Nasopharyngeal Carcinoma Cell Line CNE2 Research Articles

Nasopharyngeal carcinoma with non-squamous phenotype may be a variant of nasopharyngeal squamous cell carcinoma after inhibition of EGFR/PI3K/AKT/mTOR pathway.

Nasopharyngeal carcinoma (NPC) is a cancerous tumor that develops in the nasopharynx epithelium and typically has squamous differentiation. The squamous phenotype is evident in immunohistochemistry, with diffuse nuclear positivity for p63 and p40. Nonetheless, a few NPCs have been identified by clinicopathological diagnosis that do not exhibit the squamous phenotype; these NPCs are currently referred to as non-squamous immunophenotype nasopharyngeal carcinomas (NSNPCs). In a previous work, we have revealed similarities between the histological appearance, etiology, and gene alterations of NSNPC and conventional NPC. According to ultrastructural findings, NSNPC still falls under the category of non-keratinized squamous cell carcinoma that is undifferentiated. NSNPC has an excellent prognosis and a low level of malignancy, according to a retrospective investigation. Based on prior research, we investigated the molecular mechanism of NSNPC not expressing the squamous phenotype and its biological behavior. IHC was used to determine the expression of EGFR, PI3K, AKT, p-AKT, mTOR, p-mTOR, Notch, STAT3 and p-STAT3 in a total of 20 NSNPC tissue samples and 20 classic NPC tissue samples. We obtained human NPC cell lines (CNE-2,5-8F) and used EGFR overexpression plasmid and shRNAs to transfect them. To find out whether mRNA and proteins were expressed in the cells, we used Western blotting and qRT-PCR. Cell biological behavior was discovered using the CCK-8 assay, cell migration assay, and cell invasion assay. EGFR, PI3K, p-AKT and p-mTOR proteins were lowly expressed in NSNPC tissues by immunohistochemistry, compared with classical NPC. In the classical NPC cell lines CNE-2 and 5-8F, overexpression EGFR can up-regulate the expression of p63 through the PI3K/AKT/mTOR pathway, and promote the proliferation, migration, and invasion of nasopharyngeal carcinoma cells. At the same time, knockout of EGFR can down-regulate p63 expression through the PI3K/AKT/mTOR pathway, and inhibit the proliferation, migration, and invasion of nasopharyngeal carcinoma cells. The lack of p63 expression in NSNPC was linked with the inhibition of the EGFR/PI3K/AKT/mTOR pathway, and NSNPC may be a variant of classical NPC.

The circadian clock gene BMAL1 increases radiosensitivity in nasopharyngeal carcinoma cell CNE2

ObjectiveTo investigate the involvement of the circadian clock gene BMAL1 in regulating radiosensitivity in nasopharyngeal carcinoma (NPC). MethodsThe regulatory role of BMAL1 in NPC radiotherapy was investigated using 44 NPC cases and the NPC cell line CNE2, which had been genetically modified to either overexpress or silence BMAL1. Infected CNE2 cells were divided into four groups, including those with BMAL1 overexpression and BMAL1 RNA interference (RNAi), as well as their respective control groups. The group responses to different doses of radiotherapy were examined. ResultsThe 5-year survival rates of the BMAL1 overexpression group demonstrated a significant increase compared to those of the low expression group (p < 0.05). Furthermore, the BMAL1 overexpression group exhibited a higher tumor-growth inhibition rate (p < 0.001) and apoptotic rate (p = 0.004) following radiotherapy; a decreased proportion of S-phase cells (p < 0.001) but an increased proportion of G2/M-phase cells (p = 0.001) after 24 h of 8Gy irradiation; reduced number of cell colonies (p = 0.042), and a lower survival score (p = 0.037). ConclusionsOur findings demonstrate a positive correlation between BMAL1 expression and NPC survival, suggesting that BMAL1 promotes NPC radiosensitivity.

Near-infrared-triggered Dibenzo[a,c][1,2,5]thiadiazolo[3,4-i]phenazine heterocyclic derivatives for type-I photodynamic and photothermal therapy against human nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) remains endemic in southern China. The general therapeutic consensus is to treat the disease with radiotherapy alone or together with concurrent chemotherapy. But the treatment outcome of NPC remains disappointing with bad prognoses, low overall survival rate and severe side effects. Phototherapy can be a promising approach for treating NPC due to its excellent spatiotemporal selectivity, negligible drug resistance and minimal invasiveness. In this work, two photosensitizers (DTP-Ph and DTP-Et) with similar donor-acceptor structure were designed and constructed for the treatment of NPC. Remarkably, DTP-Ph and DTP-Et show different photo-physicochemical properties as well as mechanisms of phototherapeutic actions, especially DTP-Ph, which exhibits synergistic type-I photodynamic therapy (PDT) and photothermal therapy (PTT) effect under near-infrared (NIR) activation. DTP-Et and DTP-Ph were further encapsulated with F127 to obtain nanoparticles with good water solubility. In intro and in vivo phototherapy evaluations of DTP-Ph NPs display promising phototherapeutic activity with low IC50 value and good tumor growth inhibition (TGI) against human nasopharyngeal carcinoma cell line CNE2 and tumor-bearing xenograft model.

Inhibitory mechanism of KIF3A gene on nasopharyngeal carcinoma stem cells.

To investigate the inhibitory mechanism of the KIF3A gene on nasopharyngeal carcinoma (NPC) tumor stem cells. Set up a blank control group (BCG), NPC group, and KIF3A silence (si-KIF3A) group. The BCG cells were nasopharyngeal normal epithelial cell line NP69, without any treatment, and were cultured routinely; The NPC group cells are human NPC cell line CNE2 cells, which are not subjected to any treatment and are cultured routinely; si-KIF3A group cells were cultured in the offspring of human NPC cell line CNE2 infected with Lentivirus knockdown KIF3A gene. CCK8 was used to detect the cell proliferation ability, Western blot and qPCR were used to detect protein and related mRNA expression, while cell migration and invasion were detected using Transwell. The KIF3A protein and mRNA in the NPC group were higher than those in the BCG (P<0.05), while those in the si-KIF3A group cells were lower compared to BCG cells (P<0.05). The cell proliferation, migration and invasion activity in the si-KIF3A group were reduced than those in BCG (P<0.05). Si-KIF3A group cells HIF-1, NF-κB was reduced than that of BCG (P<0.05). The expression level of HIF-1, NF-κB protein in si-KIF3A group cells was reduced compared to BCG cells (P<0.05). Knocking down the KIF3A gene can reduce the vitality of NPC stem cells, and inhibit the malignant phenotype of NPC stem cells, via inhibiting HIF-1 and NF-κB expression.

NUCB-2/Nesfatin-1 promotes the proliferation of nasopharyngeal carcinoma cells.

The association of NUCB-2/Nesfatin-1 with nasopharyngeal carcinoma (NPC) remains unclear. We clarified the role of NUCB-2/Nesfatin-1 in the development, progression and diagnosis of NPC. In nasopharyngeal carcinoma cell lines (5-8F, 6-10B, CNE1, CNE2 and NP69), western blotting, MTT, EdU and other techniques were performed to investigate the role of NUCB-2 in nasopharyngeal carcinoma. 70 tissue samples (39 NPC and 31 rhinitis) and 140 serum samples (including NPC, rhinitis, other head and neck tumors and healthy control) were included to explore the expression of NUCB-2 and its metabolite Nesfatin-1 in tissues or serum of patients with nasopharyngeal carcinoma. NUCB-2 level in NPC tissue was higher than that in rhinitis tissue (P < 0.05). Suppression of NUCB-2 in the NPC cell line CNE2 inhibited proliferation and clone formation of the cells; on the contrary, improvement of NUCB-2 in the NPC cell line CNE1 promoted cell propagation and clone development. An elevated serum level of NUCB-2 in NPC patients was detected, compared to that in patients with other head and neck tumors, rhinitis or healthy donors. Determination of nesfatin-1 combined with EA-IgA, VCA-IgA and Rta-IgG in serum samples for NPC diagnosis reached a sensitivity of 93.6% and a specificity of 94.5%, while the positive and negative predictive value of this diagnostic model was 89.8% and 96.6%, and the accuracy yielded 94.2%. This study revealed that NUCB-2 could enhance proliferation of NPC cells and NUCB-2/nesfatin-1 has the potential to be a serological marker to aid early diagnosis of nasopharyngeal carcinoma.

In vitro evaluation of photon and carbon ion radiotherapy in combination with cisplatin in head and neck squamous cell carcinoma cell lines.

Heavy ion radiotherapy, such as carbon ion radiotherapy (CIRT), has multiple advantages over conventional photon therapy. Cisplatin, as a classic anti-tumor drugs, has been tested and discovered as a photon radiosensitizer in several cell lines, including head and neck squamous cell carcinoma (HNSCC). Hence, the aim of our study is to evaluate whether cisplatin can sensitize CIRT towards HNSCC cell lines in vitro. Human nasopharyngeal carcinoma cell line CNE-2, human tongue squamous carcinoma cell line TCA 8113 and human hypopharynx squamous carcinoma cell line FADU were all irradiated with photon beam of 2, 4, 6, 8 Gy (physical dose) and carbon ion beam of 1, 2, 3, 4 Gy (physical dose) and treated with cisplatin. Cell survival was assessed by clonogenic survival assay. CIRT showed significantly stronger cytotoxic effect than standard photon radiotherapy. The relative biological effectiveness (RBE) of carbon ion beam at 10% survival ( ) was calculated 3.07 for CNE-2, 2.33 for TCA 8113 and 2.36 for FADU. Chemoradiotherapy (both photon radiotherapy and CIRT) was more effective than radiotherapy alone. In vitro sensitizer enhancement ratios (SERs) of cisplatin in CNE-2, TCA 8113 and FA DU cell lines after photon irradiation were 1.33, 1.14 and 1.21, while after carbon ion irradiation were 1.02, 1.00 and 0.96, showed that cisplatin sensitized photon irradiation but showed no sensitization effect in carbon ion irradiation in all tested cell lines. In conclusion, high linear energy transfer (LET) CIRT was more effective than photon irradiation to prevent the proliferation of HNSCC cell lines. Additional treatment with cisplatin could sensitize photon irradiation but showed no effect on carbon ion irradiation.

Phytosphingosine inhibits cell proliferation by damaging DNA in human cell lines

Harmful cyanobacterial blooms have caused numerous biosecurity incidents owing to the production of hazardous secondary metabolites such as microcystin. Additionally, cyanobacteria also release many other components that have not been explored. We identified compounds of a toxic mixture exudated from a dominant, blooming species, Microcystis aeruginosa, and found that phytosphingosine (PHS) was one of the bioactive components. Since PHS exhibited toxicity and is deemed a hazardous substance by the European Chemicals Agency, we hypothesized that PHS is a potentially toxic compound in M. aeruginosa exudates. However, the mechanisms of PHS ecotoxicity remain unclear. We assessed the cytotoxicity of PHS using an in vitro cell model in eight human cell lines and observed that the nasopharyngeal carcinoma cell line CNE2 was the most sensitive. We exposed CNE2 cells to 0–25 µmol/L PHS for 24 hr to explore its toxicity and mechanism. PHS exposure resulted in abnormal nuclear morphology, micronuclei, and DNA damage. Moreover, PHS significantly inhibited cell proliferation and arrested cell cycle at S phase. The results of Western blot suggested that PHS increased the expression of DNA damage-related proteins (ATM, p-P53 and P21) and decreased the expression of S phase-related proteins (CDK2, CyclinA2 and CyclinE1), indicating the toxicological mechanism of PHS on CNE2 cells. These data provide evidence that PHS has genetic toxicity and inhibits cell proliferation by damaging DNA. Our study provides evidence that PHS inhibits cell proliferation by damaging DNA. While additional work is required, we propose that PHS been considered as a potentially toxic component in MaE in addition to other well-characterized secondary compounds.

Extracellular matrix stiffness mediates radiosensitivity in a 3D nasopharyngeal carcinoma model

PurposeRadiotherapy is one of the essential treatment modalities for nasopharyngeal carcinoma (NPC), however, radioresistance still poses challenges. Three-dimensional (3D) tumor culture models mimic the in vivo growth conditions of cells more accurately than 2D models. This study is to compare the tumor biological behaviors of NPC cells in 2D, On-Surface 3D and Embedded 3D systems, and to investigate the correlation between radioresistance and extracellular matrix (ECM) stiffness.MethodsThe morphology and radioresistance of the human NPC cell line CNE-1 were observed in 2D and 3D systems. The CCK-8 assay, wounding healing assays, flow cytometry, soft agar assays, and western blot analysis were used to evaluate differences in biological behaviors such as proliferation, migration, cell cycle distribution, and stem cell activity. Different ECM stiffness systems were established by co-blending collagen and alginate in varying proportions. ECM stiffness was evaluated by compressive elastic moduli measurement and colony formation assay was used to assess radioresistance of NPC cells in systems with different ECM stiffness after irradiation.ResultsCompared to 2D models, the morphology of NPC cells in 3D culture microenvironments has more in common with in vivo tumor cells and 3D cultured NPC cells exhibit stronger radioresistance. Integrin β1 but not the epithelial-to-mesenchymal transition pathway in 3D models boost migration ability. Cell proliferation was enhanced, the proportion of tumor stem cells was increased, and G1/S phase arrest occurred in 3D models. NPC cells cultured in softer ECM systems (with low alginate proportions) exhibit striking resistance to ionizing radiation.ConclusionThe tumor biological behaviors of NPC cells in 3D groups were obviously different from that of 2D. Radioresistance of NPC cells increased with the stiffness of ECM decreasing.

MiRNA profiling of human nasopharyngeal carcinoma cell lines HONE1 and CNE2 after X-ray therapy.

Radiotherapy is the main treatment for nasopharyngeal carcinoma. The radioresistance mechanism of cells is related to miRNAs. To investigate the miRNA profiling of HONE1 and CNE2 after X-ray therapy. The HONE1 and CNE2 cells were treated with X-ray at 4 Gy, 8 Gy, 16 Gy, and 20 Gy doses. The cell lines CNE2 with the best therapy effects and HONE1 with the worst therapy effects were screened out. Apoptosis and cell viability were detected with flow cytometry and Cell Counting Kit-8 (CCK-8). High-throughput sequencing was performed. A miRNA library was constructed. The miRNA annotation expression distribution, family prediction and target gene interaction, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were conducted. The 24-hour 20 Gy dose X-rays were selected as the optimal therapy conditions. The CNE2_C, CNE2_M, HONE1_C and HONE1_M miRNAs accounted for 26.5%, 31.7%, 21.3%, and 22.9% of the Cleandata reads count, respectively, and the contents of rRNAs accounted for 24.9%, 14.7%, 25.1%, and 25.1% of the Cleandata reads count, respectively. The miRNAs with differential expression between the HONE1 and CNE2 cell lines including hsa-miR-21-5p, hsa-let-7a-5p, hsa-miR-125a-5p, hsa-miR-26a-5p, hsa-let-7f-5p, hsa-miR-20a-5p, and hsa-miR-24a-3p. There were also differentially expressed miRNAs in HONE1_C vs. HONE1_M, such as hsa-miR-21-5p and hsa-let-7i-5p. The differentially expressed miRNA in CNE2_C vs. CNE2_M was hsa-miR-148b-3p. The Gene Ontology analysis showed that the differentially expressed miRNA interacting genes in HONE1_M vs. CNE2_M were mainly enriched in biological process such as negative and positive regulation of transcription from RNA polymerase II promoter, cellular component such as cytosol and molecular function such as protein binding factor. The KEGG pathway analysis revealed that the differentially expressed miRNA interacting genes in HONE1_M vs. CNE2_M were enriched in the cancer-related pathways, such as pathways in cancer, MAPK signaling pathway and Wnt signaling pathway. Twelve miRNAs and 9 genes which contribute to X-ray radiation resistance were identified. Among those with differential expression between the HONE1 and CNE2 cell lines, which played a regulatory role in multiple pathways, were hsa-miR-20a-5p, hsa-let-7a-5p, hsa-let-7f5p, hsa-let-7i-5p, hsa-miR-30e-5p, hsa-miR-148b-3p, and hsa-miR-200c-3p. The corresponding genes were MAPK1, SOS1, TGFBR1, TGFBR2, TP53, CASP3, CCNE2, PTEN, and CDK2.

Hyperthermia inhibits growth of nasopharyngeal carcinoma through degradation of c-Myc

Background Hyperthermia is a widely used adjunct treatment for different cancers including nasopharyngeal carcinoma (NPC). The protooncogene c-Myc is up-regulated in NPC and its expression is associated with poor prognosis. Objective We hypothesized that c-Myc constitutes an important hyperthermia treatment target, and we investigated its contribution to hyperthermia responses in NPC. Methods The growth of the human NPC cell lines CNE1 and CNE2 was analyzed using CCK-8 and clonogenicity assays after 43 °C hyperthermia, knockdown or overexpression of c-Myc. Flow cytometry measurements assessed cell cycle parameters and apoptosis, while levels of c-Myc together with key transcriptional targets were determined using qPCR and Western blotting. Parallel experiments were undertaken using NPC xenografts in nude mice and lastly, global transcriptomic changes were determined using ‘RNAseq’. Results Hyperthermia increased the ubiquitination and proteasomal destruction of c-Myc, causing a rapid decline in c-Myc protein levels in NPC cells. Similar to c-Myc knockdown, NPC cells treated with hyperthermia showed growth inhibition associated with the downregulation of c-Myc target genes. Moreover, low levels of c-Myc could be sustainably repressed in NPC cells through repeated hyperthermia treatments. Importantly, the key findings of growth inhibition and decreased c-Myc protein levels were reproduced in NPC tumor xenografts. Bioinformatic analyses showed that downregulation of c-Myc constituted a central node in the hyperthermia response of NPC cells. Conclusion Our study reveals that hyperthermia can readily destabilize c-Myc levels in NPC cells and inhibit tumor growth. This proposes new strategies for implementing hyperthermia to target c-Myc-driven cancers to improve therapeutic efficacy.

The Correlation Study of Circadian Clock Gene BMAL1 Regulates the Biological Behavior of Human Nasopharyngeal Carcinoma Cell After Radiotherapy

To investigate the effect of circadian clock gene BMAL1 on proliferation and cell cycle of Nasopharyngeal Carcinoma(NPC) CNE1 cells with different expression status, compare the expression level of pathway related proteins before and after radiotherapy, to further understand the mechanism of BMAL1 gene regulating the biological behavior of NPC cells, aim to explore the mechanism of BMAL1 gene regulating the expression of cell cycle related proteins through ATM/ATR pathway at the molecular level.Constructed CNE1 cell lines with BMAL1 gene overexpression group, control group, inhibitor group, blank group. Using Western blot (WB) to detect the overexpression of BMAL1 gene at protein level and the expression of ATM/ATR pathway related proteins. The absorbance of cells with the same number of plates was determined by a cell counting kit for 6 consecutive days. The effect of BMAL1 gene on the proliferation rate of CNE1 was analyzed. The effect of BMAL1 gene on cell cycle of CNE1 was studied by flow cytometry. Co-immunoprecipitation (co-IP) was used to detect the interaction between BMAL1 gene and ATM/ATR checkpoints.WB showed BMAL1 gene had a higher expression in the overexpression group. In the BMAL1 gene overexpression group, the absorbance values of CNE1 cells were lower at 1 to 6 days, the proportion of G0/G1 phase cells was higher, the relative protein expression of P21,P27 were higher. Co-IP confirmed the interaction between BMAL1 gene and the checkpoints of ATM/ATR pathway before and after radiotherapy. The protein expression of upstream genes of ATM/ATR pathway in the overexpression group was significantly higher before radiotherapy (P < 0.05). The protein expression of the upstream genes of ATM/ATR pathway increased significantly, while the expression of the downstream genes decreased significantly after radiotherapy. Comparing with the overexpression group and inhibitor group showed when the ATM/ATR pathway blocked by inhibitor KU55933, the expression of ATM/ATR pathway upstream genes decreased, and the expression of downstream genes increased. The expression of p-chk1 in blank group, control group and overexpression group after radiotherapy was significantly higher than before, all the difference were statistically significant.The circadian clock gene BMAL1 inhibits the proliferation of NPC cell line CNE1, arrests cell cycle in the G0/G1 phase by up-regulate the expression of P21,P27 and P53.The response of ATM/ATR pathway could be activated after radiotherapy, the overexpression of Bmal1 gene could up-regulate the expression of ATM/ATR pathway upstream genes such as p-chk1 and p-chk2,then inhibited the activity of downstream Cyclin-CDK complexes by phosphorylated CDC25A and CDC25C,finally achieved the function of blocking cell cycle after radiotherapy.

Epstein-Barr Virus Promotes Tumor Angiogenesis by Activating STIM1-Dependent Ca2+ Signaling in Nasopharyngeal Carcinoma.

Epstein-Barr virus (EBV) promotes tumor angiogenesis in nasopharyngeal carcinoma (NPC) by activating store-operated Ca2+ entry. Since such entry has been linked to stromal interaction molecule 1 (STIM1), we examined whether the virus acts via STIM1-dependent Ca2+ signaling to promote tumor angiogenesis in NPC. STIM1 expression was detected in NPC cell lines HK1 and CNE2 that were negative or positive for EBV. STIM1 was knocked down in EBV-positive cells using recombinant lentivirus, then cytosolic Ca2+ levels were measured based on fluorescence resonance energy transfer. Cells were also exposed to epidermal growth factor (EGF), and secretion of vascular endothelial growth factor (VEGF) was measured using an enzyme-linked immunosorbent assay. Endothelial tube formation was quantified in an in vitro angiogenesis assay. Growth of CNE2-EBV xenografts was measured in mice, and angiogenesis was assessed based on immunohistochemical staining against CD31. Paraffin-embedded NPC tissues from patients were assayed for CD31 and STIM1. EGFR and ERK signaling pathways were assessed in NPC cell lines. STIM1 expression was higher in EBV-positive than in EBV-negative NPC cell lines. STIM1 knockdown in EBV-positive NPC cells significantly reduced Ca2+ influx and VEGF production after EGF treatment. STIM1 knockdown also inhibited xenograft growth and angiogenesis. Moreover, CD31 expression level was higher in EBV-positive than EBV-negative NPC tissues, and high expression of CD31 co-localized with high expression of STIM1 in EBV-positive tissues from NPC patients. Viral infection of NPC cells led to higher levels of phosphorylated ERK1/2 after EGF treatment, which STIM1 knockdown partially reversed. Our results suggest that EBV promotes EGF-induced ERK1/2 signaling by activating STIM1-dependent Ca2+ signaling, and that blocking such signaling may inhibit EBV-promoted angiogenesis in NPC.

Forkhead Box Protein M1 Promotes Nasopharyngeal Carcinoma Cell Tumorigenesis Possibly via the Wnt/β-Catenin Signaling Pathway.

BackgroundForkhead box protein M1 (FoxM1) is an important transcription factor involved in the development and progression of various malignancies. However, its role in nasopharyngeal carcinoma (NPC) remains largely unknown. This study aimed to assess the effect of FoxM1 on NPC cell tumorigenesis as well as the underlying mechanism.Material/MethodsNPC cell lines CNE-1 and CNE-2 were treated with vehicle and FoxM1 inhibitor thiostrepton or transfected with small interfering RNA. CCK-8 assay, flow cytometric assay, and Hoechst 33258 staining were performed to assess the viability, apoptosis and nuclear morphological impairment, and cell cycle, respectively. The expression of apoptosis-related caspase-3 and caspase-9 was detected by western blot analysis The tumor growth in the mouse xenograft model of NPC treated with thiostrepton or control was assessed. The expression of Wnt/β-catenin signaling proteins p27, FoxM1, S phase kinase-associated protein 2 (SKP2), and Cyclin D1 were determined both in cells and xenograft tissues by western blot analysis.ResultsInhibition of FoxM1 by thiostrepton significantly suppressed NPC cell viability, induced apoptosis, increased cell cycle arrest, impaired nuclear morphology, and reduced NPC cell-derived tumor xenograft growth. Mechanistically, inhibition or knockdown of FoxM1 inactivated the Wnt/β-catenin signaling pathway, as demonstrated by altered expression of Wnt/β-catenin signaling-related genes, including p27, SKP2, and cyclin D1, in both NPC cells and xenograft tissues.ConclusionsWe identified FoxM1 as a novel regulator of NPC cell tumorigenesis in vitro and in vivo. Targeting FoxM1 could be a promising therapeutic strategy against NPC.

MiR-18a enhances the radiosensitivity of nasopharyngeal carcinoma cells through inducing autophagy

Objective: To explore the impacts of miR-18a overexpression or depression on the radiosensitivities of nasopharyngeal carcinoma cell line CNE1 and CNE2 and underlying mechanisms. Methods: CNE1 and CNE2 were transfected with miR-18a mimics, inhibitor and the corresponding control vectors. qRT-PCR and western blot were used to determine the ataxia telangiectasia mutated (ATM) expressions in CNE1 and CNE2. CNE1 and CNE2 with stably expressing miR-18a and miR-18a siRNA were constructed. Methyl thiazolyl tetrazolium (MTT) assay was used to detect the impacts of the miR-18a overexpression or depression combined with irradiation on the cell growth. Flow cytometry was used to detect the cell apoptosis and cell cycle. Colony formation assay was used to evaluate the raodiosensitivities of cells. Acridine orange (AO) staining and western blot were used respectively to test the autophagy and the expressions of related proteins. Independent samples t test was used to compare the mean value between groups by using SPSS 16.0. Results: ATM mRNA was decreased significantly in CNE1 and CNE2 cells transfected with 100 or 200 nmol/L miR-18a mimics for 48 hours (CNE1: RQ=0.174±0.139 and 0.003±0.001, t=9.939 and 19 470.783;CNE2: RQ=0.024±0.008 and 0.019±0.012, t=270.230 and 137.746, respectively, all P<0.001). ATM proteins were also decreased after transfected with 100 or 200 nmol/L miR-18a mimics for 72 hours. While in the cells transfected with 100 and 200 nmol/L miR-18a inhibitor for 48 hours, the expressions of ATM mRNA were upregulated significantly (CNE1: RQ=9.419±2.495 and 2.500±1.063, t=-4.427 and -41.241; CNE2: RQ=7.210±0.171 and 115.875±15.805, t=-62.789 and -12.589, all P<0.05), and the expressions of ATM proteins increased after transfected for 72 hours. The growth of cells with miR-18a overexpression plus 4 Gy irradiation were obviously inhibited compared to that of cells with the 4Gy irradiation alone; while the growth of miR-18a-inhibited cells increased compared to that of cells with 4 Gy irradiation alone (all P<0.05). CNE1 transfected with 100 nmol/L miR-18a mimics plus 4 Gy irradiation showed the higher apoptosis rate than the cells with 4 Gy irradiation alone ((22.9±2.1)% vs. (16.3±1.0)%, t=-4.870, P<0.01). Compared to the cells with 4 Gy irradiation alone, miR-18a-overexpressed cells plus 4 Gy irradiation decreased their percentages in G1 phases ((20.2±3.0)% vs. (29.8±4.4)%, t=3.119) and G2/M phases ((21.5±0.9)% vs. (33.4±3.1)%, t=6.410, P<0.05 for both), and increased their percentages in S phases ((56.7±4.9)% vs. (36.8±6.4)%, t=-4.246, P<0.05), and these cells possessed less colony number after exposure to different doses of irradiation, more autophagy-lysosome number, and more expressions of LC3 proteins (all P<0.05). There were no significant differences in the expressions of p62 expressions between different groups of cells. Conclusion: Overexpression of miR-18a can enhance the radiosensitivities of NPC cells by targeting ATM to abrogate G1/S, G2/M arrest and to induce autophagy and apoptosis.

Experimental Study on the Effect of miR-200b Regulation of Bone Morphogenetic Protein (BMP)-2 Expression on the Proliferation and Invasion of Nasopharyngeal Carcinoma Cells

BMP is an important member of the skeletal junction proteins associated with cell invasion, metastasis, and migration. MiR-200b is associated with the progression of several tumors. This study investigated whether miR-200b plays a role in regulating BMP-2 expression and affecting the proliferation and invasion of nasopharyngeal carcinoma cells. The nasopharyngeal carcinoma tissues with different TNM stages were collected and the proliferative nasal tissues alone were used as controls to detect the expression of miR-200b and BMP by RT-PCR. The nasopharyngeal carcinoma cell line CNE1 cells were divided into miR-NC group, miR-200b mimic group, siRNA-NC group, and siRNA-BMP-2 group, to detect BMP-2 level, cell invasion and proliferation ability by transwell. The BMP-2 mRNA expressed in nasopharyngeal carcinoma tissues was significantly elevated compared to controls and correlated with TNM stage. BMP-2 was higher in tumor tissues than in controls, however, the expression profile of miR-200b was opposite to BMP-2. Transfection with miR-200b mimic or siRNA-BMP-2 significantly down-regulated BMP-2 in CNE1 cells and attenuated cell invasive and proliferative capacity. Reduced expression of miR-200b is associated with elevated BMP-2 expression and increased invasive capacity of nasopharyngeal cancer cells. Overexpression of miR-200b reduces the invasive and proliferative capacity of nasopharyngeal cancer cells by targeting and inhibiting BMP-2 expression.

LncRNA CASC19 Contributes to Radioresistance of Nasopharyngeal Carcinoma by Promoting Autophagy via AMPK-mTOR Pathway.

Nasopharyngeal carcinoma (NPC) is one of the most frequent head and neck malignant tumors and is majorly treated by radiotherapy. However, radiation resistance remains a serious obstacle to the successful treatment of NPC. The aim of this study was to discover the underlying mechanism of radioresistance and to elucidate novel genes that may play important roles in the regulation of NPC radiosensitivity. By using RNA-seq analysis of NPC cell line CNE2 and its radioresistant cell line CNE2R, lncRNA CASC19 was screened out as a candidate radioresistance marker. Both in vitro and in vivo data demonstrated that a high expression level of CASC19 was positively correlated with the radioresistance of NPC, and the radiosensitivity of NPC cells was considerably enhanced by knockdown of CASC19. The incidence of autophagy was enhanced in CNE2R in comparison with CNE2 and another NPC cell line HONE1, and silencing autophagy with LC3 siRNA (siLC3) sensitized NPC cells to irradiation. Furthermore, CASC19 siRNA (siCASC19) suppressed cellular autophagy by inhibiting the AMPK/mTOR pathway and promoted apoptosis through the PARP1 pathway. Our results revealed for the first time that lncRNA CASC19 contributed to the radioresistance of NPC by regulating autophagy. In significance, CASC19 might be a potential molecular biomarker and a new therapeutic target in NPC.

LncRNA TP73-AS1 regulates miR-495 expression to promote migration and invasion of nasopharyngeal carcinoma cells through junctional adhesion molecule A.

The main obstacle to the treatment of nasopharyngeal carcinoma (NPC) is metastasis. Long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) are highly involved in the progression of NPC. In this study, we aimed to explore the regulatory role of lncRNA P73 antisense RNA 1 T (TP73-AS1) and miR-495 in migration and invasion of NPC cells. The expression levels of TP73-AS1, miR-495, and junctional adhesion molecule A (JAM-A) in NPC tissue samples and cell lines were examined by quantitative real-time PCR (qRT-PCR) and/or Western blot. NPC cells were transfected with vectors overexpressing TP73-AS1, short hairpin RNA (shRNA) against TP73-AS1, shRNA against JAM-A, miR-495 mimics, miR-495 inhibitor, and their corresponding negative controls as designated. The MTT assay, cell migration assay, and transwell assay were performed to detect cell viability, migration, and invasion, respectively. Dual-luciferase reporter assay was performed to confirm the binding of TP73-AS1 and miR-495, and miR-495 and JAM-A. TP73-AS1 and JAM-A were significantly upregulated while miR-495 was markedly downregulated in NPC tissues and cell lines compared to normal controls. The overexpression of TP73-AS1 promoted migration and invasion of NPC cell line CNE-2. TP73-AS1 targeted miR-495 and negatively regulated its expression. TP73-AS1 upregulated the expression of JAM-A through miR-495. TP73-AS1 mediated migration and invasion of CNE-2 cells via upregulating JAM-A. LncRNA TP73-AS1, miR-495, and JAM-A are involved in migration and invasion of NPC cells. The TP73-AS1/miR-495/JAM-A axis may serve as a therapeutic target for the treatment of NPC.

BIX-01294, a G9a inhibitor, suppresses cell proliferation by inhibiting autophagic flux in nasopharyngeal carcinoma cells.

G9a, a histone methyltransferase, has been found to be upregulated in a range of tumor tissues, and contributes to tumor growth and metastasis. However, the impact of G9a inhibition as a potential therapeutic target in nasopharyngeal carcinoma (NPC) is unclear. In the present study we aimed to investigate the anti-proliferative effect of G9a inhibition in the NPC cell lines CNE1 and CNE2, and to further elucidate the molecular mechanisms underlying these effects. The expression of G9a in NPC tumor tissues was significantly higher than that in normal nasopharyngeal tissues. The pharmacological inhibition of G9a by BIX-01294 (BIX) inhibited proliferation and induced caspase-independent apoptosis in NPC cells in vitro. Treatment with BIX induced autophagosome accumulation, which exacerbated the cytotoxic activity of BIX in NPC cells. Mechanistic studies have found that BIX impairs autophagosomes by initiating autophagy in a Beclin-1-independent way, and impairs autophagic degradation by inhibiting lysosomal cathepsin D activation, leading to lysosomal dysfunction. BIX was able to suppress tumor growth, possibly by inhibiting autophagic flux; it might therefore constitute a promising candidate for NPC therapy.

Exosomes derived from Taxol-resistant nasopharyngeal carcinoma (NPC) cells transferred DDX53 to NPC cells and promoted cancer resistance to Taxol.

Nasopharyngeal carcinoma (NPC) is a common cancer with high incidence in Southern China. Taxol is one of the first-line chemotherapeutic drugs for treating NPC; however, Taxol resistance has become the main difficulty for clinical treatment and the mechanisms remain not fully understood. In this study, we mainly focus on exploring whether exosomes from Taxol-resistant NPC cells played some roles in the resistance and progression of NPC. Taxol was used to treat NPC cell line CNE1 and Taxol-resistant NPC cell line CNE1-TR cells to measure cell viability and IC50 by CCK-8 assay. Exosomes from these two cells were extracted and identified by transmission electron microscopy (TEM), and special protein markers were determined by Western blot (WB) assay. Real-time PCR was performed to detect levels of mRNAs in exosomes, CNE1 and CNE1-TR cells. WB was performed to detect protein levels. The p-DDX53 and si-DDX53 were constructed and cloned into cells, resulted with DDX53 overexpression and inhibition, then resistant associated protein levels and IC50 were measured. Finally, GW4869, an inhibitor to block exosome secretion, was used to verify that the exosomes derived from CNE1-TR cells transferred DDX53 to CNE1 cells and contributed to promote NPC resistance. We found that the IC50 to Taxolin CNE1-TR was much higher than that in CNE1 cells and DDX53 was highly expressed in Taxol-resistant CNE1-TR cells. Furthermore, exosomes were successfully extracted and determined, showing high levels of DDX53 and MDR1. Thus, they could promote cell resistance for CNE1 after adding CNE1-TR exosomes into CNE1 cells. Moreover, DDX53 overexpression increased the IC50 and upregulated MDR1 in CNE1 cells, while DDX53 inhibition showed the opposite results. In addition, the DDX53 inhibition decreased the IC50 and repressed MDR1 in CNE1-TR cells. Besides, blocking exosome released from CNE1-TR by using GW4869 treatment significantly repressed the levels of DDX53 and MDR1, and the IC50 of CNE1 cells was reversed. Finally, the increased levels of MDR1 were significantly reversed following with adding DDX53 si-DDX53-CNE1-TR exosomes, and the increased IC50 to Taxol was obviously reversed. This study firstly discovered that DDX53 was highly expressed in Taxol-resistant NPC cells, which could be transferred into normal NPC cells via exosome secretion. The transferred DDX53 could upregulate the expression of MDR1 in NPC cells to promote the resistant capacity to Taxol, which provided a novel insight for understanding NPC and might be a potential therapeutic target for NPC.

The Circadian Clock Gene BMAL1 Increases Nasopharyngeal Carcinoma Radiosensitivity Through Promoting ATM Pathway-mediated G2/M Phase Cell Cycle Arrest And Apoptosis

To investigate the role and mechanism of the circadian clock gene BMAL1 in regulating nasopharyngeal carcinoma (NPC) radiosensitivity The regulatory role of BMAL1 in NPC radiotherapy was investigated using 44 NPC patients and the NPC cell line CNE2 which was genetically engineered to overexpress or silence BMAL1 gene. Its correlation with NPC survival outcome was determined using immunohistochemistry and Kaplan-Meier survival curves. Infected CNE2 cells were divided into four groups: BMAL1 overexpression and control groups; and BMAL1 RNA interference (RNAi) and control groups; and their responses to different doses of radiotherapy were examined. In addition, the association between BMAL1 and the ATM/ATR pathway was determined using western blot and co-immunoprecipitation assays. The 5-year survival rates of the BMAL1 high expression group were significantly higher than that of the low expression groups (p<0.05). Moreover, the BMAL1 overexpression group showed higher tumor growth inhibition rate (p<0.001) and apoptotic rate (p = 0.004) after radiotherapy; a lower proportion of S phase cells (p<0.001) but higher proportion of G2/M phase cells (p = 0.001) after 24 h of 8Gy irradiation; reduced number of colonies (p = 0.042), and lower survival score (p = 0.037); and upregulation of ATM/ATR pathway proteins (p<0.05). The reverse results were observed for the RNAi group. Of note, co-immunoprecipitation revealed the interaction between BMAL1 and ATM. Our findings demonstrated the positive correlation between BMAL1 expression and NPC survival and suggest that BMAL1 promotes NPC radiosensitivity by controlling cell cycle distribution of NPC cells through the ATM pathway.