[Accuracy of multi-task network based on vision Transformer in the three-dimensional upper airway analysis].

Objective: To explore the accuracy of a multi-task model based on vision Transformer for analyzing the three-dimensional (3D) upper airway and its subregions, and to evaluate its clinical applicability. Methods: According to the inclusion and exclusion criteria, cone-beam CT (CBCT) data of 10 patients [4 males and 6 females, (20.8±2.7) years] who had their first visit to the Department of Orthodontics in the Hospital of Stomatology, Wuhan University from January 2012 to January 2020 were retrospectively selected. The 3D slicer software was used to segment the upper airway and pharyngeal airway and measure their volumes as the gold standard. The Dolphin 3D software was used to segment the pharyngeal airway and its subregions and measure their volumes as the gold standard. A multi-task model based on vision Transformer developed by the research team for automatic segmentation and volume measurement of the upper airway and its subregions. All the measurements were conducted by the same attending physician. The Bland-Altman analysis and intraclass correlation coefficient (ICC) were used to evaluate the consistency between the multi-task network and the gold standard in the upper airway segmentation and volume measurements, and the paired t test was used to compare the differences between the multi-tasking model and the gold standard. Results: The mean volume deviation of the upper airway segmented by multi-task model and 3D Slicer was -979.6 mm3, and the ICC was 0.97. The mean volume deviation of the pharyngeal airway, nasopharynx, velopharynx, glossopharynx and hypopharynx segmented by multi-task network and Dolphin 3D were 2 069.5, -950.1, -823.6, -813.9 and 4 003.4 mm3, respectively. In addition, ICC in pharyngeal airway, nasopharynx, velopharynx, glossopharynx and hypopharynx were 0.97, 0.94, 0.96, 0.96 and 0.69, respectively. Conclusions: The multi-task model based on vision Transformer produced different errors in the segmentation of 3D upper airway and its subregions. The segmentation of the nasopharynx, velopharynx and glossopharynx was in good agreement with the gold standard, while the segmentation of hypopharynx was poor, suggesting that the robustness and generalization of this model should be further enhanced.

Correlations of tumour permeability parameters with apparent diffusion coefficient in nasopharyngeal carcinoma.

Background and Purpose: Functional imaging has an established role in therapeutic monitoring of cancer treatments. This study evaluated the correlations of tumour permeability parameters derived from dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) and tumour cellularity derived from apparent diffusion coefficient (ADC) in nasopharyngeal carcinoma (NPC). Material and Methods: Twenty NPC patients were examined with DCE-MRI and RESOLVE diffusion-weighted MRI (DW-MRI). Tumour permeability parameters were quantitatively measured with Tofts compartment model. Volume transfer constant (Ktrans), volume of extravascular extracellular space (EES) per unit volume of tissue (Ve), and the flux rate constant between EES and plasma (Kep) from DCE-MRI scan were measured. The time-intensity curve was plotted from the 60 dynamic phases of DCE-MRI. The initial area under the curve for the first 60 s of the contrast agent arrival (iAUC60) was also calculated. They were compared with the ADC value derived from DW-MRI with Pearson correlation analyses. Results: Among the DCE-MRI permeability parameters, Kep had higher linearity in inverse correlation with ADC value (r = -0.69, p = <0.05). Ktrans (r = -0.60, p=<0.05) and iAUC60 (r = -0.64, p = <0.05) also had significant inverse correlations with ADC. Ve showed a significant positive correlation with ADC (r = 0.63, p = <0.05). Conclusions: Nasopharyngeal tumour vascular permeability parameters derived from DCE-MRI scan were correlated linearly with tumour cellularity measured by free water diffusability with ADC. The clinical implementations of these linear correlations in the quantitative assessments of therapeutic response for NPC patients may be worth to further explore.

[A cross-sectional study of periodontal pathogens in saliva of edentulous patients].

Objective: To investigate the prevalence of five specific periodontal pathogens in the saliva of edentulous patients and to compare the differences in the saliva of dentulous individuals with various periodontal conditions. Methods: All the subjects were patients who received regular care at the Beijing Hypertension Prevention and Management Institute. Twenty-seven edentulous patients (edentulous group) were included. According to age (age gap≤5 years), gender, smoking status, diabetes status and hypertension status, each edentulous patient was paired with dentulous individuals suffering from various severity of periodontitis in the same cohort. Then, we selected 3 groups of patients (n=27 in each group) with no or mild periodontitis (mild group), moderate periodontitis (moderate group) and severe periodontitis (severe group). The whole unstimulated saliva was collected before the periodontal examination. Questionnaire survey and periodontal parameters, including plaque index (PLI), probing depth (PD), bleeding index (BI) and clinical attachment loss (CAL), were examined at mesial-buccal and distal-lingual sites of each tooth respectively. DNA was extracted from each sample of the salivary deposition. Porphyromonas gingivalis (Pg), Tannerella forsythia (Tf), Treponema denticola (Td), Campylobacter rectus (Cr) and Prevotella nigrescens (Pn) were detected by using PCR method based on 16SrRNA. The prevalence and quantity of the pathogens under various severity of periodontitis were compared. Results: One or more periodontal pathogens could be detected from the 78% (21/27) of the salivary samples in edentulous group. Thereinto, the prevalences of the five periodontal pathogens were ranked as (from high to low): Cr [56% (15/27)], Tf [44% (12/27)], Pn [26% (7/27)], Pg [22% (6/27)] and Td [11% (3/27)]. All five pathogens' prevalences and Pg, Tf, Td and Pn's quantities showed statistical differences among the four groups. The numbers of detected bacterial species in the mild, moderate and severe groups were significantly higher than that in the edentulous group (P<0.01). Furthermore, the prevalences of the red complex in three dentulous groups [96% (26/27) in each group] were significantly higher than the edentulous group [48% (13/27)] (P<0.05). The proportions of the red complex among all five pathogens (83%) in moderate and severe groups were significantly higher than that in the edentulous group (37%) (P<0.01). Conclusions: All five periodontal pathogens could be detected in most of the saliva samples from edentulous individuals. Nevertheless, the prevalence and quantity were lower than dentulous individuals.

[Expression of hepatocyte nuclear factor 4γ in gastric carcinoma and its role in cell proliferation and stemness].

Objective: To explore the role and molecular mechanism of hepatocyte nuclear factor 4γ (HNF4γ) in proliferation and stemness of gastric cancer. Methods: A total of 102 cases of paraffin-embedded gastric cancer tissues and matched adjacent gastric tissues and 42 cases of fresh-frozen tissues derived from gastric patients who received radical gastrectomy were collected from the First Affiliated Hospital of Zhengzhou University between 2012 to 2015. The expression of HNF4γ was tested by immunohistochemical staining, quantitative real-time polymerase chain reaction (qRT-PCR). HNF4γ overexpressed (AGS-HNF4γ) and shRNA silenced (HGC27-shHNF4γ) gastric cell lines were established. The effects of HNF4γ on cell proliferation and stemness were verified by XTT, clone formation and sphere formation assay. The expression of CD44 was detected by western blot. Results: The mRNA expression level of HNF4γ in fresh-frozen gastric cancer tissue was (12.43±2.702), which was significantly higher than (3.639±1.109) in normal tissue (P<0.001). The high protein expression rate of HNF4γ in paraffin-embedded gastric cancer tissues was 41.2% (42/102), which was significantly higher than 8.8% (9/102) in normal gastric mucosa tissue (P< 0.001). The protein expression of HNF4γ was closely related to the tumor differentiation, infiltration depth, lymph node metastasis and tumor stage (P<0.05). The median survival interval of patients with HNF4γ high expression was 25 months, the 3-year survival rate was 4.8% (2/42), significantly lower than 38 months and 51.7% (31/60) of patients with normal HNF4γ expression (P<0.001). The proliferation and CD44 protein expression of AGS-HNF4γ cells were significantly higher than those of the AGS-Vector cells. The number of clone formation, sphere formation rate of AGS-HNF4γ cells were 243.5±24.5 and (83.5±3.9)%, significantly higher than 81.0±16.0 and (21.8±5.6)% of AGS-Vector cells (P=0.030 and P=0.010, respectively). The proliferation and CD44 protein expression of HGC27-shHNF4 cells were significantly lower than those of the HGC27-vector cells. The number of clone formation, sphere formation rate of HGC27-shHNF4 cells were 26.0±1.0 and (20.8±8.4)%, significantly higher than 83.5±4.5 and (72.5±4.8)% of HGC27-vector cells (P=0.006 and P=0.030, respectively). Conclusions: HNF4γ is upregulated in the gastric cancer tissues and related with the poor prognosis of patients with gastric cancer. Overexpression of HNF4γ promotes the proliferation and remains the stemness of gastric cancer cells by upregulating the expression of CD44.

Prognostic and therapeutic evaluation of nasopharyngeal carcinoma by dynamic contrast-enhanced (DCE), diffusion-weighted (DW) magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS).

Nasopharyngeal carcinoma (NPC) is an aggressive head and neck malignancy, and radiotherapy (with or without chemotherapy) is the primary treatment modality. Reliable tumour assessment during the treatment phase, which can portend the efficacy of radiotherapy and early identification of potential treatment failure in radioresistant disease, has been implicit for better cancer management. Technological advancement in the last decade has fostered the development of functional magnetic resonance imaging (fMRI) techniques into a promising tool for diagnostic and therapeutic assessments in head and neck cancer. Apart from conventional morphological assessment, early detection of the physiological environment by fMRI allows a more thorough investigation in monitoring tumour response. This article discusses the relevant fMRI utilities in NPC as an early prognostic and monitoring tool for treatment. Challenges and future developments of fMRI in radiation oncology are also discussed.

[Significance of hydroxymethylglutaryl-CoA synthase 2 on prognosis of esophageal squamous cell carcinoma].

Objective: To explore the roll and function of hydroxymethylglutaryl-CoA synthase 2 (HMGCS2) in the development and progression of human esophageal squamous cell carcimoma(ESCC). Method: Using immunohistochemistry, the expression of HMGCS2 was determined in 150 primary ESCC patients from July 2002 to December 2005 in the People's Hospital of Linzhou City, Henan Province. And HMGCS2 over-expression ESCC cell lines were established to verify HMGCS2 gene function. Result: In 150 cases of ESCCs, the expression rate of HMGCS2 was 58% (87/150), which was lower than 72% (108/150) in paired normal tissues, the difference was statistically significant (P=0.013). HMGCS2 down-regulated expression was associated with tumor cell differentiation (P=0.022), pT status (P=0.036), pN status (P=0.017) and TNM stage(P=0.012). The 5-years disease-specific survival (DSS) in down HMGCS2 expression group (14 months) was poorer than those in normal expression group (20 months; P=0.002). In addition, multivariate Cox regression analysis showed that HMGCS2 expression (Wald=7.136, P=0.008) was an independent risk factor for DSS. Furthermore, functional studies demonstrated that HMGCS2 gene could suppress the tumorigenic ability of ESCC cells (OD: 0.79±0.04 vs 1.25±0.68; P=0.01), the formation of colone (number of colones: 30±10 vs 189±15, P=0.002), and cell motility (number of cells: 27±14 vs 222±40, P=0.009). Conclusion: HMGCS2 can inhibit the proliferation and migration of ESCC cells, and could be an important candidate tumor suppressor gene for ESCC.

Electrophysiological effects of melatonin on rat trigeminal ganglion neurons that participate in nociception in vitro.

OBJECTIVE: Melatonin (MT) is a hormone mainly produced by the pineal gland. It may be involved in the regulation of nociception, the mechanisms of which remain unclear. In the present study, electrophysiological effects of MT on neurons were studied. MATERIALS AND METHODS: The cultured neurons were isolated from Sprague-Dawley rats trigeminal ganglia (TG). The neuron was voltage clamped using the whole cell patch clamp technique. We recorded resting membrane potential, action potential threshold and number, action potential duration and GABA-activated inward currents in the presence of 0.01 µM, 10 µM MT, and in the absence of MT. RESULTS: In the presence of high concentration of MT, the spontaneous action potential disappeared and action potential threshold was significantly increased. GABA-activated inward currents were recorded and blocked by GABAA receptor antagonist, bicuculline, in the majority of TG neurons (91% 40/44). Continuous perfusion of MT could cause a decrease of GABA-activated currents. The inhibiting effect was dose-dependent and irreversible. CONCLUSIONS: The results suggest that MT has several electrophysiological effects on TG neurons, which may be involved in the peripheral mechanisms of orofacial pain.

LINC00963 promotes hepatocellular carcinoma progression by activating PI3K/AKT pathway.

OBJECTIVE: To explore the role of LINC00963 in the pathogenesis of hepatocellular carcinoma and its underlying mechanisms. PATIENTS AND METHODS: The expression level of LINC00963 in 48 cases of hepatocellular carcinoma (HCC) tissues and paracancerous tissues were detected by quantitative Real-time (qRT-PCR). Survival analysis was carried out based on the expression level of LINC00963. The association between the expression level of LINC00963 and clinical characteristics of these subjects was analyzed by x2-test. The proliferation and cell cycle of HCC cells after transfection of LINC00963 overexpression plasmids were evaluated by cell counting kit-8 (CCK-8) assay and flow cytometry, respectively. RESULTS: The expression level of LINC00963 in HCC tissues was remarkably higher than that in paracancerous tissues, indicating a potential diagnostic significance of LINC00963. The progression-free -with the tumor size and TNM stage, but not with age, gender, histological type and lymph node metastasis. Overexpression of LINC00963 significantly enhanced the proliferation ability of HepG2 and HCC cells and prolonged their G0/G1 phase. Furthermore, the PI3K/AKT expression was increased after overexpression of LINC00963, while AKT siRNA effectively reversed the prolonged G0/G1 phase caused by LINC00963 overexpression. CONCLUSIONS: Our data revealed that LINC00963 was upregulated in HCC, which significantly extended the G0/G1 phase of HCC cells by activating PI3K/AKT pathway and promoting the proliferative ability of HCC cells. LINC00963 may be involved in the HCC development.

DHRS2 inhibits cell growth and motility in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is highly prevailing in Asia and it is ranked in the most aggressive squamous cell carcinomas. High-frequency loss of heterozygosity occurred in chromosome 14q11.2 in many tumors including ESCC, suggesting that one or more tumor-suppressor genes might exist within this region. In this study, we identified the tumor-suppressing role of DHRS2 (short-chain dehydrogenase/reductase family, member 2) at 14q11.2 in ESCCs. Downregulation of DHRS2 occurred in 30.8% of primary ESCC tumor tissues vs paired non-tumorous tissues. DHRS2 downregulation was associated significantly with ESCC invasion, lymph nodes metastasis and clinical staging (P<0.001). Survival analysis revealed that DHRS2 downregulation was significantly associated with worse outcome of patients with ESCC. In vitro and in vivo studies indicated that both DHRS2 variants could suppress cell proliferation and cell motility. Moreover, we demonstrated that DHRS2 could reduce reactive oxygen species and decrease nicotinamide adenine dinucleotide phosphate (oxidized/reduced), increase p53 stability and decrease Rb phosphorylation; it also decreased p38 mitogen-activated protein kinase phosphorylation and matrix metalloproteinase 2. In summary, these findings demonstrated that DHRS2 had an important part in ESCC development and progression.

Identification of chemokine CXCL10 in tumor microenvironment by antibody array as a prognostic marker in hepatocellular carcinoma.

Immunological microenvironment is not only composed of multiple immune cells, but also deposited various inflammation factors that regulate immune response to tumor cells. To ascertain the crucial immune factors presented in hepatocellular carcinoma microenvironment (HCM), tumor tissue culture supernatant (TCS) and the corresponding non-tumor tissue culture supernatant (NCS) from patient with hepatocellular carcinoma (HCC) were analyzed by antibody array technology. Among the inflammation-associated cytokines assayed, high level of chemokines CXCL8/IL-8 (6.82-fold increase) and CXCL10/IP-10 (16.45-fold increase) in TCS than that in paired NCS were evidently identified. And low expression of IL-16 (0.14-fold decrease) and RANTES/CCL5 (0.17-fold decrease) in TCS were also uncovered. Especially, overexpression of CXCL10 in primary HCC compared with their non-tumor counterparts was significantly associated with serum AFP level (P = 0.004), tumor size (P = 0.021), tumor number (P < 0.001) and TNM stage (P = 0.027). In addition, Kaplan-Meier curves demonstrated that patients with higher CXCL10 expression levels had significantly poorer overall survival (P = 0.016) and disease-free survival (P = 0.022) than those with lower CXCL10 expression levels. Univariate and multivariate analyses revealed that the level of CXCL10 expression was an independent prognostic factor for overall survival in HCC patients. In summary, high concentration of CXCL10 is deposited in HCM identified by antibody array, which may contribute to the prediction of clinical outcome of HCC patients.

MicroRNA-377 suppresses initiation and progression of esophageal cancer by inhibiting CD133 and VEGF.

Esophageal cancer is one of the most lethal cancers worldwide with poor survival and limited therapeutic options. The discovery of microRNAs created a new milestone in cancer research. miR-377 is located in chromosome region 14q32, which is frequently deleted in esophageal squamous cell carcinoma (ESCC), but the biological functions, clinical significance and therapeutic implication of miR-377 in ESCC are largely unknown. In this study, we found that miR-377 expression was significantly downregulated in tumor tissue and serum of patients with ESCC. Both tumor tissue and serum miR-377 expression levels were positively correlated with patient survival. Higher serum miR-377 expression was inversely associated with pathologic tumor stage, distant metastasis, residual tumor status and chemoradiotherapy resistance. The roles of miR-377 in suppressing tumor initiation and progression, and the underlying molecular mechanisms were investigated. Results of in vitro and in vivo experiments showed that miR-377 overexpression inhibited the initiation, growth and angiogenesis of ESCC tumors as well as metastatic colonization of ESCC cells, whereas silencing of miR-377 had opposite effects. Mechanistically, miR-377 regulated CD133 and VEGF by directly binding to their 3' untranslated region. Moreover, systemic delivery of formulated miR-377 mimic not only suppressed tumor growth in nude mice but also blocked tumor angiogenesis and metastasis of ESCC cells to the lungs without overt toxicity to mice. Collectively, our study established that miR-377 plays a functional and significant role in suppressing tumor initiation and progression, and may represent a promising non-invasive diagnostic and prognostic biomarker and therapeutic strategy for patients with ESCC.

Overexpression of maelstrom promotes bladder urothelial carcinoma cell aggressiveness by epigenetically downregulating MTSS1 through DNMT3B.

We have recently identified and characterized a novel oncogene, maelstrom (MAEL) from 1q24, in the pathogenesis of hepatocellular carcinoma. In this study, MAEL was investigated for its oncogenic role in urothelial carcinoma of the bladder (UCB) tumorigenesis/aggressiveness and underlying molecular mechanisms. Here, we report that overexpression of MAEL in UCB is important in the acquisition of an aggressive and/or poor prognostic phenotype. In UCB cell lines, knockdown of MAEL by short hairpin RNA is sufficient to inhibit cell growth, invasiveness/metastasis and suppressed epithelial-mesenchymal transition (EMT), whereas ectopic overexpression of MAEL promoted cell growth, invasive and/or metastatic capacity and enhanced EMT both in vitro and in vivo. We further demonstrate that MAEL could induce UCB cell EMT by downregulating a critical downstream target, the metastasis suppressor 1 (MTSS1) gene, ultimately leading to an increased invasiveness of cancer cells. Notably, overexpression of MAEL in UCB cells substantially enhanced the enrichment of DNA methyltrans-ferase (DNMT)3B and histone deacetylase (HDAC)1/2 on the promoter of the MTSS1, and thereby epigenetically suppressing the MTSS1 transcription. Downregulation of MTSS1 by MAEL in UCB cells is partially dependent on DNMT3B. Furthermore, we identify that beside the gene amplification of MAEL, miR-186 is a key negative regulator of MAEL and downregulation of miR-186 is another important mechanism for MAEL overexpression in UCBs. These data suggest that overexpression of MAEL, caused by gene amplification and/or decreased miR-186, has a critical oncogenic role in UCB pathogenesis by downregulation of MTSS1, and MAEL could be used as a novel prognostic marker and/or effective therapeutic target for human UCB.

Decreased expression of H3K27me3 in human ovarian carcinomas correlates with more aggressive tumor behavior and poor patient survival.

It has been confirmed that trimethylation of lysine 27 on histone H3 (H3K27me3) plays an important role in epigenetic process of tumorigenesis. However, the status of H3K27me3 in ovarian cancer and its impact on patients' clinicopathologic characteristics and prognosis are unclear. In the present study, the immunohistochemistry (IHC) was utilized to detect protein expression of H3K27me3 in 12 normal ovaries, 26 ovarian cystadenomas, 31 borderline ovarian tumors and 168 ovarian carcinomas by tissue microarray. The association between H3K27me3 expression with clinicopathologic features and patient prognosis were also evaluated using various statistical models. The expression of H3K27me3 was decreased in 2 of 12 (16.7%) cases of the normal ovaries, 8 of 26 (30.8%) cases of cystadenomas, 12 of 31 (38.7%) cases of borderline ovarian tumors, and 93 of 168 (55.4%) cases of primary ovarian carcinomas, respectively (P<0.05). Further correlation analysis suggested that decreased expression of H3K27me3 in ovarian carcinomas was significantly correlated with more advanced pM and FIGO stages (P<0.05). In addition, a significant association between decreased expression of H3K27me3 and shortened patient survival (mean 66 months versus 101 months, p=0.019) was demonstrated by univariate survival analysis of the ovarian carcinoma cohorts. Importantly, H3K27me3 expression provided a significant independent prognostic factor in multivariate analysis (p=0.028). These findings confirmed that decreased expression of H3K27me3 in primary ovarian cancer might be correlated with the acquisition of an invasive and/or aggressive phenotype of tumor, and might serve as an independent biomarker for poor prognosis in patients with ovarian carcinoma.

Knock-down of p300 decreases the proliferation and odontogenic differentiation potentiality of HDPCs.

AIM: To investigate the role of p300 in the regulation of proliferation and odontogenic differentiation of human dental pulp cells (HDPCs). METHODOLOGY: The recombinant lentiviral vector pshRNA-copGFP was used to knock-down p300 expression in HDPCs. Protein level of acetylated H3 was detected. The proliferation of HDPCs was measured using the CCK8 assay. The cell cycle and apoptosis were analysed using flow cytometry and TUNEL staining, respectively. The expression levels of Cdc25A, p21(waf1) and the cleaved products of caspase 3 and caspase 7 were determined utilizing real-time quantitative polymerase chain reaction and Western blotting analysis. The alkaline phosphatase (ALP) activity was measured, and the formation of mineralized nodules was assessed using alizarin red staining after the induction of odontogenic differentiation of HDPCs. The expression levels of the odontogenic differentiation markers DMP-1, DSPP and DSP were detected utilizing real-time quantitative polymerase chain reaction and Western blotting analysis. RESULTS: After p300 was knocked down in HDPCs, p300 was significantly down-regulated at both the mRNA and protein levels, and histone H3 acetylation was reduced. The proliferation capacity of HDPCs was suppressed in p300 knock-down groups. The cells were arrested in the G0/G1 phase of the cell cycle, and cell apoptosis was triggered. ALP activity, the formation of mineralized nodules and the expression levels of DMP-1, DSPP and DSP were all decreased in p300-knock-down HDPCs undergoing odontogenic differentiation. CONCLUSION: Knocking down p300 restrains the proliferation and odontogenic differentiation potentiality of HDPCs.

Anti-angiogenic pathway associations of the 3p21.3 mapped BLU gene in nasopharyngeal carcinoma.

Zinc-finger, MYND-type containing 10 (ZMYND10), or more commonly called BLU, expression is frequently downregulated in nasopharyngeal carcinoma (NPC) and many other tumors due to promoter hypermethylation. Functional evidence shows that the BLU gene inhibits tumor growth in animal assays, but the detailed molecular mechanism responsible for this is still not well understood. In current studies, we find that 93.5% of early-stage primary NPC tumors show downregulated BLU expression. Using a PCR array, overexpression of the BLU gene was correlated to the angiogenesis network in NPC cells. Moreover, expression changes of the MMP family, VEGF and TSP1, were often detected in different stages of NPC, suggesting the possibility that BLU may be directly involved in the microenvironment and anti-angiogenic activity in NPC development. Compared with vector-alone control cells, BLU stable transfectants, derived from poorly-differentiated NPC HONE1 cells, suppress VEGF165, VEGF189 and TSP1 expression at both the RNA and protein levels, and significantly reduce the secreted VEGF protein in these cells, reflecting an unknown regulatory mechanism mediated by the BLU gene in NPC. Cells expressing BLU inhibited cellular invasion, migration and tube formation. These in vitro results were further confirmed by in vivo tumor suppression and a matrigel plug angiogenesis assay in nude mice. Tube-forming ability was clearly inhibited, when the BLU gene is expressed in these cells. Up to 70-90% of injected tumor cells expressing increased exogenous BLU underwent cell death in animal assays. Overexpressed BLU only inhibited VEGF165 expression in differentiated squamous NPC HK1 cells, but also showed an anti-angiogenic effect in the animal assay, revealing a complicated mechanism regulating angiogenesis and the microenvironment in different NPC cell lines. Results of these studies indicate that alteration of BLU gene expression influences anti-angiogenesis pathways and is important for the development of NPC.

Transcriptome sequencing of sea cucumber (Apostichopus japonicus) and the identification of gene-associated markers.

Sea cucumber (Apostichopus japonicus) is an ecologically and economically important species in East and South-East Asia. This project aimed to identify large numbers of gene-associated markers and differentially expressed genes (DEGs) after lipopolysaccharides (LPS) challenge in A. japonicus using high-throughput transcriptome sequencing. A total of 162 million high-quality reads of 174 million raw reads were obtained by deep sequencing using Illumina HiSeq™ 2000 platform. Assembly of these reads generated 94 704 unigenes, with read length ranging from 200 to 16 153 bp (average length of 810 bp). A total of 36 005 were identified as coding sequences (CDSs), 32 479 of which were successfully annotated. Based on the assembly transcriptome, we identified 142 511 high-quality single nucleotide polymorphisms (SNPs). Among them, 33 775, 63 120 and 45 616 were located in sequences without predicted CDS (non-CDSs), CDSs and untranslated regions (UTRs), respectively. These putative SNPs included 82 664 transitions and 59 847 transversions. Totally, 89 375 (59.1%) were distributed in 15 473 known genes. A total of 6417 microsatellites were detected in 5970 unigenes, 3216 of which were annotated and 2481 were successfully subjected for primer design. The numbers of simple sequence repeats (SSRs) identified in non-CDSs, CDSs and UTRs were 2367, 2316 and 1734. These potential SNPs and SSRs are expected to provide abundant resources for genetic, evolutionary and ecological studies in sea cucumber. Transcriptome comparison revealed 1330, 1347 and 1291 DEGs in the coelomocytes of A. japonicus at 4 h, 24 h and 72 h after LPS challenge, respectively. Approximately 58.4% (1802) of total DEGs have been successfully annotated.

Hyperthyroid monkeys: a nonhuman primate model of experimental Graves' disease.

Graves' disease (GD) is a common organ-specific autoimmune disease with the prevalence between 0.5 and 2% in women. Several lines of evidence indicate that the shed A-subunit rather than the full-length thyrotropin receptor (TSHR) is the autoantigen that triggers autoimmunity and leads to hyperthyroidism. We have for the first time induced GD in female rhesus monkeys, which exhibit greater similarity to patients with GD than previous rodent models. After final immunization, the monkeys injected with adenovirus expressing the A-subunit of TSHR (A-sub-Ad) showed some characteristics of GD. When compared with controls, all the test monkeys had significantly higher TSHR antibody levels, half of them had increased total thyroxine (T4) and free T4, and 50% developed goiter. To better understand the underlying mechanisms, quantitative studies on subpopulations of CD4+T helper cells were carried out. The data indicated that this GD model involved a mixed Th1 and Th2 response. Declined Treg proportions and increased Th17:Treg ratio are also observed. Our rhesus monkey model successfully mimicked GD in humans in many aspects. It would be a useful tool for furthering our understanding of the pathogenesis of GD and would potentially shorten the distance toward the prevention and treatment of this disease in human.

Overexpression of matrix metalloproteinase 10 is associated with poor survival in patients with early stage of esophageal squamous cell carcinoma.

Matrix metallopeptidase 10 (MMP10) is frequently expressed and correlates closely with metastasis and poor prognosis in various human cancers. However, the significance of MMP10 expression in esophageal squamous cell carcinoma (ESCC) and its role in ESCC progression remains unclear. In this report, upregulation of MMP10 mRNA was detected in 39/60 (65.0%) of primary ESCC tissues compared with their paired nontumor esophageal tissues. Tissue microarray (TMA) study found protein overexpression of MMP10 in 188/239 (78.7%) of primary ESCC tissues but not in their corresponding nontumor esophageal tissues, suggesting that overexpression of MMP10 may play important roles in ESCC development and progression. Although the overexpression of MMP10 was not significantly associated with disease-specific survival rate (P= 0.182) for all tested ESCCs, it was significantly associated with poorer disease-specific survival (P= 0.001) in early stage of ESCCs (I-IIA). In addition, multivariate analysis found that MMP10 expression in tumor tissues was evaluated as a potential independent prognostic factor for early stage ESCC patients. These findings suggest that MMP10 plays an important role in ESCC progression in the early stage, and overexpression of MMP10 in tumor tissues could be used as a potential prognostic marker for patients with early clinical stage of ESCC.

EZH2 supports nasopharyngeal carcinoma cell aggressiveness by forming a co-repressor complex with HDAC1/HDAC2 and Snail to inhibit E-cadherin.

The enhancer of zeste homolog 2 (EZH2) is upregulated and has an oncogenic role in several types of human cancer. However, the abnormalities of EZH2 and its underlying mechanisms in the pathogenesis of nasopharyngeal carcinoma (NPC) remain unknown. In this study, we found that high expression of EZH2 in NPC was associated closely with an aggressive and/or poor prognostic phenotype (P<0.05). In NPC cell lines, knockdown of EZH2 by short hairpin RNA was sufficient to inhibit cell invasiveness/metastasis both in vitro and in vivo, whereas ectopic overexpression of EZH2 supported NPC cell invasive capacity with a decreased expression of E-cadherin. In addition, ablation of endogenous Snail in NPC cells virtually totally prevented the repressive activity of EZH2 to E-cadherin, indicating that Snail might be a predominant mediator of EZH2 to suppress E-cadherin. Furthermore, co-immunoprecipitation (IP), chromatin IP and luciferase reporter assays demonstrated that in NPC cells, (1) EZH2 interacted with HDAC1/HDAC2 and Snail to form a repressive complex; (2) these components interact in a linear fashion, not in a triangular fashion, that is, HDAC1 or HDAC2 bridge the interaction between EZH2 and Snail; and (3) the EZH2/HDAC1/2/Snail complex could closely bind to the E-cadherin promoter by Snail, but not YY1, to repress E-cadherin. The data provided in this report suggest a critical role of EZH2 in the control of cell invasion and/or metastasis by forming a co-repressor complex with HDAC1/HDAC2/Snail to repress E-cadherin, an activity that might be responsible, at least in part, for the development and/or progression of human NPCs.