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.

Micro-CT study of the human cochlear aqueduct.

OBJECTIVE: The anatomic structure of the cochlear aqueduct (CA) in human temporal bone specimens was observed using micro-computed tomography (CT). MATERIALS AND METHODS: Micro-CT scanning of 18-µm-thick slices was performed on 30 slides of human temporal bone specimens to observe the CA structure and its relationship with its surroundings. The length, internal and external apertures, and the narrowest width of the CA were measured. The differences in CAs were compared between high jugular bulb (HJB) specimens and normal specimens. RESULTS: A large number of CA images were acquired using Micro-CT scanning, which clearly displayed the basic anatomic structures, stereotactic localizations, and adjacent relationships of the CAs. The whole course of a CA was 12.31 ± 3.60 mm, the diameter of the internal aperture was 465 ± 242 µm, the diameter of the external aperture was 2.88 ± 1.06 mm, the narrowest diameter was 601 ± 335 µm, the diameter of the opening of inferior cochlear vein (ICV) was 151 ± 50 µm, the distance between the internal aperture and ICV was 270 ± 197 µm, and the distance between the inferior margin of the internal acoustic meatus (IAM) and the top most part of the external aperture of the CA was 6.783 ± 2.15 mm. No bony obstruction of the CA or CA enlargement was observed in the specimens. A total of 28 CAs had one accompanying bony canal in the surroundings. The length and travelling of the CA were not affected by the level of the jugular bulb (JB). The variation of the travelling of the ICV was larger than that of the CA. CONCLUSION: Micro-CT adequately displayed the bony CA canal and provided a new method for anatomical studies of the CA and a basis for functional studies.

A study of Epstein-Barr virus infection in the Chinese tree shrew(Tupaia belangeri chinensis).

BACKGROUND: Epstein-Barr virus (EBV) is closely associated with many human diseases, including a variety of deadly human malignant tumours. However, due to the lack of ideal animal models,the biological characteristics of EBV, particularly its function in tumourigenesis, have not been determined. Chinese tree shrews (Tupaia belangeri chinensis), which are similar to primates, have been used to establish a variety of animal models and have recently received much attention. Here, we established tree shrews as a model for EBV infection by intravenous injection. METHODS: Ten tree shrews were inoculated with EBV by intravenous injection,and blood was collected at regular intervals thereafter from the femoral artery or vein to detect EBV markers. RESULTS: Eight of 10 tree shrews showed evidence of EBV infection. In the 8 EBV-infected tree shrews, EBV copy number increased intermittently or transiently, EBV-related gene expression was detected, and anti-EBV antibodies increased to varying degrees. Macroscopic hepatomegaly was observed in 1 tree shrew, splenomegaly was observed in 4 tree shrews, and enlarged mesenteric lymph nodes were observed in 3 tree shrews. Haematoxylin and eosin (HE) staining showed splenic corpuscle hyperplasia in the spleens of 4 tree shrews and inflammatory cell infiltration of the liver of 1 tree shrew and of the mesenteric lymph nodes of 3 tree shrews. EBER in situ hybridization(ISH) and immunohistochemical (IHC) staining showed that EBER-, LMP1- and EBNA2- positive cells were present in the spleens and mesenteric lymph nodes of some tree shrews. Western blotting (WB) revealed EBNA1-positive cells in the spleens of 4 tree shrews. EBV markers were not detected by HE, EBER-ISH or IHC in the lung or nasopharynx. CONCLUSIONS: These findings suggest that EBV can infect tree shrews via intravenous injection. The presented model offers some advantages for exploring the pathophysiology of EBV infection in humans.

Auditory brainstem responses after electrolytic lesions in bilateral subdivisions of the medial geniculate body of tree shrews.

This study aimed to establish a tree shrew model of bilateral electrolytic lesions in the medial geniculate body (MGB) to determine the advantages of using a tree shrew model and to assess the pattern of sound processing in tree shrews after bilateral electrolytic damage in different parts of the MGB. The auditory brainstem responses (ABRs) of a normal control group (n = 30) and an electrical damage group (n = 30) were tested at 0 h, 24 h, 48 h, 72 h, 7 days, 15 days, and 30 days after surgery. (1) The bilateral ablations group exhibited a significant increase in the ABR threshold of the electrolytic damage group between pre- and post-operation. (2) There were significant increases in the I-VI latencies at 0 h after MGBd and MGBm lesions and at 24 h after MGBv lesion. (3) The amplitudes of wave VI were significantly decreased at 24 h and 48 h after MGBd lesion, at 72 h and 7 days after MGBm lesion, and at 24 h, 48 h, 72 h, and 7 days after MGBv lesion. (1) The electrolytic damage group suffered hearing loss that did not recover and appeared to be difficult to fully repair after bilateral ablation. (2) The latencies and amplitudes of responses in the MGB following bilateral electrolytic lesion were restored to pre-operation levels after 15-30 days, suggesting that a portion of the central nuclei lesion was reversible. (3) The tree shrew auditory animal model has many advantages compared to other animal models, such as greater complexity of brain structure and auditory nuclei fiber connections, which make the results of this experiment more useful for clinical diagnoses compared with studies using rats and guinea pigs.

14-Thienyl methylene matrine (YYJ18), the derivative from matrine, induces apoptosis of human nasopharyngeal carcinoma cells by targeting MAPK and PI3K/Akt pathways in vitro.

BACKGROUND/AIMS: Nasopharyngeal carcinoma (NPC) is a distinctive type of head and neck cancer with the highest incidence in South China. Previous studies have proved that matrine, a main alkaloid isolated from Sophora flavescens Ait, has antitumor activity against NPC. However, the effect is not so pronounced and the underlying mechanism remains largely unclear. Here we investigated whether 14-thienyl methylene matrine (YYJ18) that was derived from matrine could exert more effective suppression activity on NPC, along with the underlying mechanism. METHODS: NPC cell lines CNE1, CNE2 and HONE1 were treated with YYJ18. Cell proliferation and apoptosis were determined by MTT assay and flow cytometry. Activation of mitogen-activated protein kinases (MAPK) and phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) pathways were determined by Western blotting and quantitative RT-PCR. RESULTS: YYJ18 remarkably inhibited proliferation and induced apoptosis of all three NPC cell lines in a dose-dependent manner, especially in CNE2 cells. Furthermore, YYJ18 treatment significantly suppressed phosphorylation of p38 in CNE2 cells, but upregulated phosphorylation of extracellular signal-regulated kinase1/2 (ERK1/2) and Akt. Next, alterations in downstream signaling were found, including activation of BCL2-associated X protein (Bax), caspase-3 and inactivation of B-cell CLL/lymphoma 2 (Bcl-2). CONCLUSION: We demonstrate the potent inhibitory effects of 14-thienyl methylene matrine on NPC cells for the first time, which could be mediated by modulation of MAPK and PI3K/Akt pathways.

The effect of RAD51 135 G>C and XRCC2 G>A (rs3218536) polymorphisms on ovarian cancer risk among Caucasians: a meta-analysis.

Genetic polymorphisms of RAD51 135 G>C and XRCC2 G>A (rs3218536) have been reported to change the risk of ovarian cancer, but the results are controversial. To get a more precise result, a meta-analysis was performed. A comprehensive literature search in PubMed, Excerpta Medica Database, and China National Knowledge Infrastructure was carried out to get case-control studies published up to November 2013. The pooled odds ratio (OR) and its corresponding 95 % confidence interval (CI) were conducted to estimate the effect of RAD51 135 G>C and XRCC2 G>A (rs3218536) polymorphisms on ovarian cancer risk. A total of 13 independent case-control studies with 5,927 cases and 10,303 controls were included in this meta-analysis. There was no significant association between RAD51 135 G>C polymorphism and risk of ovarian cancer. However, the result of total studies indicated the XRCC2 G>A (rs3218536) polymorphism could reduce the risk of ovarian cancer (heterozygote model AG vs. GG: OR=0.877, 95 % CI=0.770-0.999, P=0.048; dominant model AA/AG vs. GG: OR=0.864, 95 % CI=0.763-0.979, P=0.022). The result was still significant after Hardy-Weinberg equilibrium-violating studies were excluded (allele contrast A vs. G: OR=0.836, 95 % CI=0.74-0.943, P=0.004; homozygote model AA vs. GG: OR=0.562, 95 % CI=0.317-0.994, P=0.048; heterozygote model AG vs. GG: OR=0.859, 95 % CI=0.753-0.98, P=0.023; dominant model AA/AG vs. GG: OR=0.842, 95 % CI=0.74-0.958, P=0.009). In the stratified analysis by ethnicity, significantly reduced risk was observed among Caucasians in dominant model (AA/AG vs. GG: OR=0.867, 95 % CI=0.764-0.984, P=0.027). No significant association was found between the RAD51 135G>C polymorphism and the risk of ovarian cancer. Interestingly, XRCC2 G>A (rs3218536) polymorphism might reduce the risk of ovarian cancer. Larger-scale and well-designed studies are needed to further clarify the association.

Some Macrophages With High Expression of CHOP Undergo Necroptosis in Chronic Rhinosinusitis.

BACKGROUND: Endoplasmic reticulum stress (ER stress) is activated in chronic sinusitis with nasal polyps (CRSwNP) and leads to increased expression of C/EBP homologous protein (CHOP). However, the role of CHOP in the pathogenesis of CRSwNP remains unclear. METHODS: CHOP expression was detected by immunohistochemistry staining in nasal mucosa of control and CRSwNP patients. Co-localization of CHOP and cleaved caspase3, p-MLKL, and CD68 was detected by immunofluorescence staining in CRSwNP patients. TNFα, IFNγ, IL1ß, LPS, and tunicamycin were added to primary dispersed nasal polyp cells (DNPCs) to explore their roles in cell death. Western blot, CCK8 assay, and flow cytometry were employed to detect cell death. RESULTS: CHOP was specifically activated in CRSwNP compared to controls. It was mainly macrophages that highly expressed CHOP, some of which underwent apoptosis and the other underwent necroptosis. IL1ß induced increased CHOP and apoptosis, and a slight p-MLKL. In addition, ER stress could also promote p-MLKL expression. Whereas classical TNFα plus IFNγ and LPS did not induce increased necroptosis in DNPCs. CONCLUSION: IL1ß induced the apoptotic pathway and minor necroptosis. And ER stress also plays a role in the occurrence of necroptosis in CRSwNP.

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].

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.

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.

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.