CXCL11 secreted by cancer-associated fibroblasts promotes nasopharyngeal carcinoma progression via CXCR3/PD-L1 axis

This study explored whether the migration, invasion, and apoptosis of nasopharyngeal carcinoma (NPC) cells were affected by the CXCR4/CXCR7-CXCL12 axis and if this mechanism was related to G-protein signaling pathway. A total of 72 NPC patients admitted in our hospital between April 2013 and February 2015 were incorporated in this study. Immunohistochemistry was performed to compare the expression levels of CXCR4, CXCR7, and CXCL12 between NPC tissues and adjacent normal tissues. Then, the correlation analysis was implemented to assess the association among CXCR4, CXCR7, and CXCL12 expressions. Jellyfish glow protein experiment was carried out after the cultivation of CNE-2Z cell lines in order to observe the intracellular calcium mobilization resulted from G-protein activation contributed by CXCR4/CXCR7-CXCL12 axis. The impact of CXCR4/CXCR7-CXCL12 axis on the migration and invasion of NPC cells was explored using transwell experiments. Finally, the anti-apoptosis effects of CXCR4/CXCR7-CXCL12 axis on NPC cells were investigated by the splicing of poly ADP-ribose polymerase (PARP). Compared to NPC patients with low-grade (stage I-II) tumor node metastasis (TNM) and those without lymph node metastasis, the expression of CXCR4, CXCR7, and CXCL12 were significantly higher in NPC patients with high-grade (stage III-IV) TNM and those with lymph node metastasis (P < 0.05). Moreover, there was significant positive correlation between the expression level of CXCL12 and CXCR7 (r s = 0.484, P < 0.001) as well as the expression level of CXCL12 and CXCR4 (r s = 0.414, P < 0.001). As suggested by cellular experiments using CNE-2Z, the calcium mobilization degree induced by CXCR4-CXCL12 axis in activating G proteins seemed to be slightly more effective than that induced by CXCR4/CXCR7-CXCL12 axis, while the CXCR7-CXCL12 axis could hardly activate calcium mobilization. Furthermore, the transwell experiment showed that CXCR4/CXCR7-CXCL12 axis could exacerbate the migration and invasion of NPC cells (P < 0.05). The transwell experiment also suggested that the CXCR4/CXCR7-CXCL12 axis was associated with the expression of matrix metallo proteinase 9 (MMP9) which is a substance in the downstream of G-protein pathways (P < 0.05). Results from PARP shear zone also indicated that the CXCR4/CXCR7-CXCL12 axis could suppress NPC cell apoptosis (P < 0.05). The expressional levels of CXCR4, CXCR7, and CXCL12 significantly varied with clinical stages and status of lymph node metastasis of NPC patients. This revealed potential indicators which can be used for NPC prognosis. Additionally, the CXCR4/CXCR7-CXCL12 axis may regulate the expression of downstream proteins (e.g., MMP-9) through the activation of G-protein signaling pathways. These conclusions may provide key evidence for NPC aetiology which can be further investigated to develop novel molecular targets for NPC treatments.

Peptidyl arginine deiminase 4 (PADI4), an enzyme that converts arginine residues to citrulline residues in the presence of calcium ions, affects the biochemical activities of proteins. The biological function of PADI4 as well as its mechanism in nasopharyngeal carcinoma (NPC) necessitates further investigation. PADI4 expression in NPC tissues and cells was detected using Western blot. qRT-PCR was used to determine the expression of miR-335-5p and PADI4 mRNA in NPC tissues and cells. BrdU assay and CCK-8 assay were employed to detect cell proliferation. Cell migration and invasion were evaluated using Transwell assay. NPC cells were exposed to different doses of radiation in vitro, and then colony formation assays were used to detect colony survival. The target relationship between miR-335-5p and PADI4 was verified using Western blot, qRT-PCR, and dual-luciferase reporter gene assays. Compared with normal mucosal epithelial tissues and cell lines, the expression level of PADI4 in NPC tissues and cells was significantly up-regulated. PADI4 overexpression promoted the proliferation, migration, and invasion of NPC cells. Under radiation, NPC cell survival was significantly promoted by the up-regulation of PADI4. Conversely, knock-down of PADI4 suppressed the above-mentioned malignant phenotypes. MiR-335-5p could bind with the 3' UTR of PADI4 mRNA, and suppressed the expression of PADI4. PADI4 down-regulated the expression of p21 and activated the mTOR signaling pathway. PADI4, which is negatively regulated by miR-335-5p, promotes the proliferation, migration, invasion and radioresistance of NPC cells by regulating the p21 and mTOR signaling pathways.

Nasopharyngeal carcinoma (NPC) is a highly invasive epithelial malignant tumor with unique geographical and ethnic distribution characteristics. NPC is mostly found in south China and Southeast Asia, and its treatment mainly depends on radiotherapy and chemotherapy. However, NPC is usually found in the late stage, and local recurrence and distant metastasis are common, leading to poor prognosis. The receptor tyrosine kinase AXL is up-regulated in various tumors and it is involved in tumor proliferation, migration, invasion, and other processes, which are associated with poor prognosis of tumors. This study aims to detect the expression of AXL in NPC cell lines and tissues, and to investigate its biological function of AXL and the underlying molecular mechanisms in regulation of NPC. The expression levels of AXL in normal nasopharyngeal epithelial tissues and NPC tissues were analyzed by GSE68799, GSE12452, and GSE53819 data sets based on Gene Expression Omnibus (GEO) database. The Cancer Genome Atlas (TCGA) database was used to analyze the relationship between AXL and prognosis of head and neck squamous cell carcinoma (HNSC). The indicators of prognosis included overall survival (OS), disease-free interval (DFI), disease-specific survival (DSS), and progression-free interval (PFI). Western blotting assay was used to detect the AXL protein expression levels in normal nasopharyngeal epithelial cell line and NPC cell lines. Immunohistochemical method was used to detect AXL expression levels in normal nasopharyngeal epithelial tissues and NPC tissues. Cell lines with stable AXL knockdown were established by infecting 5-8F and Fadu cells with lentivirus interference vector, and cell lines with stable AXL overexpression were established by infecting C666-1 and HK-1 cells with lentivirus expression vector. Real-time PCR and Western blotting were used to detect the efficiency of knockdown and overexpression in stable cell lines. The effects of AXL knockdown or overexpression on proliferation, migration, and invasion of NPC cells were detected by CCK-8, plate colony formation, and Transwell assays, and the effect of AXL knockdown on tumor growth in nude mice was detected by subcutaneous tumor formation assay. The sequence of AXL upstream 2.0 kb promoter region was obtained by UCSC online database. The PROMO online database was used to predict AXL transcription factors with 0% fault tolerance, and the JASPAR online database was used to predict the binding sites of ETS1 to AXL. Real-time PCR and Western blotting were used to detect the effect of ETS1 on AXL protein and mRNA expression. The AXL upstream 2.0 kb promoter region was divided into 8 fragments, each of which was 250 bp in length. Primers were designed for 8 fragments. The binding of ETS1 to AXL promoter region was detected by chromatin immuno-precipitation (ChIP) assay to determine the direct regulatory relationship between ETS1 and AXL. Rescue assay was used to determine whether ETS1 affected the proliferation, migration, and invasion of NPC cells through AXL. Bioinformatics analysis showed that AXL was highly expressed in NPC tissues (P<0.05), and AXL expression was positively correlated with OS, DFI, DSS, and PFI in HNSC patients. Western blotting and immunohistochemical results showed that AXL was highly expressed in NPC cell lines and tissues compared with the normal nasopharyngeal epithelial cell line and tissues. Real-time PCR and Western blotting results showed that knockdown and overexpression efficiency in the stable cell lines met the requirements of subsequent experiments. The results of CCK-8, plate colony formation, Transwell assays and subcutaneous tumor formation in nude mice showed that down-regulation of AXL significantly inhibited the proliferation, migration, invasion of NPC cells and tumor growth (all P<0.05), and the up-regulation of AXL significantly promoted the proliferation, migration, and invasion of NPC cells (all P<0.05).As predicted by PROMO and JASPAR online databases, ETS1 was a transcription factor of AXL and had multiple binding sites in the AXL promoter region. Real-time PCR and Western blotting results showed that knockdown or overexpression of ETS1 down-regulated or up-regulated AXL protein and mRNA expression levels. ChIP assay result showed that ETS1 bound to AXL promoter region and directly regulate AXL expression. Rescue assay showed that AXL rescued the effects of ETS1 on proliferation, migration and invasion of NPC cells (P<0.05). AXL is highly expressed in NPC cell lines and tissues, which can promote the malignant progression of NPC, and its expression is regulated by transcription factor ETS1.

This study investigated the underlying mechanism of miR-18a-5p regulating the proliferation, invasion, and metastasis of nasopharyngeal carcinoma (NPC) cells in vitro and in vivo to indicate the pathogenesis of NPC. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) was utilized to determine miR-18a-5p expression level in NPC tissues and cell lines. Besides, 2,5-diphenyl-2 H-tetrazolium bromide (MTT) and colony formation assays were employed to detect the effect of miR-18a-5p expression level on NPC cell proliferation. Wound healing and Transwell assays were utilized to detect the effect of miR-18a-5p on NPC cell invasion and migration. The expression levels of epithelial-mesenchymal transition (EMT)-related proteins (Vimentin, N-cadherin, and E-cadherin) were identified by Western blot assay. After collecting exosomes from CNE-2 cells, it was found that exosomal miR-18a-5p secreted from NPC cells promoted NPC cell proliferation, migration, invasion, and EMT, whereas inhibition of miR-18a-5p expression level led to the opposite results. The dual-luciferase reporter assay showed that BTG anti-proliferation factor 3 (BTG3) was the target gene of miR-18a-5p, and BTG3 could overturn the effect of miR-18a-5p on NPC cells. Xenograft mouse model of NPC nude mice showed that miR-18a-5p promoted NPC growth and metastasis in vivo. This study revealed that exosomal miR-18a-5p derived from NPC cells promoted angiogenesis via targeting BTG3 and activating the Wnt/β-catenin signaling pathway.

BackgroundLong noncoding RNAs (lncRNAs) are known as key regulators in many cancer types, but their biological functions in nasopharyngeal carcinoma (NPC) remain largely unknown. In the present study, we aim to explore the role of the lncRNA ZNRD1-AS1 in NPC tumor development.MethodsThe role of ZNRD1-AS1 in NPC tissues and cells was explored by using quantitative real-time PCR assay. Cellular behavioral experiments were used in testing NPC cell proliferation, invasion, and migration. Luciferase reporter assay, RNA-binding protein immunoprecipitation, and Western blot analysis were used in estimating the associations among ZNRD1-AS1, miR-335, and ROCK1.ResultsZNRD1-AS1 expression was elevated in the NPC tissues and cells, and ZNRD1-AS1 overexpression was positively correlated with advanced TNM stage and the presence of lymph node metastasis. Our biological experiments indicated that ZNRD1-AS1 knockdown reduces NPC cell invasion and metastasis. Further analyses revealed that ZNRD1-AS1 as a ceRNA promotes the migration and invasion of NPC cells by sponging miR-335. We provided evidence that ZNRD1-AS1 facilitates the invasion and metastasis of NPC cells via the miR-335–ROCK1 axis.ConclusionOur data shed light on the oncogenic role of ZNRD1-AS1 in NPC tumor development, and a promising therapeutic target for NPC was identified.

BackgroundIn recent years, miR-152 has been dysregulated in a variety of tumors and used as a tumor suppressor. Nevertheless, its role in nasopharyngeal carcinoma (NPC) remains unidentified.Materials and methodsReal-time quantitative PCR (polymerase chain reaction) was performed to analyze the expression of miR-152 in NPC cell lines. MiR-152 expression profiles in NPC tissues were obtained from Gene Expression Omnibus (GEO GSE36682). The effect of miR-152 on the invasion and proliferation of NPC cells was determined through cell invasion, wound healing, and cell viability assays. Apoptosis was examined by flow cytometry, and Western blot was performed to measure expression of the target gene. Pyrosequencing was used to detect the methylation level of NPC cells.ResultsIn this study, miR-152 was downregulated in the NPC tissues and cell lines. When miR-152 was enhanced, the invasion and migration of NPC cells were inhibited. However, miR-152 had no effect on the proliferation of NPC cells. Luciferase reporter gene analysis was performed, and the results showed that DNMT1 (DNA (cytosine-5)-methyltransferase 1) is a direct target of miR-152 in NPC cells. DNMT1 downregulation and miR-152 overexpression both reversed the effects of miR-152 inhibition on the NPC cells. In addition, miR-152 expression increased as a result of the inhibition of the methylation level of miR-152 when DNMT1 expression was downregulated.ConclusionThe overexpression of miR-152 inhibited the migration and invasion of NPC cells by targeting DNMT1. Furthermore, DNMT1 regulated miR-152 expression through DNA methylation. Overall, the novel miR-152-DNMT1 regulatory circuit may provide better understanding of the pathogenesis of NPC and new epigenetic therapeutic target in NPC.

BackgroundE2F transcription factor 3 (E2F3) is oncogenic and dysregulated in various malignancies. Complex networks involving microRNAs (miRNAs) and E2F3 regulate tumorigenesis and progression. However, the potential roles of E2F3 and its target miRNAs in nasopharyngeal carcinoma (NPC) are rarely reported.MethodsE2F3 expression was detected in human NPC tissues and cell lines through quantitative real-time PCR. NPC cell proliferation, migration, and invasion were evaluated in vitro by colony forming, cell counting kit-8, wound healing, and Transwell invasion assays. Publicly available database software was used to explore the target miRNAs of E2F3. Dual-luciferase reporter assay was performed to identify the direct relationship. The function of miRNAs in vivo was investigated by using a tumor xenograft model.ResultsE2F3 was upregulated in NPC cell lines and tissues, and its exotic expression promoted NPC cell invasion and migration. E2F3 was identified as a target of miR-432, which restrained NPC cell invasion and migration in vitro and in vivo. Further experiments revealed that miR-432 repressed the invasion and migration potential of NPC cells by modulating E2F3 expression.ConclusionmiRNA-432 suppressed the malignant biological behavior of NPC cells by targeting E2F3. This study provided further insights into NPC prognosis and treatment.

The emerging concept of generating cancer stem cells from epithelial-mesenchymal transition has attracted great interest; however, the factors and molecular mechanisms that govern this putative tumor-initiating process remain largely elusive. We report here that miR-200a not only regulates epithelial-mesenchymal transition but also stem-like transition in nasopharyngeal carcinoma cells. We first showed that stable knockdown of miR-200a promotes the transition of epithelium-like CNE-1 cells to the mesenchymal phenotype. More importantly, it also induced several stem cell-like traits, including CD133(+) side population, sphere formation capacity, in vivo tumorigenicity in nude mice, and stem cell marker expression. Consistently, stable overexpression of miR-200a switched mesenchyme-like C666-1 cells to the epithelial state, accompanied by a significant reduction of stem-like cell features. Furthermore, in vitro differentiation of the C666-1 tumor sphere resulted in diminished stem-like cell population and miR-200a induction. To investigate the molecular mechanism, we demonstrated that miR-200a controls epithelial-mesenchymal transition by targeting ZEB2, although it regulates the stem-like transition differentially and specifically by β-catenin signaling. Our findings reveal for the first time the function of miR-200a in shifting nasopharyngeal carcinoma cell states via a reversible process coined as epithelial-mesenchymal to stem-like transition through differential and specific mechanisms.

Nasopharyngeal carcinoma (NPC) is one of the most severe types of malignant cancer of the head and neck as it is difficult to treat. Ezrin is highly expressed in numerous types of cancer. However, the role of Ezrin in NPC has not been fully investigated and further studies are required in order to uncover its therapeutic potential in the treatment of NPC. The aim of the present study was to investigate the expression of Ezrin in human NPC and to evaluate the effect of knockdown of Ezrin using small interfering (si)-RNA on NPC cell migration and invasion. The expression levels of Ezrin were determined using reverse transcription-quantitative polymerase chain reaction, immunohistochemical staining and western blotting. Following transfection of Ezrin-siRNA into NPC cells, cell invasion and migration were analyzed and the mRNA expression levels of matrix metalloproteinase(MMP)-2 and MMP9 were determined. The results revealed that the expression of Ezrin was markedly increased in human NPC tissue samples compared with normal adjacent nasopharyngeal tissue samples. Ezrin was also highly expressed in the NPC cell lines 6–10B and C6661 when compared with the normal nasopharyngeal cell line NP69. Transfection of NPC cell lines with siRNA targeting Ezrin significantly inhibited NPC cell migration and invasion, and downregulated the mRNA expression level of MMP2; however, no effect was observed on MMP9 mRNA expression. At the same time, knockdown of Ezrin significantly decreased the expression levels of phosphatidylinositol 3-kinase (PI3K) and phosphorylated protein kinase B (Akt), which downregulated the mRNA expression of MMP2. In conclusion, the results revealed that knockdown of Ezrin suppressed NPC migration and invasion by reducing the mRNA expression of MMP2 via the PI3K/Akt signaling pathway. These results highlight the important role of Ezrin in NPC cell migration and invasion. In addition, they indicate that silencing of Ezrin may serve as a potential therapeutic strategy to treat human NPC.

Deuterium-depleted water (DDW) inhibits the proliferation and migration of nasopharyngeal carcinoma cells in vitro

High expression levels of the forkhead box M1 (FoxM1) transcription factor are associated with metastasis and poor prognosis of malignancies. However, little is known concerning its function in nasopharyngeal carcinoma (NPC). The present study aimed to investigate the impact of FoxM1 inhibition on the migration and invasion of NPC cells and the potential mechanisms. The effects of FoxM1 inhibitor treatment and FoxM1 silencing on the proliferation, migration and invasion of NPC CNE-1 and CNE-2 cells were examined by CCK-8, Transwell migration/invasion and colony formation assays. The effects of stable FoxM1 silencing on the growth and lung metastasis of implanted NPC were evaluated. The relative levels of FoxM1, zinc finger E-box binding homeobox 2 (ZEB2), Snail2 and E-cadherin in the different groups of NPC cells and tumors were determined by quantitative real-time PCR, western blotting and immunohistochemical assays. Treatment with thiostrepton, FoxM1 inhibitor, significantly reduced the survival of NPC cells. Treatment with thiostrepton and/or knockdown of FoxM1 inhibited the anchorage-independent proliferation, migration and invasion of NPC cells. Inhibition of FoxM1 also increased the relative levels of E-cadherin, but reduced ZEB2 and Snail2 expression in NPC cells. Stable FoxM1 silencing inhibited the growth and lung metastasis of implanted NPC in vivo, which was associated with increased levels of E-cadherin, but decreased ZEB2 and Snail2 expression in the NPC tumors. In conclusion, our data clearly indicate that knockdown of FoxM1 inhibited the growth and metastasis of human NPC by modulating epithelial-to-mesenchymal transition (EMT), and FoxM1 may be a potential target for the intervention of NPC.

Recent studies have revealed the important role of long noncoding RNAs (lncRNAs) in the progression of tumorigenesis. This study aimed to identify the biological function of lncRNA small nucleolar RNA host gene 7 (SNHG7) in the progression of nasopharyngeal carcinoma (NPC). LncRNA SNHG7 expressions in NPC cell lines and 50 paired NPC tissue samples were detected by Real-time quantitative polymerase chain reaction (RT-qPCR). Transwell assay, wound healing assay and proliferation assay were conducted to evaluate the in vitro function of SNHG7 in NPC cells. Xenograft model was established for determining the in vivo effect of SNHG7 on tumor formation and metastasis of NPC. The underlying mechanism of SNHG7 in mediating the progression of NPC was explored by RT-qPCR and Western blot. SNHG7 expression was remarkably downregulated in NPC tissues compared with that in adjacent normal samples. Knockdown of SNHG7 attenuated proliferation, invasion and migration of NPC cells. Moreover, tumor size and the number of metastatic nodules were reduced in mice administrated with NPC cells transfected with sh-SNHG7. Knockdown of SNHG7 downregulated ROCK1 at mRNA and protein level. Besides, the expression of ROCK1 in tumor tissues was positively correlated to SNHG7 expression. Knockdown of SNHG7 inhibits migration, invasion and proliferation of NPC cells through downregulating ROCK1, which may offer a new therapeutic intervention for NPC patients.

Accumulating evidences highlight the critical roles of long noncoding RNAs (lncRNAs) in a variety of cancers. LncRNA PXN‐AS1‐L was previously shown to exert oncogenic roles in hepatocellular carcinoma. However, the expression, role, and molecular mechanism of PXN‐AS1‐L in nasopharyngeal carcinoma (NPC) malignancy remain unknown. Here, we determined that PXN‐AS1‐L is upregulated in NPC tissues and cell lines. Increased expression of PXN‐AS1‐L predicts worse prognosis of NPC patients. PXN‐AS1‐L overexpression promotes NPC cell proliferation, migration, and invasion in vitro, and NPC tumor growth in vivo. PXN‐AS1‐L silencing suppresses NPC cell proliferation, migration, and invasion in vitro. Mechanistically, PXN‐AS1‐L directly interacts with SAPCD2 mRNA 3′‐untranslated region, prevents the binding of microRNAs‐AGO silencing complex to SAPCD2 mRNA, and upregulates the mRNA and protein level of SAPCD2. SAPCD2 is also increased in NPC tissues. The expression of SAPCD2 is significantly positively associated with that of PXN‐AS1‐L in NPC tissues. Gain‐of‐function and loss‐of‐function experiments demonstrated that SAPCD2 also promotes NPC cell proliferation, migration, and invasion. Furthermore, depletion of SAPCD2 significantly reverses the roles of PXN‐AS1‐L in promoting NPC cell proliferation, migration, and invasion in vitro, and NPC tumor growth in vivo. In conclusion, lncRNA PXN‐AS1‐L is upregulated in NPC and promoted NPC malignancy by upregulating SAPCD2 via direct RNA‐RNA interaction.

Epithelial-to-mesenchymal transition (EMT) process is prevalent during the progression of tumors. Nasopharyngeal carcinoma (NPC) is no exception. High-mobility group box 1 (HMGB1) was reported to have the effect of inducing EMT in malignancy. However, the impact of HMGB1-induced EMT in NPC is unclear. Resolvin D1 (RvD1) was reported to regress the progression of inflammation and apoptosis of phagocytes. The effect of RvD1 in the EMT is largely unknown. The current research explored the role of RvD1 on HMGB1-induced EMT in NPC. EMT markers were investigated in 10 NPC and 10 nasopharyngitis (NPG) patients using immunohistochemistry and Western blot. In vitro, expression of EMT markers and HMGB1 in CNE1 and CNE2 cells was assessed with immunohistochemical, Western blot, and confocal microscopy after treatment with recombinant human HMGB1 (rhHMGB1) or HMGB1 gene silencing or RvD1. The invasion and migration of NPC cells were detected by scratch test and transwell assay. Overexpression and gene silencing of lipoxin A4 receptor/formyl peptide receptor 2 (ALX/FPR2) and G protein-coupled receptor 32 (GPR32) in CNE2 cells confirmed the effect of RvD1 using Western blots. N-cadherin, vimentin, and HMGB1 were found up-regulated in NPC samples compared with NPG samples, while ZO-1 and E-cadherin were down-regulated in NPC tissues. RhHMGB1-induced EMT in CNE1 and CNE2 cells in a dose-dependent way. CNE2 cell lines treated with rhHMGB1 possessed greater invasion and migration ability, which was confirmed by gene silencing. RvD1 suppressed HMGB1-induced EMT in NPC cells via ALX/FPR2 and GPR32 receptors. These results showed that EMT was obvious in NPC. HMGB1 played a key role in inducing EMT. RvD1 inhibited HMGB1-induced EMT and might have potential application in the area of NPC treatment. Impact statement Nasopharyngeal carcinoma has a high incidence in China. Discussing the molecular mechanism of nasopharyngeal carcinoma is important because of high recurrent rate and low quality of life after treatment. HMGB1, as an important inflammatory factor, promotes the process in many cancers. But little is known about how HMGB1 affects the progress of nasopharyngeal carcinoma cells. In our research, we assessed the role of HMGB1 on metastasis and invasion of nasopharyngeal carcinoma cells. The result of study indicates HMGB1-induced EMT in nasopharyngeal carcinoma cells. Furthermore, we observed that RvD1, which plays an actively protective role in many diseases, controls the migration and invasion of nasopharyngeal carcinoma cells by inhibiting the HMGB1-induced EMT. RvD1 can be further studied as a protective factor for nasopharyngeal carcinoma.

625 Comparison of gene expression profiles in breast cancer cells treated with 5-fluorouracil, cisplatin or etoposide using cDNA microarray