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Abstract Introduction Nuclear receptor subfamily five group A member two (NR5A2) plays a key role in the development of many tumor types, while it is uncertain in cutaneous squamous cell carcinoma (cSCC). The aim of this work was to determine the role of NR5A2 in cSCC proliferation, and to determine whether NR5A2 mediates the effect of cisplatin in cSCC. Methods We performed a systematic study of existing data and conducted a preliminary bioinformatics analysis of NR5A2 expression in cSCC using bioinformatics databases. Immunohistochemical staining was performed on cSCC tissues of seven patients to study NR5A2 expression. NR5A2 expression was examined in human keratin‐forming cells (HaCaT) and human cSCC cells (A431, Colo‐16, SCL‐1, SCL‐2, and HSC‐5). Stable A431 and SCL‐2 cell lines consisting of sh‐RNA‐NR5A2 were constructed to detect changes in cell proliferation, cell cycle, apoptosis, and to determine the key proteins in the Wnt/β‐catenin pathway. We also investigated changes in the effects of cisplatin on cSCC cells by CCK‐8, clone formation assay, and Flow apoptosis assay after NR5A2 knockdown. Results NR5A2 showed enhanced expression in cSCC tissues than in healthy tissues. Downregulation of NR5A2 in cSCC cells led to the formation of a less malignant phenotype. In contrast, the proliferative capacity of the cSCC cells was enhanced posttreatment with RJW100, an NR5A2 agonist. Additionally, NR5A2 knockdown led to a decrease in the expression level of the proteins in the Wnt/β‐catenin pathway, and this inhibition was reversed by LiCl and recombinant antibody, Wnt3a. Moreover, NR5A2 knockdown resulted in diminished proliferative capacity and increased apoptotic cells after the addition of cisplatin. Conclusion NR5A2 plays a crucial role in the progression of cSCC, and the Wnt/β‐catenin signaling pathway may be involved in the regulation of NR5A2‐mediated cSCC. Knockdown of NR5A2 enhanced both the proliferation inhibiting and apoptosis promoting effects of cisplatin on cSCC.

Objective To screen a key molecule, leucine-rich glioma inactivated protein 4 (LGI4), which participates in endothelial cell-mediated osteosarcoma metastasis, and to further analyze its function and molecular mechanism in osteosarcoma metastasis. Methods A co-culture model of human umbilical vein endothelial cells (HUVECs)-osteosarcoma cells was established, and the genes with significant fold change were screened by whole transcriptome sequencing in combination with relevant literature. After, the candidate target gene, LGI4 was verified with real-time quantitative PCR (RT-qPCR) and Western blotting, the expression of LGI4 at mRNA and protein levels was also determined. Osteosarcoma cell line 143B and MTF LGI4 were transfected with the overexpression plasmid of LGI4 to construct the cells with stable overexpression of LGI4. Then the experiments included 5 groups of osteosarcoma cells, including negative control group (normally cultured), co-culture group (co-cultured with HUVECs), co-culture medium group (treated with the HUVECs medium), vector control group (transfected with control plasmid) and LGI4 group (overexpression of LGI4). Transwell assay and wound healing test were used to detect the migration and invasion abilities of osteosarcoma cells. A tumor metastasis model in nude mice was used to verify the effect of LGI4 overexpressed osteosarcoma cells on distant metastasis in vivo. Bioinformatics analysis was applied to analyze the potential downstream target signaling pathways of LGI4, and recovery experiments were performed with related inhibitors to clarify the role of target signaling pathways in the regulation of osteosarcoma metastasis by LGI4. Results The expression levels of LGI4 were markedly decreased in 143B and MTF cells after co-culture with HUVECs (P < 0.05). Compared with the negative control group, the cell migration and invasion abilities of the co-culture group and co-medium group were significantly enhanced, whereas the cell migration and invasion rates of the LGI4 overexpression group were significantly lower than that of the vector control group (P < 0.05). The nude mice metastasis model revealed that the metastasis ability of osteosarcoma cells was suppressed with LGI4 overexpression. Further analysis revealed that NOTCH4 signaling pathway was the downstream target signaling pathway of LGI4, and functional recovery experiments confirmed that LGI4 resulted in osteosarcoma metastasis by inhibiting NOTCH4 signaling pathway (P < 0.05). Conclusion Endothelial cells activate NOTCH4 signaling pathway and thus promote tumor metastasis by inhibiting the expression of LGI4 in osteosarcoma cells.

Objective: To determine the quality changes of the quality of pork ribs in simultaneous combination of steam and microwave cooking. Methods: The physicochemical quality, texture, fatty acids, amino acids, total thiol group, total carbonyl and vitro protein digestibility of pork ribs treated with simultaneous combination of steam and microwave were investigated. Results: The simultaneous combination of steam and microwave could significantly reduce the cooking time compared with the single steaming. And the combined cooking of microwave with 500 W and steam with 1 300 W for 13 min (MS-13) could reduce 48% of the cooking time. Compared with other groups, the pork ribs of MS-13 group had the highest moisture content, protein content and sulfhydryl content, which were higher than those of the individual steamed group for 5.91%, 5.85% and 101.60%, respectively, which were higher than the microwave group for 14.55%, 4.90% and 19.78%, respectively. The pork ribs of MS-13 group had the lowest fat content and carbonyl content, which were 91.87% and 45.02% of the individual steamed group, and 95.48% and 67.18% of the individual microwave group. Compared with fresh pork ribs, the relative contents of unsaturated fatty acids of MS-13 group increased significantly. Conclusion: It can be seen that the pork ribs cooked by microwave with 500 W and steam with 1 300 W for 13 min had better sensory and nutritional quality.

Objective To investigate the effect of inhibitor of differentiation-1 (ID1) on angiogenesis in osteosarcoma cell line 143B by regulating vascular endothelial growth factor A (VEGF-A), and verify the correlation of ID1 expression with angiogenesis and VEGF-A expression in human osteosarcoma tissues. Methods We investigated the expression patterns of ID1 and microvessel density (MVD) in 33 human osteosarcoma tissues by immunohistochemistry (IHC) and further analyzed the correlation between ID1 and VEGF-A expression in osteosarcoma by IHC and bioinformatics analysis. Lentiviral vector and siRNA transfection were employed to make ID1 differential expressed in osteosarcoma 143B cells. And the conditional medium (CM) were collected. Transwell assay and tube formation assay were used to determine the effect of CM collected from ID1 differential expressed 143B cells on abilities of the migration and tube formation in human umbilical vein endothelial cells (HUVECs) in vitro. PCR, ELISA and Western blot assay were adopted to detect the responses of VEGF-A expression in 143B cells in vitro, with or without ID1 differential expression. Results Bioinformatics analysis indicated that ID1 was involved in many pathways related to the angiogenesis in osteosarcoma, and the expression of VEGF-A was positively correlated with the mRNA level of ID1. In the human osteosarcoma tissues, these with positive ID1 expression had higher MVD than those with negative expression (P < 0.05), and the protein level of ID1 had a positive correlation with that of VEGF-A (r=0.464 3, P=0.006 5). In vitro study showed that the CM collected from ID1 overexpression 143B cells significantly enhanced the abilities of migration and tube formation in HUVECs than the CM from control cells (P < 0.05), and the CM from ID1-silencing 143B cells obviously suppressed the abilities (P < 0.05). The expression and secretion of ID1 was enhanced in the ID1 overexpression 143B cells, but reduced in the ID1-silencing 143B cells (P < 0.05). Conclusion ID1 promotes angiogenesis in human osteosarcoma by up-regulating VEGF-A.

Objective To evaluate the practical value of detecting folate receptor (FR)-positive circulating tumor cells (CTC) in clinical diagnosis of benign and malignant lung nodules. Methods A total of 288 patients with pulmonary nodules admitted in our hospital from May 2017 to April 2018 were enrolled in this study. FR-targeted CTC technology was used to quantitatively detect CTC content in peripheral blood samples. The results were analyzed with the postoperative pathological results to evaluate the accuracy and feasibility of the detection in the auxiliary diagnosis of lung malignant tumors. Results With a cut-off value of 8.7 Folate Units/3 mL as the threshold, the CTC content was significantly higher in the 136 patients with pulmonary malignant tumor than the 152 patients with pulmonary benign disease (P < 0.05). Among the 136 patients with pathologically diagnosed pulmonary malignant tumors, FR-positive CTC were observed in 106 patients. In the 152 patients with benign nodules, 44 cases showed false positive CTC results. In general, the sensitivity and specificity of FR-positive CTC detection was 78.7% and 71.1%, respectively in the detection of pulmonary malignant tumors. Furthermore, the sensitivity reached 76.9% in stage I lung cancer (70/91), and all 9 cases of lung cancer in situ were positive in the detection. Compared with tumor markers and CT examinations, FR-positive CTC detection was of higher auxiliary diagnostic value for early lung cancer. Conclusion FR-positive CTC detection can be used to screen benign and malignant lung nodules, and has a good value in the diagnosis of early-stage pulmonary malignant tumors.

Objective To investigate the effect of overexpression of Ran-binding protein 9 (RanBP9) on apoptosis of osteosarcoma cells and the underlying mechanism. Methods Real-time PCR and Western blotting were used to detect the mRNA and protein expression of RanBP9 in cultured osteoblasts and different osteosarcoma cell lines (SaOS2, MG63, U2OS and HOS). In a SaOS2 cell model with stable RanBP9 overexpression induced by transfection with a lentiviral vector, the cell apoptotic rate was determined using flow cytometry and TUNEL assay, and the changes in mitochondrial transmembrane potential were analyzed. Results RanBP9 was positively expressed in cultured osteoblasts and in the 4 osteosarcoma cell lines. RanBP9 expression was significantly higher in the osteoblasts than in the osteosarcoma cells, and was significantly higher in osteosarcoma cell lines MG63 and HOS than in SaOS2 and U2OS cell lines that had higher levels of malignancy. In SaOS2 cells, overexpression of RanBP9 obviously suppressed cell proliferation and promoted cell apoptosis and significantly increased the expression levels of caspase-3 and Cytc and the ratio of Bax/Bcl-2, resulting also in the dissipation of mitochondrial transmembrane potential and activation of the mitochondrial pathway to induce cell apoptosis. Conclusion RanBP9 promotes osteosarcoma cell apoptosis via the endogenous mitochondrial pathway.

The prognosis of hepatocellular carcinoma (HCC) is closely associated with the occurrence of distant metastases, which is likely due to circulating tumor cells (CTCs). However, the low number of CTCs is the main obstacle limiting research of the mechanism of CTC metastasis. Here, We evaluated the role of ubiquitin-specific protease 1 (USP1) in promoting CTC survival during blood-borne metastases. We observed that USP1 was frequently upregulated in CTCs and correlated with metastasis and a reduced overall survival rate of patients. Additionally, genetic knockout of USP1 the survival rate of CTCs. Further analyses showed that USP1 mediates oncogenic activity by deubiquitinating and stabilizing transducin β-like 1 X-linked receptor 1 (TBLR1), which plays essential roles in regulating Wnt signaling. These results demonstrated that USP1 may act as an essential factor in promoting the survival of CTCs and suggest that inhibition of USP1 is a potential strategy for HCC treatment.

The anisotropy and microstructures during aging treatment for 2A66 Al-Li alloy were studied by Brinell hardness, tensile testing, optical microscope(OM), scanning electron microscope(SEM) and transmission electron microscope (TEM). The main factors which influence the anisotropy of mechanical properties were discussed. The results indicate that, before 165℃ peak-aged, the anisotropy of the mechanical properties of the 2A66 Al-Li alloy decreases gradually with the extension of aging time. When the alloy is over-aged, the anisotropy of the alloy increases; the anisotropy of ductility is more serious than that of strength. The IPA values of σb, σ0.2 and δ of the alloy reach the lowest value at 3.0%, 3.0% and 12.2% respectively at the time of peak aging (64h), and the alloy is also obtained with good plasticity and axial tensile properties. σb, σ0.2 and δ of the alloy are 526.5, 448.9MPa, 10.1% respectively. Under different heat treatment conditions, the general behavior of the anisotropy of 2A66 Al-Li alloy is as follows: longitudinal (0°) and transverse (90°) have the highest strength, 45° direction is the lowest strength; 45° direction specimen has the highest elongation, vertical and horizontal direction has the minimum elongation.

Kai Yang, Fu-Jun Zhang, Hong Tang, Cheng Zhao, Yu-An Cao, Xiao-Qiang Lv, Dan Chen, Ya-Dong LiDepartment of Oral and Maxillofacial Surgery, The First Affiliated Hospital, Chongqing Medical University, Chongqing, ChinaObjectives: The purpose of this study was to investigate in-vivo visible imaging of oral squamous cell carcinoma (OSCC) by targeting epidermal growth factor receptor (EGFR) with near-infrared quantum dots.Materials and methods: Quantum dots with an emission wavelength of 800 nm (QD800) were conjugated to monoclonal antibodies against EGFR, resulting in the probe designated as QD800-EGFR Ab. OSCC cell line (BcaCD885) expressing high levels of EGFR was transplanted subcutaneously into nude mice cheeks to develop an OSCC animal model. QD800-EGFR Ab containing 100 pmol equivalent of QD800 was intravenously injected into the animal model, and in-situ and in-vivo imaging of cheek squamous cell carcinoma was analyzed at 10 different time points.Results and conclusion: In-vivo imaging and immunohistochemical examination of the tumors showed that intravenously injected QD800-EGFR Ab probe could bind EGFR expressed on BcaCD885 cells. Fluorescence signals of BcaCD885 cells labeled with QD800-EGFR Ab probe could be clarly detected, and these fluorescence signals lasted for 24 hours. The most complete tumor images with maximal signal-to-noise ratio were observed from 15 minutes to 6 hours after injection of the probe. To the best of the authors' knowledge, this is the first study that has obtained clear in-situ and in-vivo imaging of head and neck cancer by using QD800-EGFR Ab probe. The authors conclude that the combination of near-infrared quantum dots that are highly penetrating for tissues with EGFR monoclonal antibody has promising prospects in in-vivo imaging of OSCC and development of personalized surgical therapies.Keywords: oral cancer, head and neck cancer, near-infrared fluorescence, visual in-vivo imaging, epidermal growth factor receptor, nanotechnology

Abstract Background Vascular endothelial growth factor (VEGF) is a signal protein produced by cells that stimulates vasculogenesis and angiogenesis. VEGF is believed to implicate poor prognosis in various cancers. The overexpression of VEGF may be an early step in the process of metastasis. Methods ELISA was used to investigate the levels of VEGF, bFGF and IL8 in human bone metastatic LNCaP-derivative C4-2B prostate cancer cell line and its parental cell line, LNCaP and to determine the effect of bevacizumab on reducing the level of VEGF. Cell proliferation assay, invasion assay and in vitro angiogenesis assay were performed under the condition with bevacizumab or control IgG. Results Human bone metastatic LNCaP-derivative C4-2B prostate cancer cell line expressed a higher level of VEGF than its parental primary prostate cancer cell line LNCaP. The effect of bevacizumab is dose-dependent and time-dependent: 100 μg/mL of bevacizumab and 3-day treatment was more effective than low-dose and lesser-day treatment for decreasing the level of VEGF. Bevacizumab is able to suppress cell proliferation, angiogenesis and invasion in human bone metastatic C4-2B prostatic cancer cell line. Conclusions The overexpression of VEGF can be inhibited by bevacizumab in human bone metastatic cancer cell line. The behaviors of metastasis involving proliferation, angiogenesis and invasion are suppressed by anti-VEGF therapy.

Abstract Background Epstein-Barr virus (EBV) establishes its latency in EBV-associated malignancies, accompanied by occasionally reactivated lytic cycle. Promoter CpG methylation of EBV genome plays an essential role in maintaining viral latency. Two immediate-early (IE) genes, BZLF1 and BRLF1, induce the switch from latent to lytic infection. Studies of methylation-dependent binding of BZLF1 and BRLF1 to EBV promoters have been well reported, but little is known about the methylation status of BZLF1 and BRLF1 promoters (Zp and Rp) in tumor samples. Methods We evaluated the methylation profiles of Zp and Rp by methylation-specific PCR (MSP) and bisulfite genomic sequencing (BGS), as well as BZLF1 and BRLF1 expression by semiquantitative reverse transcription (RT)-PCR in tumors of epithelial, NK- and B-cell origins. Results We found that both Zp and Rp were hypermethylated in all studied EBV-positive cell lines and tumors of lymphoid (B- or NK cell) or epithelial origin, while unmethylated Zp and Rp alleles were detected in cell lines expressing BZLF1 and BRLF1. Following azacytidine treatment or combined with trichostatin A (TSA), the expression of BZLF1 and BRLF1 was restored along with concomitant promoter demethylation, which subsequently induced the reactivation of early lytic gene BHRF1 and late lytic gene BLLF1. Conclusions Hypermethylation of Zp and Rp mediates the frequent silencing of BZLF1 and BRLF1 in EBV-associated tumors, which could be reactivated by demethylation agent and ultimately initiated the EBV lytic cascade.

Abstract Background Expression of kappa gene is under the control of distinct cis-regulatory elements, including the kappa intron enhancer (iEκ) and the kappa 3' enhancer (3'Eκ). The active enhancers and expression of immunoglobulin is generally considered to be restricted to B lymphocytes. However, accumulating evidence indicated that epithelial cancer cells, including nasopharyngeal carcinoma (NPC) cell lines, express immunoglobulins. The mechanisms underlying the expression of Igs in nonlymphoid cells remain unknown. On the basis of our previous finding that expression of kappa light chain in NPC cells can be upregulated by EBV-encoded latent membrane protein 1(LMP1) through the activation of NF-κB and AP-1 signaling pathways, we thus use NPC cells as model to further explore the molecular mechanisms of nonlymphoid cells expressing Ig kappa. Results In this study, luciferase reporter plasmid containing human wild-type iEκ, and its derivative plasmids containing mutant binding sites for transcription factor NF-κB or AP-1 were constructed. Luciferase reporter assays demonstrate iEκ is active in Igκ-expressing NPC cells and LMP1 expression can upregulate the activity of iEκ in NPC cells. Mutation of the NF-κB or AP-1 site within and downstream the iEκ, inhibition of the NF-κB and AP-1 pathways by their respective chemical inhibitor Bay11-7082 and SP600125 as well as stable or transient expression of dominant-negative mutant of IκBα (DNMIκBα) or of c-Jun (TAM67) indicate that both sites are functional and LMP1-enhanced iEκ activity is partly regulated by these two sites. Gel shift assays show that LMP1 promotes NF-κB subunits p52 and p65 as well as AP-1 family members c-Jun and c-Fos binding to the κNF-κB and the κAP-1 motifs in vitro, respectively. Both chemical inhibitors and dominant negative mutants targeting for NF-κB and AP-1 pathways can attenuate the LMP1-enhanced bindings. Co-IP assays using nuclear extracts from HNE2-LMP1 cells reveal that p52 and p65, c-Jun and c-Fos proteins interact with each other at endogenous levels. ChIP assays further demonstrate p52 and p65 binding to the κB motif as well as c-Jun and c-Fos binding to the AP-1 motif of Ig kappa gene in vivo. Conclusion These results suggest that human iEκ is active in Igκ-expressing NPC cells and LMP1-stimulated NF-κB and AP-1 activation results in an augmenting activation of the iEκ. LMP1 promotes the interactions of heterodimeric NF-κB (p52/p65) and heterodimeric AP-1 (c-Jun/c-Fos) transcription factors with the human iEκ enhancer region are important for the upregulation of kappa light chain in LMP1-positive nasopharyngeal carcinoma cells.