The IRF2/CENP-N/AKT signaling axis promotes proliferation, cell cycling and apoptosis resistance in nasopharyngeal carcinoma cells by increasing aerobic glycolysis.

BACKGROUND: Centromere protein N (CENP-N) has been reported to be highly expressed in malignancies, but its role and mechanism in nasopharyngeal carcinoma (NPC) are unknown. METHODS: Abnormal CENP-N expression from NPC microarrays of GEO database was analyzed. CENP-N expression level was confirmed in NPC tissues and cell lines. Stable CENP-N knockdown and overexpression NPC cell lines were established, and transcriptome sequencing after CENP-N knockdown was performed. In vitro and in vivo experiments were performed to test the impact of CENP-N knockdown in NPC cells. ChIP and dual luciferase reporter assays were used to verify the combination of IRF2 and CENP-N. Western blot analysis, cellular immunofluorescence, immunoprecipitation and GST pulldown assays were used to verify the combination of CENP-N and AKT. RESULTS: CENP-N was confirmed to be aberrantly highly expressed in NPC tissues and cell lines and to be associated with high 18F-FDG uptake in cancer nests and poor patient prognosis. Transcriptome sequencing after CENP-N knockdown revealed that genes with altered expression were enriched in pathways related to glucose metabolism, cell cycle regulation. CENP-N knockdown inhibited glucose metabolism, cell proliferation, cell cycling and promoted apoptosis. IRF2 is a transcription factor for CENP-N and directly promotes CENP-N expression in NPC cells. CENP-N affects the glucose metabolism, proliferation, cell cycling and apoptosis of NPC cells in vitro and in vivo through the AKT pathway. CENP-N formed a complex with AKT in NPC cells. Both an AKT inhibitor (MK-2206) and a LDHA inhibitor (GSK2837808A) blocked the effect of CENP-N overexpression on NPC cells by promoting aerobic glycolysis, proliferation, cell cycling and apoptosis resistance. CONCLUSIONS: The IRF2/CENP-N/AKT axis promotes malignant biological behaviors in NPC cells by increasing aerobic glycolysis, and the IRF2/CENP-N/AKT signaling axis is expected to be a new target for NPC therapy.

Overexpression of Notch2 enhances radiosensitivity via inhibition of the AKT/mTOR signaling pathway in nasopharyngeal carcinoma.

Our previous study found that in nasopharyngeal carcinoma (NPC) cells, overexpression of Notch2 can inhibit epithelial-mesenchymal transition (EMT), which plays a vital role in mediating radiosensitivity. The purpose of this study was to explore the radiosensitizing efficacy of the Notch2 gene in NPC cells and its potential mechanism. We used the recombinant plasmid transfection technique to establish Notch2-overexpressing 5-8 F and CNE-2 NPC cells. Cell proliferation, radiosensitivity, apoptosis and cell cycle distribution were assessed by cell counting kit-8 (CCK-8) experiments, colony formation experiments and flow cytometry. The levels of proteins related to cell cycle, apoptosis, and the AKT/mTOR signaling pathway were evaluated by using Western blotting. The results suggested that Notch2 overexpression increased the radiosensitivity of NPC cells, with sensitizing enhancement ratios (SERs) of 1.24 (5-8 F cells) and 1.34 (CNE-2 cells). Flow cytometry indicated that the level of apoptosis and percentage of cells in G2/M-phase were highest in NPC cells overexpressing Notch2 and treated with radiotherapy compared to cells overexpressing Notch2 alone or administered radiotherapy alone. Western blotting showed that compared to that of cells treated with Notch2 overexpression or radiotherapy alone, the levels of γH2AX, Bax, Bcl-2, Cyclin D1 and AKT/mTOR signaling pathway-related proteins were modified in NPC cells overexpressing Notch2 and treated with radiotherapy. These findings showed that overexpression of Notch2 can increase the radiosensitivity of NPC cells by inhibiting the AKT/mTOR pathway.AbbreviationsNPC: Nasopharyngeal carcinoma; EMT: Epithelial-mesenchymal transition; CCK8: Cell counting kit-8; EBV: Epstein-Barr virus; FBS: Fetal bovine serum; PE: Plating efficiency; SF: Survival fraction; SER: Sensitizing enhancement ratio; DSBs: DNA double-strand breaks[Figure: see text].

PLAC8 gene knockout increases the radio-sensitivity of xenograft tumors in nude mice with nasopharyngeal carcinoma by promoting apoptosis.

In vitro cell experiments showed that knocking out the placenta-specific protein 8 (PLAC8) gene significantly increased the sensitivity of tumor cells to radiation. This study used two nude mouse models of nasopharyngeal carcinoma (NPC) to investigate the radio-sensitization and molecular mechanism of PLAC8 knockout in vivo. The expression of PLAC8 in 120 NPC tissues and 30 nasopharyngitis (NPG) tissues was detected by immunohistochemistry (IHC) to analyze the relationship between PLAC8 and neck lymph node metastasis and prognosis in NPC patients. The mRNA expression level of PLAC8 in several NPC cell lines was detected by semi-quantitative RT-PCR. The PLAC8 gene was knocked out in CNE-2 cells using CRISPR/Cas9. The effect of PLAC8 gene knockout on the radiotherapy sensitivity of NPC cells was analyzed by establishing model 1 and model 2 tumor-bearing nude mouse models with two different irradiation methods. The expression of γH2AX, Bax, Bcl-2, Caspase-3 and cleaved Caspase-3 was detected by immunofluorescence (IF), IHC and western blot analysis. PLAC8 expression was significantly increased in NPC tissue samples and NPC cell lines compared with NPG tissue samples and normal cell lines (P<0.01). PLAC8 upregulation was associated with lymph node metastasis and a poor prognosis in patients with NPC (P<0.01). Both animal models showed that radiotherapy after PLAC8 knockout significantly slowed tumor growth and reduced tumor volume, with tumor inhibition rates of 100% and 66.04%, respectively. In model 2, PLAC8 knockout with radiotherapy increased the expressions of γH2AX, Bax, Caspase-3 and cleaved Caspase-3 but decreased the expression of Bcl-2 (P<0.01). In model 1, there was no tumor formation at the site where the cancer cells were injected. The expression levels of γH2AX, Bax, Caspase-3 and cleaved Caspase-3 in skin tissues taken at the injection site were lower than those in NPC tissues treated with radiotherapy, while the expression level of Bcl-2 was higher (P<0.01). PLAC8 expression is closely related to neck metastasis and the prognosis of NPC. PLAC8 gene knockout significantly increases the radio-sensitivity of NPC cells in vivo by promoting apoptosis, which is an effective strategy for the radiotherapy sensitization of NPC.

Integrated analysis of deregulation microRNA expression in head and neck squamous cell carcinoma.

ABSTRACT: MicroRNAs (miRNAs) play critical roles in carcinogenesis and development of cancers. In this study, we analyzed the eccentrically expressed miRNAs in head and neck squamous cell carcinoma (HNSCC) tissues based on the miRNA-Seq data of HNSCC patients available in the Cancer Genome Atlas database. Aberrant expression of 2589 miRNAs was detected in HNSCC tissues (1128 downregulated and 1461 upregulated). The differential expression levels of the miRNAs were further validated by analysis of 25 HNSCC samples and paired control tissues and compared with the Gene Expression Omnibus database to determine the candidate miRNAs. Quantitative reverse transcription polymerase chain reaction was used to compare the expression of these candidate miRNAs between 22 fresh HNSCC tissue samples and 11 control samples. In addition, the relationship between the expression of these candidate miRNAs and Tumor, Node, Metastases staging of HNSCC was analyzed. Compared with the expression in control tissues, the levels of hsa-miR-410-3p, hsa-miR-411-5p, hsa-miR-125b-2-3p, and hsa-miR-99a-3p were significantly lower in HNSCC. According to the Cancer Genome Atlas dataset analyzed, all 4 miRNAs were shown to inhibit tumor progression (T stage), positive lymph node metastasis (N stage), and distant metastasis (M stage) in HNSCC. Kyoto Encyclopedia of Genes and Genomes analysis showed that genes regulated by these 4 miRNAs were enriched in certain pathways, including the transforming growth factor-ß signaling pathway and the Hippo pathway. Enriched gene ontology terms mainly included regulation of transcription, cell proliferation, and apoptosis, which are well-characterized functions of miRNAs. Moreover, all 4 miRNAs inhibited the progression of primary tumors (T stage) and metastasis of regional lymph nodes (N stage). The top 4 aberrantly expressed miRNAs identified in this study have great clinical value in developing strategies for early diagnosis and treatment of HNSCC. More intensive studies are required to elucidate the mechanism underlying the roles of these miRNAs in HNSCC.

Plac8-mediated autophagy regulates nasopharyngeal carcinoma cell function via AKT/mTOR pathway.

To explore the relationship between autophagy and cell function, we investigated how PLAC8-mediated autophagy influences proliferation, apoptosis and epithelial-mesenchymal transition (EMT) in NPC. Colony formation analyses and CCK8 assays were used to assess the proliferative capacity of NPC cells. Transmission electron microscopy (TEM) was used to identify autophagosomes. Autophagic flux was monitored using the tandem monomeric RFP-GFP-tagged LC3 (tfLC3) assay. The rate of apoptosis in NPC cells was analysed by flow cytometry. Western blot analysis was used to evaluate the activation of autophagy and the signalling status of the AKT/mTOR pathway. Our study reveals that knocking out PLAC8 (koPLAC8) induces autophagy and apoptosis, while suppressing NPC cell proliferation and EMT. However, inhibition of autophagy with 3-methyladenine or by knocking down Beclin-1 reverses the cell proliferation, apoptosis and EMT influenced by koPLAC8. We find that koPLAC8 inhibits the phosphorylation of AKT and its downstream target, mTOR. Moreover, immunofluorescence and co-immunoprecipitation reveal complete PLAC8/AKT colocalization and PLAC8/AKT interaction, respectively. Furthermore, knockout of PLAC8 induced autophagy and inactivated AKT/mTOR signalling pathway of NPC xenografts. Overall, our findings demonstrate that koPLAC8 induces autophagy via the AKT/mTOR pathway, thereby inhibiting cell proliferation and EMT, and promoting apoptosis in NPC cells.

The diagnostic value of measurement of cochlear length and height in temporal bone CT multiplanar reconstruction of inner ear malformation.

CONCLUSION: The cochlear length (CL) and cochlear height (CH) measured through MPR will provide for more accurate quantitative diagnosis of inner ear malformation, and are subsequently convenient for calculating cochlear duct length (CDL) before cochear implant. OBJECTIVES: Qualitative and quantitative diagnosis of inner ear malformation in deaf patients through multiplanar reconstruction (MPR) was performed to provide a reference for cochlear implants. METHODS: One hundred and two cases without sensorineural deafness and 560 patients with sensorineural deafness had MPR of temporal bone computed tomography performed to obtain the standardized cochlear-view and oblique coronal-view images. The inner ear radial lines were measured to formulate normal values for inner ear malformation diagnosing, and the CDL was estimated based on CL. RESULTS: The normal range values of inner ear radial lines were measured and formulated, of which CL was 8.1-9.59 mm and CH was 3.28-3.90 mm. According to inner ear morphology and the normal values measured above, 61 cases of incomplete partition-type II (IP-II) and a high percentage (27/110, 24.5%) of hypoplasia of cochlea (HC) were diagnosed. The HC group was further divided into 1-turn, 1.5-turn, and 2-turn sub-groups, which had CDL of 15.98 ± 1.48 mm, 21.36 ± 0.96 mm, and 26.56 ± 0.60 mm, respectively.