Ability Of Nasopharyngeal Carcinoma Cells Research Articles

Circadian Clock Gene BMAL1 and Hypoxia Inducible Factor HIF-1α Study on the Effect on Proliferation, Migration and Radiosensitivity of Nasopharyngeal Carcinoma Cells

<h3>Purpose/Objective(s)</h3> To investigate the relationship between biological clock gene BMAL1 and hypoxia inducible factor-1α at the protein level and their effects on the proliferation, migration and radiosensitivity of nasopharyngeal carcinoma cells. <h3>Materials/Methods</h3> The cell line was constructed by infecting lentivirus. Western blot analysis was used to verify the infection efficiency and the relationship of BMAL1 gene and HIF-1α in nasopharyngeal carcinoma cells. A cell counting kit cell proliferation test was used to detect the proliferation ability of nasopharyngeal carcinoma cells, scratch test was used to detect the migration ability of nasopharyngeal carcinoma cells, flow cytometry was used to detect apoptosis, and clone formation test was used to detect the effects of BMAL1 and HIF-1α on the radiosensitivity of hone1 nasopharyngeal carcinoma after different doses (0, 2, 4, 6gy) of X-ray irradiation. <h3>Results</h3> Compared with the control group, overexpression of BMAL1 can inhibit the protein expression of HIF-1α in nasopharyngeal carcinoma cells, silencing BMAL1 can promote the protein expression of HIF-1α in nasopharyngeal carcinoma cells. The mechanism of BMAL1 may be related to HIF-1α. The cell counting kit cell proliferation test and scratch test showed that BMAL1 overexpressed or HIF-1α gene silencing could reduce the proliferation and migration of nasopharyngeal carcinoma cells (P <0.05). The apoptosis rate after radiotherapy in the interference group was higher than that in the control group (P<0.05). The apoptosis rate of BMAL1 overexpression group or HIF-1α Interference group after radiotherapy was higher than that of control group (P< 0.05). Clone formation test showed that the clone formation rate and survival score of BMAL1 overexpression group or HIF-1α Interference group were lower than those of negative control group. The radiosensitization ratio of BMAL1 overexpression group was 1.381 and the radiosensitization ratio of HIF-1α interference group was 1.063, the difference was statistically significant. <h3>Conclusion</h3> BMAL1 may affect the protein expression of HIF-1α in protein level. High expression of BMAL1 or interference with HIF-1α can inhibit the proliferation and migration of nasopharyngeal carcinoma cells, increase apoptosis after radiotherapy and improve radiosensitivity.

Circadian clock gene BMAL1 inhibits the proliferation and tumor-formation ability of nasopharyngeal carcinoma cells and increases the sensitivity of radiotherapy

ABSTRACT BMAL1 is a core circadian clock gene that is expressed rhythmically in a variety of tumor cells and is related to cancer cell proliferation and chemoradiotherapy sensitivity. Radiotherapy plays an important role in the treatment of nasopharyngeal carcinoma (NPC). However, the rhythmicity of BMAL1 in NPC, as well as its precise role in radiotherapy, remains unclear. We assessed changes in BMAL1 expression over 48 h in NPC cells and normal nasopharyngeal epithelial cells NP69 using real-time quantitative polymerase chain reaction (RT-PCR) and western blotting (WB). Then, we induced the overexpression and knocked-down the levels of BMAL1 in NPC cells, and subsequently used Cell Counting Kit-8 assays to assess the proliferation of NPC cells. Xenograft tumour growth was used to evaluate the effect of BMAL1 in vivo. Immunohistochemical staining was used to detect the expression of BMAL1 protein in transplanted tumors. Gene Set Enrichment Analysis (GSEA) was performed to explore the biological signaling pathway. Finally, RT-PCR and WB were used to detect the expressions of BMAL1, p53 and p21. The results showed that the mRNA expression levels of circadian clock gene BMAL1 fluctuated rhythmically with time, and the expression levels of BMAL1 also changed depending on the protein levels in NPC and NP69 cells. Overexpression of BMAL1 inhibited the proliferation of NPC cells, while knockdown BMAL1 had the opposite effects. In a xenograft model, we observed that the upregulation of BMAL1 inhibited tumor growth and enhanced the sensitivity of NPC cells to radiotherapy. Ultimately, the downregulation of BMAL1 promoted tumor growth and decreased radiosensitivity. GSEA analysis suggested that BMAL1 significantly affected the p53 pathway. Overexpression of BMAL1 promoted the expression of p53 and p21, while the knockdown of BMAL1 inhibited the expression of p53 and p21. We speculate that BMAL1 has the potential to be a prognostic biomarker and therapeutic target for NPC.

MFSD4A inhibits the malignant progression of nasopharyngeal carcinoma by targeting EPHA2

DNA Methylation can lead to abnormal gene expression. In the present study, we investigated whether the expression of methylated MFSD4A (major facilitator superfamily domain containing 4 A) was downregulated in nasopharyngeal carcinoma (NPC) and whether it is associated with malignant progression and poor prognosis of NPC. Bioinformatic analysis, bisulfite pyrosequencing, quantitative real-time reverse transcription PCR, and western blotting assays were performed to explore the relationship between hypermethylation of MFSD4A and its expression in NPC. The role of MFSD4A in NPC was verified by Cell Cycle Kit 8, transwell assays and flow cytometry in vitro and by animal experiments in vivo. Mass spectrometry, co-immunoprecipitation, and immunofluorescence assays were applied to explore the mechanism by which MFSD4A inhibits NPC. The prognostic significance of MFSD4A or EPHA2 was investigated by immunohistochemical analysis of clinical specimens. Hypermethylation of the promoter region of MFSD4A led to decreased expression of MFSD4A. When MFSD4A expression was upregulated or downregulated, the proliferation, apoptosis, migration, and invasion abilities of NPC cells were altered accordingly. Mechanistically, MFSD4A could specifically bind to and degrade EPH receptor A2 (EPHA2) by recruiting ring finger protein 149 (RNF149), which led to alterations in the EPHA2-mediated PI3K-AKT-ERK1/2 pathway and epithelial-mesenchymal transition (EMT), thereby affecting NPC progression. Clinically, high MFSD4A expression or low-EPHA2 expression was associated with better prognosis for patients with NPC. In all, reduced MFSD4A expression in NPC is caused by promoter hypermethylation. MFSD4A or EPHA2 expression is associated with the malignant biological behavior and prognosis of NPC. MFSD4A is a promising potential therapeutic target for NPC.

Nogo-B promotes invasion and metastasis of nasopharyngeal carcinoma via RhoA-SRF-MRTFA pathway

Distant metastasis remains the major cause for treatment failure in patients with nasopharyngeal carcinoma (NPC). Thus, it is necessary to investigate the underlying regulation mechanisms and potential biomarkers for NPC metastasis. Nogo-B (neurite outgrowth inhibitor B), encoded by reticulon-4, has been shown to be associated with the progression and advanced stage of several cancer types. However, the relationship between Nogo-B and NPC remains unknown. In this study, we found that higher expression of Nogo-B was detected in NPC cells and tissues. Higher expression of Nogo-B was statistically relevant to N stage, M stage, and poor prognosis in NPC patients. Further functional investigations indicated that Nogo-B overexpression could increase the migration, invasion, and metastasis ability of NPC cells in vitro and in vivo. Mechanistically, Nogo-B promoted epithelial-mesenchymal transition (EMT) and enhanced the invasive potency by interacting directly with its receptor NgR3 in NPC. Additionally, overexpression of Nogo-B could upregulate the protein levels of p-RhoA, SRF, and MRTFA. A positive relationship was found between the expression of Nogo-B and the p-RhoA in NPC patients as well as in mouse lung xenografts. Nogo-Bhigh p-RhoAhigh expression was significantly associated with N stage, M stage, and poor prognosis in NPC patients. Notably, CCG-1423, an inhibitor of the RhoA-SRF-MRTFA pathway, could reverse the invasive potency of Nogo-B and NgR3 in NPC cell lines, and decrease the expression of N-Cadherin, indicating that CCG-1423 may be a potential target drug of NPC. Taken together, our findings reveal that Nogo-B enhances the migration and invasion potency of NPC cells via EMT by binding to its receptor NgR3 to regulate the RhoA-SRF-MRTFA pathway. These findings could provide a novel insight into understanding the metastasis mechanism and targeted therapy of advanced NPC.

Influences of S100A8 and S100A9 on Proliferation of Nasopharyngeal Carcinoma Cells through PI3K/Akt Signaling Pathway.

Objective To investigate the effects of S100A8 and S100A9 on proliferation in nasopharyngeal carcinoma cells and the regulatory effects of PI3K/Akt signaling pathway. Methods Nasopharyngeal carcinoma cells (CNE1) were cultured and randomly divided into three groups: control group, S100A8/S100A9 overexpression group, and siRNA S100A8/S100A9 group. CCK-8 method was used to detect the effect of S100A8 and S100A9 on the viability of nasopharyngeal carcinoma cells. The effects of S100A8 and S100A9 on the colony forming ability of nasopharyngeal carcinoma cells were detected by colony forming assay. The effects of S100A8 and S100A9 on the proliferation of nasopharyngeal carcinoma cells were detected by EdU staining. The mRNA levels of PI3K and Akt were detected by RT-PCR. The expression levels of PI3K and Akt in NPC cells were detected by Western blot. Wortmannin, an inhibitor of PI3K/Akt pathway, was used to inhibit the activation of PI3K/Akt pathway. Results Compared with the control group, the cell viability, the number of plate clones, the positive rate of EdU staining, and the mRNA and protein levels of PI3K and Akt were increased in the overexpression group. Compared with the control group, the cell viability, the number of plate clones, the positive rate of EdU staining, and the mRNA and protein levels of PI3K and Akt were decreased in the siRNA group. After inhibiting the activation of PI3K/Akt pathway, the viability of NPC cells in the overexpression group decreased significantly at 48 h and 72 h, while that in the siRNA group increased significantly. Conclusion SiRNA S100A8 and S100A9 could inhibit the proliferation of nasopharyngeal carcinoma cells, and the underlying mechanism may be related to the inhibition of PI3K/Akt signaling pathway.

The Overexpression of NMHC IIA Promoted Invasion and Metastasis of Nasopharyngeal Carcinoma Cells.

Background: Nasopharyngeal carcinoma (NPC) is a kind of head and neck squamous cell carcinoma (HNSCC) with a strong tendency for metastasis and recurrence. Non-muscle myosin heavy chain IIA (NMHC IIA) plays important roles in recurrence and metastasis of cancers. However, the function and mechanism of NMHC IIA expression in NPC remain unclear.Methods: A receiver operating characteristic (ROC) curve was constructed for 141 specimens of HNSCC tissues and 44 control samples from The Cancer Genome Atlas (TCGA) database. Co-expressed genes with MYH9 were identified using LinkedOmics. Transcription factors (TFs) and miRNA regulation network were constructed using Networkanalyst. The migration and invasion ability of nasopharyngeal carcinoma cells were evaluated by in vitro migration and matrigel invasion assays, respectively.Results: The public microarray results showed that MYH9 expression levels were upregulated in HNSCC tissues compared with the matched adjacent normal tissues in this study (p<0.0001). The AUC of MYH9 reached up to 0.8303 at a cutoff value of 175.2, with a sensitivity and specificity of 70.21% and 86.36%, respectively. MYH9 expression was increased in lymph node metastasis HNSCC tumors compared with that in tumors without lymph node metastasis (p<0.05) and showed a strong positive association with expression of FLNA. High MYH9 and FLNA expression were related with poorer overall survival in HNSCC. MYH9 with positively associated genes regulated focal adhesion, cell-substrate junction assembly and cell morphogenesis were involved in differentiation using GO and KEGG analysis. MYH9 was correlated with a network of TFs including SP1, SRF, JUN and FOS in HNSCC. The suppression of endogenous NMHC IIA decreased cellular migration and invasion in HNE1 cells and reduced the expression of phosphorylation of EGFR, AKT and ERK. The over-expression of NMHC IIA increased cellular migration and invasion in COS-7 cells and increased the expression of phosphorylation of EGFR, AKT and ERK.Conclusion: Expression of NMHC IIA mRNA was higher in HNSCC than in the adjacent normal tissues. NMHC IIA expression was increased in lymph node metastasis HNSCC tumors compared with tumors without lymph node metastasis. High MYH9 was association with poorer overall survival in HNSCC. NMHC IIA expression increased the invasion and metastasis abilities of the nasopharyngeal cancer cell line in vitro by augmenting the expression of phosphorylation of EGFR, AKT and ERK. These findings will be beneficial for providing an effectively therapeutic strategy for NPC.

Identified IGSF9 association with prognosis and hypoxia in nasopharyngeal carcinoma by bioinformatics analysis

BackgroundDespite improvements in nasopharyngeal carcinoma (NPC) treatment, patients with recurrence and metastasis still have a poor prognosis. Thus, the identification of novel biomarkers is urgently needed to predict outcomes and tailor treatment for NPC.MethodsFour data sets were downloaded from Gene Expression Omnibus, and one data set GSE68799 of which was applied to filtrate key modules and hub genes by construction of a co-expression network. Other data sets (GSE12452 and GSE53819) were used to verify hub genes. The data set GSE102349 was devoted to identify prognostic hub genes by survival analysis. To explored whether prognostic hub genes are related to hypoxia signatures in NPC, correlation analysis was carried out, and followed by functional verification experiments of those genes in vitro.ResultsBy co-expression network analysis, blue module was regarded as a key module in the benign and malignant group, and IGSF9 of the blue module was identified as a prognostic hub gene. Moreover, IGSF9 is expected to be a innovative hypoxia-related gene in NPC based on the strong associativity between expression of IGSF9 and hypoxia scores of three signatures (99-gene, 26-gene and 15-gene). Further functional studies verified that down-regulated expression of IGSF9 could reduce the proliferation, migration and invasion ability of NPC cells, and hypoxia could induce the expression of IGSF9.ConclusionIGSF9 was identified to be relevant to prognosis and involved in hypoxia in NPC. IGSF9 might serve as one novel prognostic indicator of NPC in the future.

LAPTM4B promotes the progression of nasopharyngeal cancer

Lysosomal protein transmembrane 4 beta (LAPTM4B) is a protein that contains four transmembrane domains. The impact of LAPTM4B on the malignancy of nasopharyngeal carcinoma (NPC) remains unclear. In the present study, we aimed to investigate the role of LAPTM4B in NPC. NPC tissue samples were used to evaluate the expression of LAPTM4B and its relationship with patient prognosis. Furthermore, we inhibited the expression of LAPTM4B in NPC cell lines and examined the effects of LAPTM4B on NPC cell proliferation, migration, and invasion. We found that LAPTM4B protein was mainly localized in the cytoplasm and intracellular membranes of NPC cells. LAPTM4B protein was upregulated in NPC tissues and cell lines. High LAPTM4B expression was closely related to pathological subtypes and disease stages in NPC patients. NPC patients with high LAPTM4B expression had a worse prognosis. LAPTM4B knockdown inhibited the proliferation, migration, and invasion ability of NPC cells. LAPTM4B plays a cancer-promoting role in the progression of NPC and may be a potential target for NPC therapy.

Effects of NOX4 on radiosensitivity of nasopharyngeal carcinoma cells by activating the PI3K/AKT pathway

Objective: To explore the relationship between NOX4 and radiosensitivity of nasopharyngeal carcinoma cells. Methods: Western blot was used to test the expression of NOX4 in nasopharyngeal carcinoma cells (CNE1, CNE2 and HONE1) and normal nasopharyngeal epithelial cells (NP69). The lentiviral vectors for RNA interference and overexpression of NOX4 gene were constructed and nasopharyngeal carcinoma cells were transfected. After treatment with radiation or/and PI3K/AKT inhibitor LY294002, the expressions of related proteins in cells were tested by Western blot, and the cell proliferation was detected by CCK-8 assay and the cell apoptosis was determined by flow cytometry. GraphPad Prism 5 was used for statistical analysis, and P<0.05 was statistically significant. Results: The expressions of NOX4 in nasopharyngeal carcinoma cells were higher than those in normal nasopharyngeal epithelial cells. Compared with the siNC group, the siNOX4 group of nasopharyngeal carcinoma cell had lower proliferation capacity [72 h absorbance (A) value:1.16 vs. 0.75] and higher apoptosis rate (2.9% vs. 10.0%). In contrast,compared with the vector group, the NOX4 group of nasopharyngeal carcinoma cell had higher proliferation capacity [72 h absorbance (A) value: 1.01 vs. 1.32] and lower apoptotic rate (1.7% vs. 1.1%).Treatment with LY294002 for nasopharyngeal carcinoma cells of NOX4 overexpression,compared with the NOX4 group, the proliferation ability of nasopharyngeal carcinoma cells in the NOX4+LY294002 group was reduced (72 h absorbance (A) value: 1.32 vs. 0.77), while the apoptotic rate was increased (1.1% vs. 3.1%).Treatment with radiotherapy, compared with the siNC/Vector group, the proliferation ability of nasopharyngeal carcinoma cells in the siNOX4 group was reduced (72 h absorbance (A) value: 0.72 vs. 0.33), and the apoptotic rate was increased (7.8% vs. 17.3%). However, in the NOX4 group, the proliferation of nasopharyngeal carcinoma cells was enhanced (72 h absorbance (A) value:0.65 vs. 0.78), and the apoptotic rate was reduced (8.1% vs. 3.8%). Compared with the NOX4+radiation group, the proliferation ability of nasopharyngeal carcinoma cells in the NOX4+radiation+LY294002 group was reduced (72 h absorbance (A) value: 0.79 vs. 0.56), while the apoptotic rate was increased (3.8% vs. 8.1%). Conclusion: NOX4 can inhibit radiosensitivity of nasopharyngeal carcinoma cells possibly by activating PI3K/AKT pathway.

LncRNA HOTAIR upregulates COX-2 expression to promote invasion and migration of nasopharyngeal carcinoma by interacting with miR-101

BackgroundNasopharyngeal carcinoma (NPC) is the most common type of head and neck cancers which is notable for its distinctive pattern of geographical distribution. HOTAIR has been reported to regulate nasopharyngeal carcinoma tumorigenesis and progression. However, the detailed mechanism underlying HOTAIR-promoted nasopharyngeal carcinoma remains not fully understood. MethodsWe used RT-qPCR approach to examine genes expression and mRNA level. MTT assay and soft agar assay were used to detect cell growth rate in culture and under suspended condition, respectively. Besides, we employed wound healing assay and transwell invasion assay to determine migration and invasion ability of nasopharyngeal carcinoma cells. We predicted direct downstream targets of miR-101 by bioinformatic analysis, which was confirmed by dual luciferase reporter assay. ResultsHOTAIR was upregulated in NPC tissues and cells. miR-101 inhibitor greatly enhanced HOTAIR knockdown-regulated cell proliferation, migration and invasion of CNE1 and CNE2 cells. miR-101 was shown to directly bind 3′-UTR of COX-2 and downregulate COX-2 expression. Finally, COX-2 overexpression was demonstrated to rescue the tumor phenotypes of nasopharyngeal carcinoma cells attenuated by HOTAIR knockdown or miR-101 mimic. ConclusionsHere, we highlight the importance of HOTAIR/miR-101/COX-2 axis in progression of nasopharyngeal carcinoma cells. Our findings provide a novel mechanism for explaining HOTAIR-induced nasopharyngeal carcinoma and help developing the therapeutical strategies by targeting HOTAIR.

In vitro study of joint intervention of E-cad and Bmi-1 mediated by transcription activator-like effector nuclease in nasopharyngeal carcinoma

To explore the effect of intervention of E-cadherin (E-cad) and B-lymphoma Moloney murine leukemia virus insertion region-1 (Bmi-1) mediated by transcription activator-like effector nuclease (TALEN) on the biological behaviors of nasopharyngeal carcinoma cells. Methods: Multi-locus gene targeting vectors pUC-DS1-CMV-E-cad-2A-Neo-DS2 and pUC-DS1-Bmi-1 shRNA-Zeo-DS2 were constructed, and the E-cad and Bmi-1 targeting vectors were transferred with TALEN plasmids to CNE-2 cells individually or simultaneously. The integration of target genes were detected by PCR, the expressions of E-cad and Bmi-1 were detected by Western blot. The changes of cell proliferation were detected by cell counting kit-8 (CCK-8) assay. The cell cycle and apoptosis were detected by flow cytometry. The cell migration and invasion were detected by Transwell assay. Results: The E-cad and Bmi-1 shRNA expression elements were successfully integrated into the genome of CNE-2 cells, the protein expression level of E-cad was up-regulated, and the protein expression level of Bmi-1 was down-regulated. The intervention of E-cad and Bmi-1 didn't affect the proliferation, cell cycle and apoptosis of CNE-2 cells, but it significantly inhibited the migration and invasion ability of CNE-2 cells. Furthermore, the intervention of E-cad and Bmi-1 together significantly inhibited the migration ability of nasopharyngeal carcinoma cells compared with the intervention of E-cad or Bmi-1 alone (all P<0.01). Conclusion: The joint intervention of E-cad and Bmi-1 mediated by TALEN can effectively inhibit the migration and invasion of nasopharyngeal carcinoma cells in vitro, which may lay the preliminary experimental basis for gene therapy of human cancer.

Study the effect of CTSD interaction proteins in invasion and metastasis of nasopharyngeal carcinoma

Background: Cathepsin D is a lysosomal aspartyl protease. Our previous studies have suggested that cathepsin D plays an important role in invasion and metastasis of nasopharyngeal carcinoma (NPC). Methods: To identify proteins that interact with cathepsin D and gain an insight into the role of cathepsin D in invasion and metastasis of NPC, co-immunoprecipitation (co-IP) combined with mass spectrometry (MS)-based proteomics approach was used. Cathepsin D associated proteins were identified by MS. Protein-protein interaction network were analyzed by bioinformatics including gene ontology (GO), function clustering. And co-IP and western blotting confirmation were for protein-protein interaction. The expression of cathepsin D, epidermal growth factor receptor (EGFR) and heat-shock protein 90A (HSP90A) in NPC were detected by immunohistochemistry (IHC). And the invasion and metastasis capability were detected by Transwell invasion assay. Results: One hundred and forty-one cathepsin D associated proteins were identified, including EGFR and HSP90A, proteins clearly associated with tumor invasion and metastasis. The interaction of these two proteins with cathepsin D was further validated by co-IP followed by western blotting. The 141 proteins were classified into 12 function-related groups. Protein-protein interaction network analysis indicated that cathepsin D might have a significance on invasion and metastasis of NPC by interacting with EGFR and HSP90A. Indeed, cathepsin D/EGFR/HSP90A could form complexes in NPC cells. In addition, overexpression of cathepsin D in NPC cells not only up-regulated EGFR and HSP90A, but also increased the invasive ability of NPC cells. Conclusions: Cathepsin D could enhance the invasion and metastasis capability of NPC cells may through binding to EGFR and HSP90A and triggering the activation of the signaling pathways.

High YBX1 expression indicates poor prognosis and promotes cell migration and invasion in nasopharyngeal carcinoma

Y-box binding protein-1 (YBX1) is a multifunctional protein and often acts as an indicator of poor prognosis in cancers. Increasing evidence has shown that the levels of YBX1 protein were closely associated with multidrug resistance, relapse, metastasis and poor prognosis in cancers. However, its role in nasopharyngeal carcinoma (NPC) metastasis remains unknown. In our study, we discovered that the expression of YBX1 was increased in nasopharyngeal carcinoma tissues. YBX1 protein levels positively correlated with T stage and metastasis of NPC patients. Moreover, expression of YBX1 was negatively correlated with membrane E-cadherin levels and positively correlated with Vimentin expression. In vitro, the expression of YBX1 was closely related to the invasive and migratory ability of nasopharyngeal carcinoma cells. Knockdown of YBX1 inhibited migration and invasion in 5–8F cells, and over-expression of YBX1 promoted CNE1 cells migration and invasion. Transforming growth factor-β1 (TGF-β1) treatment led to epithelial-to-mesenchymal transition (EMT) in CNE1 cells accompanied by elevated YBX1 expression. On the contrary, knockdown of YBX1 partially inhibited the TGF-β1-induced CNE1 cell migration, together with changes of EMT-associated markers. Our study revealed that TGF-β1/YBX1 signaling might be one of novel mechanisms mediating EMT in NPC, providing a new target for the treatment of nasopharyngeal carcinoma.

Effect of ShRNA targeting silencing of VTCN1 gene on nasopharyngeal carcinoma cells

Objective:The aim of this study is to investigate the expression of VTCN1 in nasopharyngeal carcinoma cells and targeting silencing of VTCN1 gene by infected with lentiviral vector to inhibit the proliferation and invasion in nasopharyngeal carcinoma cells. Method:The VTCN1 expression level of different nasopharyngeal carcinoma cells was detected by RT-PCR and Western blotting, the cell lines of the most expression level were seleted to conduct the Subsequent experiments; The lentiviral vector of silenced VTCN1 was transfected into HNE2 cells with VTCN1 expression by lipofectamine 2000, and stable cell lines were screened. Then, the silencing efficiency was detect by RT-PCR and Western blotting; The proliferation and invasion abilities of nasopharyngeal carcinoma cells after VTCN1 gene silencing were detected by ckk-8 and Transwell invasion assay, respectively. The phosphorylation levels of JAK/STAT proteins in nasopharyngeal carcinoma cells with VTCN1 gene silencing were detected by Western blotting. Result:RT-PCR and Western blotting detected that stable transfection of VTCN1ShRNA into HNE2 cells resulted significantly declined expression of VTCN1 (P<0.05); The proliferation and invasion abilities of HNE2 cells were significantly decreased (P<0.05) and phosphorylation level of the JAK, STAT proteins were significantly decreased (P<0.05). Conclusion:VTCN1 ShRNA can effectively silence the expression of VTCN1, and significantly inhibits the proliferation and invasion of HNE2 cells. It may be related to down regulation of protein activity in JAK/STAT signaling pathway..

Annexin A1 can inhibit the in vitro invasive ability of nasopharyngeal carcinoma cells possibly through Annexin A1/S100A9/Vimentin interaction.

Annexin A1 is a member of a large superfamily of glucocorticoid-regulated, calcium- and phospholipid-binding proteins. Our previous studies have shown that the abnormal expression of Annexin A1 is related to the occurrence and development of nasopharyngeal carcinoma (NPC). To understand the roles of Annexin A1 in the tumorigenesis of NPC, targeted proteomic analysis was performed on Annexin A1-associated proteins from NPC cells. We identified 436 proteins associated with Annexin A1, as well as two Annexin A1-interacted key proteins, S100A9 and Vimentin, which were confirmed by co-immunoprecipitation. Gene function classification revealed that the Annexin A1-associated proteins can be grouped into 21 clusters based on their molecular functions. Protein–protein interaction analysis indicated that Annexin A1 /S100A9/Vimentin interactions may be involved in the invasion and metastasis of NPC because they can form complexes in NPC cells. The down-regulation of Annexin A1 in NPC may lead to the overexpression of S100A9/Vimentin, which may increase the possibility of the invasion ability of NPC cells by adjusting the function of cytoskeleton proteins. Results suggested that the biological functions of Annexin A1 in NPC were diverse, and that Annexin A1 can inhibit the in vitro invasive ability of NPC cells through Annexin A1 /S100A9/Vimentin interaction.

Liquiritigenin Enhances the Radiosensitivity of Nasopharyngeal Carcinoma Cell by Affecting Its Autophagy

Objective: To investigate the effect of licorice on radiosensitization of nasopharyngeal carcinoma cells and its mechanism. Methods: MTT assay was used to detect the effect of different concentrations of glycyrrhizin on the activity of nasopharyngeal carcinoma cells. The changes of autophagy were observed by transmission electron microscopy (TEM) in the treatment of nasopharyngeal carcinoma cells. Western blotting was used to detect the effect of glycyrrhizin on the level of autophagy protein in nasopharyngeal carcinoma. Flow cytometry was used to detect the changes of apoptosis rate of nasopharyngeal carcinoma cell line after radiotherapy. Results: After successfully constructing CNE-2-RR radiation resistant cell line, MTT assay was used to detect the best inhibition rate of 20 mmol/L glycyrrhizin on nasopharyngeal carcinoma (58.86 ± 5.02)%. Transmission electron microscopy showed that the number of autophagosomes increased and the mitochondria and nuclei were abnormal in the treatment of nasopharyngeal carcinoma cells. Western Blotting was used to detect the increase of LC3-II level and the decrease of LC3-I level in nasopharyngeal carcinoma cells of 20 mmol/L concentration group. Conclusion: Glycyrrhizin enhances its sensitivity to radiotherapy by influencing autophagy and DNA repair ability of nasopharyngeal carcinoma cells.

Epigenetic inactivation of follistatin-like 1 mediates tumor immune evasion in nasopharyngeal carcinoma.

Follistatin like-1 (FSTL1) is a secreted glycoprotein involved in a series of physiological and pathological processes. However, its contribution to the development of cancer, especially the pathogenesis of nasopharyngeal carcinoma (NPC), remains to be elucidated. We aimed to investigate the dysregulation of FSTL1 and its possible function in NPC. FSTL1 was frequently downregulated in NPC cell lines and primary tumor biopsies by promoter hypermethylation. Ectopic expression of FSTL1 significantly suppressed the colony formation, proliferation, migration and invasion ability of NPC cells and induced cell apoptosis. Overexpression of FSTL1 decreased the tumorigenicity of NPC cells in vivo. In addition, the proliferation of NPC cells in vitro was inhibited by treatment with soluble recombinant FSTL1 protein. The protein level of FSTL1 was decreased in primary NPC tumors and was associated with downregulated interleukin 1β (IL-1β) and tumor necrosis factor α (TNF-α). Furthermore, recombinant human FSTL1 protein induced secretion of IL-1β and TNF-α in macrophage cultures, therefore FSTL1 might activate macrophages and attenuate the immune evasion of NPC cells. In conclusion, the epigenetic downregulation of FSTL1 may suppress the proliferation and migration of NPC cells, leading to dysfunctional innate responses in surrounding macrophages.

Cucurbitacin I elicits anoikis sensitization, inhibits cellular invasion and in vivo tumor formation ability of nasopharyngeal carcinoma cells

Nasopharyngeal carcinoma (NPC) is an Asian-prevalent head and neck cancer with high invasiveness. Although several important risk factors for NPC development have been identified, there is currently no preventive strategy for NPC, even in endemic regions. Signal transducer and activator of transcription 3 (STAT3) has been implicated in NPC carcinogenesis, which may serve as a potential target for cancer prevention. Here, we examined the chemopreventive potential of Cucurbitacin I, a natural-occurring selective inhibitor of JAK/STAT3, in NPC models. We hypothesized that Cucurbitacin I would prevent NPC invasion and tumor formation. Our data demonstrated that brief exposure of NPC cells to Cucurbitacin I was sufficient to significantly reduce the in vitro clonogenicity and in vivo tumorigenicity of NPC cells. The chemopreventive potential of Cucurbitacin I was further demonstrated by pre-dosing of the animals with Cucurbitacin I prior to tumor inoculation, which was found to be able to suppress tumor growth up to 7 days post-inoculation. The anti-proliferation activity of Cucurbitacin I was accompanied by downregulation of phospho-STAT3 and STAT3 target gene expression (e.g. cyclin D1 and Mcl-1). Cucurbitacin I also reduced the invasiveness of invasive NPC cell lines with elevated STAT3 activation. Furthermore, our data demonstrated for the first time that Cucurbitacin I harbored potent anoikis-sensitization activity (i.e. sensitizing cancer cells to detachment-induced cell death) against human cancer. Taken together, our results suggested that Cucurbitacin I may be a potent chemopreventive agent for NPC with anti-invasion and anoikis-sensitizing activities.

The expression of ezrin in NPC and its interaction with NGX6, a novel candidate suppressor

Ezrin, the linker between membrane protein and cytoskeleton, plays an important role in the cellular morphology, cytoskeleton reorganization, adhesion, invasion and metastasis. In this study, ezrin was found to express in high levels either in nasopharyngeal carcinoma tissues or in nasopharyngeal carcinoma 5-8F cells and the knockdown of ezrin expression in the 5-8F cells by RNA interferance could reduce invasive ability, suggesting that ezrin is involved in the progression and invasion of nasopharyngeal carcinoma. Nasopharyngeal carcinoma-associated gene 6 is a novel candidate suppressor gene of tumor metastasis, which was originally cloned in nasopharyngeal carcinoma high-frequency heterozygosity loss region 9p21-22 and is down-regulated in nasopharyngeal carcinoma. In the present study, we hypothesize that nasopharyngeal carcinoma-associated gene 6 plays an inhibitory role in the migration and invasion of nasopharyngeal carcinoma cells via modulating the function of ezrin. Firstly, different mutants of NGX6 were constructed and transfected into nasopharyngeal carcinoma 5-8F cells. The invasion and migration of 5-8F cells overexpressing nasopharyngeal carcinoma-associated gene 6 or mutants were measured. The results showed that enhanced expression of nasopharyngeal carcinoma-associated gene 6 could reduce invasive and migratory abilities of 5-8F cells, and the cytoplasmic domain was essential for nasopharyngeal carcinoma-associated gene 6 to modulate cell migration and invasion. Further experiment results showed that nasopharyngeal carcinoma-associated gene 6 protein was associated with ezrin by its cytoplasm region, and it could down-regulate the expression level of ezrin. These results demonstrated that nasopharyngeal carcinoma-associated gene 6 was probably involved in the modulation of invasive and adhesive ability of nasopharyngeal carcinoma cells by down-regulating the expression level of ezrin.