-3 gene silencing inhibits migration and invasion of human tongue cancer cells in vitro via downregulating ß-catenin.

AIM: Galectin-3 (Gal-3) is a member of the carbohydrate-binding protein family that contributes to neoplastic transformation, tumor survival, angiogenesis, and metastasis. The aim of this study is to investigate the role of Gal-3 in human tongue cancer progression. METHODS: Human tongue cancer cell lines (SCC-4 and CAL27) were transfected with a small-interfering RNA against Gal-3 (Gal-3-siRNA). The migration and invasion of the cells were examined using a scratch assay and BD BioCoat Matrigel Invasion Chamber, respectively. The mRNA and protein levels of ß-catenin, Akt/pAkt, GSK-3ß/pGSK-3ß, MMP-9 in the cells were measured using RT-PCR and Western blotting, respectively. RESULTS: Transient silencing of Gal-3 gene for 48 h significantly suppressed the migration and invasion of both SCC-4 and CAL27 cells. Silencing of Gal-3 gene significantly decreased the protein level of ß-catenin, leaving the mRNA level of ß-catenin unaffected. Furthermore, silencing Gal-3 gene significantly decreased the levels of phosphorylated Akt and GSK-3ß, and suppressed the mRNA and protein levels of MMP-9 in the cells. CONCLUSION: Our data suggest that Gal-3 mediates the migration and invasion of tongue cancer cells in vitro via regulating the Wnt/ß-catenin signaling pathway and Akt phosphorylation.

Effects of allogenous periosteal-derived cells transfected with adenovirus-mediated BMP-2 on repairing defects of the mandible in rabbits.

PURPOSE: This report describes the effect of periosteal-derived cells transfected with adenovirus-mediated bone morphogenetic protein-2 (BMP-2) on the repair of mandibular defects in rabbits. MATERIALS AND METHODS: Periosteal-derived cells were transfected with a replication-defective adenoviral vector encoding BMP-2, and the expression of BMP-2 was examined in transfected cells using in situ hybridization and enzyme-linked immunosorbent assay. In addition, the proliferation ability and activity of alkaline phosphatase of transfected cells were examined using the 3-[4,5-dimethylthiazol-2-Yl]-2,5-diphenyltetrazolium bromide method and enzymology, respectively. In vitro critical-size defects (about 10 × 6 mm) were made bilaterally in each rabbit mandible, and individual sites were implanted with tissue-engineered bone modified with an adenovirus construct encoding the recombinant human BMP-2 gene (Ad-BMP-2), tissue-engineered bone without modification, single bioactive glass ceramic, or no implants (control). New bone formation was evaluated by histochemical stain. RESULTS: BMP-2 expression in the supernate of infected cells was detected from the first day after Ad-BMP-2 transfection and remained at a high level for at least 2 weeks. Alkaline phosphatase expression in transfected cells was significantly greater than in uninfected cells. The group of Ad-BMP-2-modified periosteal-derived cells formed more new bone than the other group at any time point. CONCLUSION: Gene-modified tissue-engineered bone grafts have greater osteogenic potential than single tissue-engineered bone and single bioactive glass ceramic graft. Ex vivo Ad-BMP-2 transfer to periosteal-derived cells can increase bone formation in critical-size bone defects. Further studies are needed to determine if modified engineered cells can be developed for safe and effective clinical applications.

Co-inhibition of adenosine 2b receptor and programmed death-ligand 1 promotes the recruitment and cytotoxicity of natural killer cells in oral squamous cell carcinoma.

Adenosine promotes anti-tumor immune responses by modulating the functions of T-cells and natural killer (NK) cells in the tumor microenvironment; however, the role of adenosine receptors in the progression of oral squamous cell carcinoma (OSCC) and its effects on immune checkpoint therapy remain unclear. In this study, we obtained the tumor tissues from 80 OSCC patients admitted at the Shandong University Qilu Hospital between February 2014 and December 2016. Thereafter, we detected the expression of adenosine 2b receptor (A2BR) and programmed death-ligand 1 (PD-L1) using immunohistochemical staining and analyzed the association between their expression in different regions of the tumor tissues, such as tumor nest, border, and paracancer stroma. To determine the role of A2BR in PD-L1 expression, CAL-27 (an OSCC cell line) was treated with BAY60-6583 (an A2BR agonist), and PD-L1 expression was determined using western blot and flow cytometry. Furthermore, CAL-27 was treated with a nuclear transcription factor-kappa B (NF-κ B) inhibitor, PDTC, to determine whether A2BR regulates PD-L1 expression via the NF-κ B signaling pathway. Additionally, a transwell assay was performed to verify the effect of A2BR and PD-L1 on NK cell recruitment. The results of our study demonstrated that A2BR and PD-L1 are co-expressed in OSCC. Moreover, treatment with BAY60-6583 induced PD-L1 expression in the CAL-27 cells, which was partially reduced in cells pretreated with PDTC, suggesting that A2BR agonists induce PD-L1 expression via the induction of the NF-κ B signaling pathway. Furthermore, high A2BR expression in OSCC was associated with lower infiltration of NK cells. Additionally, our results demonstrated that treatment with MRS-1706 (an A2BR inverse agonist) and/or CD274 (a PD-L1-neutralizing antibody) promoted NK cell recruitment and cytotoxicity against OSCC cells. Altogether, our findings highlight the synergistic effect of co-inhibition of A2BR and PD-L1 in the treatment of OSCC via the modulation of NK cell recruitment and cytotoxicity.

Dysbiotic tumor microbiota associates with head and neck squamous cell carcinoma outcomes.

BACKGROUND: The need for an effective tool to predict prognosis of head and neck squamous cell carcinoma (HNSCC) patients is critical and unmet. Microbiota has recently been found involved in tumor progression and response to immunotherapy. However, the association of microbiota with the prognosis of HNSCC patients remains obscure. This study aims to investigate the association between tumor microbiota and outcomes of HNSCC patients. METHODS: A retrospective study including 129 primary tumors of HNSCC was conducted. Using 16S rRNA sequencing, the profile and the composition of tumor microbiota were measured and their associations with overall survival (OS) and disease free survival (DFS) were examined. RESULTS: We observed a reduced richness and enriched abundances of genera Schlegelella and Methyloversatilis in tumor microbiota of HNSCC patients with poor prognosis. However, a richer tumor microbiota with greater abundances of genera Bacillus, and Lactobacillus and Sphingomonas was characterized in the patients with favorable prognosis.The ratio of these differentially abundant taxa, microbial dysbiosis index (MDI), was significantly associated with OS (hazard ratio [HR], 4.67, 95% confidence interval [CI], 2.51 to 8.69,P < 0.001) and DFS (HR, 2.89; 95% CI, 1.74 to 4.80, P < 0.001) independently of age, tumor size, lymph node metastasis, differentiation and p16 status. The risk score of multivariate Cox regression exhibited an excellent performance for estimating three-year OS (AUC of 0.826). We also found a richer tumor microbiota was correlated with moderate peritumoral inflammatory infiltration. CONCLUSION: These results indicate that tumor microbiota associates with outcomes of HNSCC patients.

OPN promotes survival of activated T cells by up-regulating CD44 in patients with oral lichen planus.

Oral lichen planus (OLP) is a chronic inflammatory disorder of oral mucosa, which represents cell-mediated autoimmune diseases. Pathological study demonstrated that abundant T lymphocytes infiltrated the oral mucosa, in which the activated T cells that trigger apoptosis of oral epithelial cells is an important mechanism for OLP. However, to date the molecular mechanisms underlying the T lymphocytes infiltration and accumulation in OLP remain unclear. In this paper, we found that the levels of plasma OPN were elevated and were associated with the up-regulated expressions of CD44 in OLP patients. In vitro, the addition of exogenous OPN can suppress the apoptosis of activated CD8(+) T cells via CD44, and this T cell resistance to apoptosis may be attributed to the reduction of endogenous mature granzyme B. Our results suggested that the abnormally elevated levels of OPN may contribute to the abnormal infiltration and accumulation of the activated T cells by up-regulating CD44 in OLP.

The possible roles of OPN-regulated CEACAM1 expression in promoting the survival of activated T cells and the apoptosis of oral keratinocytes in oral lichen planus patients.

Oral lichen planus is a chronic inflammatory disorder of the oral mucosa that represents T cell-mediated autoimmune diseases. The regulation and roles of carcinoembryonic antigen-related cellular adhesion molecule 1 (CEACAM1), a novel immune molecule, in the immunopathogenesis of T cell-mediated autoimmune diseases remain unclear. In the current paper, CEACAM1 was found to be overexpressed in peripheral T cells and epithelial cells in oral lichen planus patients. A fraction of infiltrating inflammatory mononuclear cells in the lamina propria of the oral lichen planus mucosa also expressed CEACAM1. Importantly, for the first time, CEACAM1 expression in T cells and in normal human oral keratinocytes was demonstrated to be regulated differently by osteopontin in vitro. Furthermore, the apoptosis of oral keratinocytes and activated T cells can be markedly suppressed by CEACAM1-specific monoclonal antibodies. In conclusion, OPN-regulated CEACAM1 expression may play a critical role in the immunopathogenesis of oral lichen planus.

Silencing Id-1 with RNA interference inhibits adenoid cystic carcinoma in mice.

BACKGROUND: The helix-loop-helix (HLH) protein Id-1 (inhibitor of DNA binding/differentiation) has been demonstrated to play an important role in tumor development. Our previous in vitro research has shown that Id-1 is a potential target in the treatment of human adenoid cystic carcinoma (ACCM). The purpose of this study was to analyze the influence of Id inhibition on ACCM in mice. MATERIALS AND METHODS: To suppress the expression of Id-1 gene, we used lentivirus-mediated RNA interference to silence the Id-1 gene post-transcriptionally in ACCM models that stably express GFP in mice. Tumor development was evaluated by size measurement. Effects of Id-1 siRNA on mRNA and protein expression of Id-1 were analyzed using quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting respectively. Ki-67 expression was measured by immunohistochemistry. In vitro studies of Hoechst staining for cell apoptosis, Boyden-chamber assay for cell invasion, and MTT-tests for cell growth were performed as well. RESULTS: Id-1 knockdown resulted in inhibition of tumor growth in mice. Id-1 siRNA significantly decreased not only Id-1 in mRNA and protein level, but also Ki-67 expression. In addition, apoptosis was induced and cell proliferation activity and invasion were significantly reduced. CONCLUSIONS: Lentivirus-mediated gene knockdown by silencing Id-1 constitute a valid methodological approach, which may represent an attractive, potent and specific therapeutic tool for the treatment of ACCM.

Over-expression of Gadd45a enhances radiotherapy efficacy in human Tca8113 cell line.

AIM: To investigate the effect of the growth arrest- and DNA damage-inducible Gadd45a gene on the radiosensitivity of human tongue squamous cell carcinoma cell line to ionizing radiation (IR). METHODS: Short interfering ribonucleic acid (si-RNA) targeting Gadd45a or an irrelevant mRNA (nonsense si-RNA) was chemically synthesized. The constructed si-RNAs were transfected into Tca8113 cells and Gadd45a expression was determined using quantitative real-time PCR and Western-blot. After 24-h exposure to IR at a dose rate of 4 Gy/min, apoptosis of Tca8113 cells was detected using flow cytometry, and radiosensitivity was measured using MTT assays. RESULTS: IR apparently increased the expression of Gadd45a at mRNA and protein levels in Tca8113 cells. The effect was efficiently inhibited by transfection with Gadd45a si-RNA (P<0.01). Furthermore, silencing Gadd45a gene significantly increased cell viability and decreased the percentage of apoptotic cells during irradiation, which indicated that IR-induced Gadd45a over-expression could increase the radiosensitivity of Tca8113 cells. CONCLUSION: These results indicated that targeting Gadd45a may have important therapeutic implications in sensitizing Tca8113 cells to IR.

BMP2 and VEGF promote angiogenesis but retard terminal differentiation of osteoblasts in bone regeneration by up-regulating Id1.

Inadequate vascularization limits the repair of bone defects. In order to improve angiogenesis and accelerate osteogenesis, the synergism of co-cultured cells with genetic modification in bone regeneration was investigated in this study. Endothelial progenitor cells (EPCs) and bone marrow stem cells (BMSCs) were transfected with the genes of vascular endothelial growth factor (VEGF) and bone morphogenetic protein 2 (BMP2) by adenovirus, respectively. The co-cultured cells, designated as four groups including BMSC + EPC, Ad-BMP2-BMSC + EPC, BMSC + Ad-VEGF-EPC, and Ad-BMP2-BMSC + Ad-VEGF-EPC groups, were seeded on an alginate gel and then implanted into rat intramuscularly to evaluate the effects on angiogenesis and osteogenesis. Both VEGF and BMP2 could induce the overexpression of inhibitor of DNA-binding 1(Id1) gene which significantly promoted tube formation in vitro and increase the amount of blood vessels in the Ad-BMP2-BMSC + Ad-VEGF-EPC group after implantation. Nevertheless, overexpression of Id1 retarded the terminal differentiation of osteoblasts and the bone formation. Later, osteogenic gene expression at transcriptional level, calcium nodules, and alkaline phosphatase (ALP) activity showed a gradual decrease and the amount of newly formed osteogenesis area exhibited a small increase in the Ad-BMP2-BMSC + Ad-VEGF-EPC group. This finding suggests that a balanced regulation of Id1 expression in VEGF-EPCs and BMP2-BMSCs may be critical to cell-based and gene-based approaches for bone regeneration.

Fucoidan increases TNF-alpha-induced MMP-9 secretion in monocytic cell line U937.

OBJECTIVE: To investigate the effect of fucoidan on the expression of matrix metalloproteinase-9 (MMP-9) from monocytes. METHODS: Human monocytic cell line U937 was purchased from ATCC. During the experiment, FBS-free 1640 was used and U937 was cultivated with 20 ng/ml TNF-alpha and/or different concentrations of fucoidan for 24 h. RT-PCR experiments were used to determine the MMP-9 mRNA expression. ELISA and gelatin zymography detected MMP-9 amounts and activity in the supernatant. The intracellular level of MMP-9 was assayed by Western blot, and the level of CD44 on the surface was assayed by FACS. RESULTS: In this study, we showed that pro-inflammatory cytokine TNF-alpha up-regulated U937 MMP-9 mRNA and protein levels (P < 0.05). Fucoidan can increase the TNF-alpha-induced MMP-9 secretion from U937 (P < 0.05), but no significant difference was observed in MMP-9 mRNA. The intracellular level of MMP-9 treated with TNF-alpha and fucoidan was lower (P < 0.05) than that treated with TNF-alpha alone. In addition, we demonstrated that fucoidan downregulated the surface level of CD44, the main molecule to which MMP-9 attaches. CONCLUSIONS: We demonstrated that fucoidan post-translationally regulated MMP-9 secretion from U937. Reduced intracellular level and decreased membrane attachment may contribute to the increase in MMP-9 secretion.

Octreotide inhibits choroidal neovascularization in rats.

AIMS: Octreotide exhibits anti-angiogenic activity in animal models of retinopathy of prematurity and in clinical cases of proliferative diabetic retinopathy. In this study, we tested the applicability of using octreotide for inhibiting experimental choroidal neovascularization (CNV) in rats. METHODS: Of 15 adult rats used, 3 served as non-laser-treated controls. CNV was induced in the right eye of the remaining 12 rats by laser photocoagulation. These 12 rats were divided into two groups (n = 6 each) which were retrobulbarly injected with octreotide (50 miucrog/kg) or phosphate-buffered saline (PBS) (0.15 ml) immediately and on day 3 after the first injection. Fluorescein angiography and quantitative analysis of the retinal pigment epithelium (RPE)-choroidal flat mounts were performed 14 days after the operation to evaluate the changes in the CNV lesions. Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) was used to compare the changes shown by the octreotide-, PBS-, and non-laser-treated rats (n = 3 each) with regard to mRNA levels of the vascular endothelial growth factor (VEGF) and insulin-like growth factor (IGF)-1 in the RPE-choroidal complex. RESULTS: Octreotide treatment significantly inhibited fluorescein leakage and the extent of neovascularization as was observed in the flat mounts of choroidal tissue derived from the rats with induced CNV. The VEGF and IGF-1 mRNA levels were reduced following treatment with octreotide. CONCLUSION: The retrobulbar administration of octreotide may be an effective new therapeutic strategy for CNV.

Preoperative neutrophil lymphocyte ratio but not platelet lymphocyte ratio predicts survival and early relapse in patients with oral, pharyngeal, and lip cancer.

BACKGROUND: To evaluate the prognostic value of preoperative neutrophil lymphocyte ratio (NLR) and platelet lymphocyte ratio (PLR) in oral, pharyngeal, and lip cancer for survival and relapse. METHODS: Clinic-pathologic and hematological records were retrospectively retrieved. Patients completed follow-up period were included for survival and relapse analysis. RESULTS: The preoperative NLR value was a prognostic factor for both overall survival and relapse-free survival. The high NLR group demonstrated higher total relapse rate, higher local relapse rate, and higher relapse rate within 12 months. However, the preoperative PLR did not associate with survival or relapse. CONCLUSIONS: The preoperative NLR, not PLR, is an independent prognostic indicator of survival. It also exhibits predictive value for relapse, particularly early relapse within 12 months. The preoperative NLR value might be recommended as a useful tool for predicting the outcomes and stratifying patients for different management strategies.

Kif4A mediate the accumulation and reeducation of THP-1 derived macrophages via regulation of CCL2-CCR2 expression in crosstalking with OSCC.

Crosstalk between tumor infiltrating macrophages and tumor cells is thought to play an indispensable role in oral squamous cell carcinomas (OSCC) by induction and maintenance of tolerance microenvironment. High infiltration of M2 macrophages and increasing expression of Kinesin family member 4A (Kif4A) in primary OSCC have been proved to correlate with greater tumoral size and poor clinical outcome. However, linkage between Kif4A and infiltrating macrophages in tumorigenesis and progression remains unclear. In the present study, we show that, the interaction between THP-1derived macrophage and OSCC cell line Cal-27 may up-regulate the Kif4A expression in both of them. Additionally, elevated soluble CCL2 in medium and more expression of CCR2 on macrophage were observed during the crosstalk. SiRNA of Kif4A and neutralizing antibody of CCL2 were utilized to identify that; increasing Kif4A can promote the recruitment of macrophages towards Cal-27 and educate them to M2 polarized macrophages via regulating CCL2/CCR2. In combination, the results of the present study may provide interesting clues to understanding the Kif4A-CCL2/CCR2-macrophage axis as a novel therapeutic target to improve the clinical outcome of OSCC.

Kif4 regulates the expression of VEGFR1 through the PI3K/Akt signaling pathway in RAW264.7 monocytes/macrophages.

Kinesin superfamily protein 4 (Kif4), a microtubule-based motor protein, has been shown to participate in a number of critical cellular processes, such as cell division, the intracellular transport of membranous vesicles and signal transduction. However, whether KIF4 regulates vascular endothelial growth factor (VEGF) receptor 1 (VEGFR1) expression remains unknown. Thus, in this study, in order to examine the effects of Kif4 on the expression of VEGFR1 in RAW264.7 monocytes/macrophages, Kif4 was silenced using siRNA. RT-qPCR, western blot analysis and ELISA were used to assess the expression of Kif4 and VEGFR1 up- and downstream signaling molecules, including VEGF-A, VEGFR1, soluble form of VEGFR1 (sVEGFR1), phosphorylated (p-)Akt and Akt. The silencing Kif4 inhibited the mRNA expression of VEGF (P<0.01) and p-Akt (P<0.05); however, the level of VEGF-A was increased (P<0.05) compared with the negative control siRNA-transfected group. The silencing of Kif4 decreased the VEGFR1 mRNA (P<0.05), VEGFR1 protein and sVEGFR1 levels in the cell supernatant (P<0.01). Following the application of insulin-like growth factor-1 (100 ng/ml), the specific agonist of PI3K/Akt in the Kif4 siRNA-transfected group, the VEGFR1 mRNA levels (P<0.001), the VEGFR1 protein levels and the sVEGFR1 (P<0.01) levels significantly increased; however, the levels of VEGF in the cell supernatant were decreased (P<0.05). Taken together, these findings suggest that Kif4 regulates the expression of VEGFR1 in RAW264.7 cells and that the PI3K/Akt pathway is involved in this process.