miR-9 targets CXCR4 and functions as a potential tumor suppressor in nasopharyngeal carcinoma.

MicroRNA deregulation and pathway alterations have been implicated in nasopharyngeal carcinoma (NPC), a highly invasive and metastatic cancer widely prevalent in Southern China. In this study, we report that miR-9 is commonly downregulated in NPC specimens and NPC cell lines with important functional consequences. The reduced expression of miR-9 was inversely correlated with clinical stages and marked the progression from locoregional to metastatic tumors. The CpG island hypermethylation contributed to miR-9 silencing in NPC cell lines and tissues. Ectopic expression of miR-9 dramatically inhibited the proliferative, migratory and invasive capacities of NPC cells in vitro and in vivo. We found that miR-9 strongly reduced the expression of CXCR4 in NPC cells. Luciferase assay demonstrated that miR-9 could directly bind to the 3' untranslated region of CXCR4. Similar to the restoring miR-9 expression, CXCR4 downregulation inhibited cell growth, migration and invasion, whereas CXCR4 overexpression rescued the suppressive effect of miR-9. Mechanistic investigations revealed that CXCR4 functionally mediated the SDF-1-stimulated activation of p38 mitogen-activated protein kinase pathway in NPC cells with miR-9 downregulation or CXCR4 overexpression. In clinical specimens, CXCR4 and phospho-p38 were widely overexpressed, and the levels increased with the progression from locoregional to metastatic tumors in NPC tissues. The levels of CXCR4 were inversely correlated with miR-9 or phospho-p38 expression. Taken together, our results indicate that miR-9 functions as a tumor-suppressive microRNA in NPC, and that its suppressive effects are mediated chiefly by repressing CXCR4 expression.

Epstein-Barr virus nuclear antigen 1 (EBNA1) protein induction of epithelial-mesenchymal transition in nasopharyngeal carcinoma cells.

BACKGROUND: The Epstein-Barr virus (EBV)-encoded EB nuclear antigen 1 (EBNA1) protein is required for maintenance and transmission of the viral episome in EBV-infected cells. The objective of this study was to investigate the role of EBNA1 protein in nasopharyngeal carcinoma (NPC). METHODS: Tissue samples from 48 patients with NPC and 12 patients with chronic nasopharyngitis were subjected to immunohistochemical analysis of EBNA1 expression. EBNA1 combinational DNA was used to overexpress EBNA1 protein in NPC cell lines to assess tumor cell epithelial-mesenchymal transition (EMT), colony formation, migration and invasion, and gene expression. RESULTS: EBNA1 protein was highly expressed in NPC tissue specimens, and its expression was associated with NPC lymph node metastasis. EBNA1 expression affected NPC cell morphology and the expression of EMT markers in vitro. Furthermore, overexpression of EBNA1 inhibited the expression of microRNA 200a (miR-200a) and miR-200b and, in turn, up-regulated expression of their target genes, zinc finger E-box binding homeobox 1 ( ZEB1) and ZEB2, which are well known mediators of EMT. In addition, EBNA1-regulated miR-200a and miR-200b expression was mediated by transforming growth factor-ß1. CONCLUSIONS: The current findings provided novel insight into the vital role of EBNA1 in manipulating a molecular switch of EMT in EBV-positive NPC cells.

Positive lymph node ratio predicts adverse prognosis for patients with lymph nodes metastatic hypopharyngeal squamous cell carcinoma after primary surgery.

Background: Positive lymph node ratio (LNR) is associated with the prognosis of many cancers. However, its prognostic value in patients with hypopharyngeal squamous cell carcinoma (HSCC) is unclear due to the rarity of HSCC. This study aimed to investigate the prognostic value of LNR in HSCC using the Surveillance, Epidemiology, and End Results (SEER) database. Methods: Data spanning 2004 to 2015 of eligible HSCC patients were retrospectively retrieved from the SEER database. Clinicopathological data, including age at diagnosis, race, gender, marital status, primary tumor site, tumor size, tumor grade, Tumor-Lymph Node-Metastasis (TNM) stage, surgical type, postoperative adjuvant therapy (POAT) record, the number of lymph nodes (LNs) examined, the number of positive LNs, survival time, and death classification were collected and dichotomized through the receiver operating characteristic (ROC) curve. The LNR was defined as the ratio of positive LNs to the total number of LNs examined. The Kaplan-Meier method and Cox regression models were used to assess the association between LNR vs. cancer-specific survival (CSS) and overall survival (OS). Results: The 5-year CSS and OS rates of the 391 patients were 44% and 33.7%, respectively. The median LNR was 0.083 [interquartile range (IQR), 0.043-0.179], and the optimal cut-off value of LNR was 0.23. Kaplan-Meier curves showed that patients with LNR ≥0.23 had significantly shorter CSS and OS than LNR <0.23. In multivariable analysis, large tumor size [hazard ratio (HR): 1.012, P=0.016], N3 stage (HR: 2.113, P=0.040), M1 stage (HR: 2.458, P=0.041), with POAT (HR: 0.559, P=0.001), and LNR ≥0.23 (HR: 1.795, P=0.001) independently predicted CSS, while old age (HR: 1.019, P=0.009), large tumor size (HR: 1.012, P=0.006), M1 stage (HR: 3.422, P=0.001), with POAT (HR: 0.610, P=0.001), and LNR ≥0.23 (HR: 1.667, P=0.001) independently predicted OS. The subgroup analysis showed that patients with LNR ≥0.23 shared worse CSS and OS in either N2 or N3 subgroups than those with LNR <0.23. Furthermore, POAT provided an independent protective factor in the LNR ≥0.23 group, while it had no significant effect in the LNR <0.23 group. Conclusions: This study demonstrates a strong association between LNR and prognosis in patients with LNs metastatic HSCC. Further, it provides an alternative tool for providing supplemental information regarding prognosis.

Correction to "Highly Efficient Super-Continuum Generation on an Epsilon-Near-Zero Surface".

[This corrects the article DOI: 10.1021/acsomega.9b04026.].

Highly Efficient Super-Continuum Generation on an Epsilon-Near-Zero Surface.

The efficient super-continuum (SC) generation on a surface via a high-order photo-electron interaction is a great challenge for integrated optics because the surficial nonlinear optical efficiency is usually limited by finite light-matter interaction length and electric field intensity. Nowadays, epsilon-near-zero (ENZ) materials, showing infinite enhanced electronic field in theory, provide a kind of new platform to obtain a giant nonlinear response on the surface. Here, under the irradiation of a multiwavelength laser, an exotic and efficient SC generation from 406 to 1100 nm on the ENZ aluminum-doped zinc oxide surface was experimentally demonstrated by diversified nonlinear processes, including second harmonic generation, third harmonic generation, four wavelength mixing, and cascading stimulated Raman scattering. Particularly, an unprecedented nonlinear conversion efficiency of 3.94% W-1, 16 orders of magnitude higher than the common surface case (about 10-16% W-1), was presented.

Surface Nonlinear Optics on Centrosymmetric Dirac Nodal-Line Semimetal ZrSiS.

Gapless surface states (SSs) are features of topological semimetals and are extensively observed. Nowadays, the emerging question is whether the SSs possess exotic and applicable properties. Here, associated with the symmetrical selection rule for nonlinear optical materials, the surface nonlinear optics on a centrosymmetric Dirac nodal-line semimetal ZrSiS crystal is studied and it is found that the SSs bring record nonlinear susceptibilities. The unprecedented conversion efficiencies for second and third harmonic generations are 0.11‰ and 0.43‰, respectively, more than ten orders of magnitude larger than the typical surface second harmonic generation. This work discovers a new route toward studying the SSs for applications in nonlinear photonics.

Establishment of Immortalized Laryngeal Epithelial Cells Transfected with Bmi1.

Primary laryngeal epithelial cells are essential to exploring the mechanisms of laryngeal and voice disorders; however, they are difficult to study and apply because of their limited life span. The purpose of this study was to develop a stable and reliable in vitro model for the comprehensive study of the pathogenesis of laryngeal and voice diseases. The pLVTHM-Bmi1 plasmid was constructed and used to immortalize primary laryngeal epithelial cells by lentiviral infection. The expressions of Bmi1, human telomerase reverse transcriptase (hTERT), p53, and pRB pathway proteins were detected by western blotting. Functional characteristics of the immortalized cell lines were verified by cell senescence ß-galactosidase staining, 5-ethynyl-2'-deoxyuridine cell proliferation test, and flow cytometry. We successfully introduced Bmi into human subglottic (hSG) cells and human ventricle (hV) cells. Both the human immortalized subglottic Bmi1 (hSG-Bmi1) cell line and the human immortalized ventricle Bmi1 (hV-Bmi1) cell line maintained normal epithelial morphology and divided successfully after more than 20 culture passages. As Bmi1 was overexpressed in these cells, the expression of human telomerase reverse transcriptase (hTERT) and phosphorylated Rb increased while p16 and p21 decreased. Following Bmi1-mediated immortalization, cell senescence decreased significantly, and cell proliferation was accelerated. Tumor formation was not observed for hSG, hV, or hSG-Bmi1, and hV-Bmi1 cells in nude mice. hSG-Bmi1 cells dominated by stratified squamous epithelium and hV-Bmi1 cells dominated by columnar cells were established. The new cell lines lay a foundation for the study of the pathogenic mechanisms of laryngeal and voice diseases.

[A preliminary study on the assessment of swallowing function after supracricoid partial laryngectomy].

OBJECTIVE: To investigate a reliable and easy assessment method for swallowing function by evaluating objectively the recovery process of swallowing function in patients six months after supracricoid partial laryngectomy. METHODS: The swallowing function of patients who underwent supracricoid partial laryngectomy was evaluated six months after operation in Nanfang Hospital of Southern Medical University between January 2013 and February 2014 with two methods, the modified barium swallow (MBS) and fiberoptic endoscopic evaluation of swallowing (FEES), combined with modified penetration aspiration scale (MPAS). Furthermore, the feasibility, reliability and accuracy of these two methods were compared. RESULTS: Eleven patients were enrolled. MPAS equals score 1 for solid food, semiliquid food, and liquid food was defined as a criteria of normal swallowing function. By MBS evaluation, the numbers of patients with normal swallowing function were two cases at day 16-30 postoperation, two cases at day 31-45 postoperation, five cases at day 46-90 postoperation, and six cases at day 91-180 postoperation, respectively. By FEES evaluation, the above numbers were three cases, four cases, six cases and eight cases, respectively. When the aspiration was minimal and ejected completely and MPAS was less than or equal to score 4 for solid food, semiliquid food, and liquid food, the gastric tube could be removed. According to this standard, the gastric tube was removed in all cases, and the mean time was (21.7 ± 9.8) days. A good correlation was obtained between these two methods when evaluating solid and semiliquid food, and the Kappa values were 0.802 and 0.844, respectively. However, a little agreement was obtained between these two methods when evaluating liquid food, and the Kappa value was 0.529. CONCLUSIONS: Patients who underwent supracricoid partial laryngectomy could restore good swallowing function in six months after the operation. Both the MBS and FEES are valuable procedures for evaluating objectively the swallowing function in patients after supracricoid partial laryngectomy. The FEES is much better than MBS, because FEES is a simple operation performed alone by otolaryngologists with no radiation.

Proteomic identification of alpha-2-HS-glycoprotein as a plasma biomarker of hypopharyngeal squamous cell carcinoma.

Hypopharyngeal squamous cell carcinoma (HSCC) has very poor prognosis compared with other head and neck squamous cell carcinomas. Late-stage diagnosis of HSCC increases mortality. Therefore, more effective biomarkers for early diagnosis of HSCC are necessary. Unfortunately, appropriate biomarkers for clinical diagnosis and prognosis have not been identified yet. However, recent progresses in quantitative proteomics have offered opportunities to identify plasma proteins as biomarkers for HSCC. In the present study, plasma samples were analyzed by two-dimensional differential gel electrophoresis (2D-DIGE), and differentially expressed proteins were identified by matrix assisted laser desorption ionization-time of flight/time of flight mass spectrometry (MALDI-TOF/TOF MS). A total of 26 proteins representing 12 unique gene products were identified. The up-regulation proteins were alpha-2-HS-glycoprotein (AHSG), complement C4-B, haptoglobin, C-reactive protein, and ceruloplasmin, whereas the down-regulation proteins were serum albumin, angiotensinogen, alpha-1-antichymotrypsin, Ig gamma-3 chain C region, fibrinogen gamma chain, apolipoprotein A-I, and Ig kappa chain C region. Among all the differentially expressed proteins, AHSG was validated by western blot and ELISA. The results were consistent with the data from 2D-DIGE, further suggesting that AHSG may be employed as a potential biomarker for the early diagnosis of HSCC. In summary, this study was the first to use 2D-DIGE and MALDI-TOF/TOF platform to identify the potential plasma biomarkers for HSCC. The plasma AHSG showed great potential for HSCC screening.

Levels of plasma Epstein-Barr virus DNA prior and subsequent to treatment predicts the prognosis of nasopharyngeal carcinoma.

The level of Epstein-Barr virus DNA (EBV-DNA) in the plasma prior and subsequent to treatment is a reliable biomarker for the screening, diagnosis, monitoring and prognosis of nasopharyngeal carcinoma (NPC). The present retrospective study aimed to determine whether pre- and post-treatment levels of plasma EBV-DNA were predictive of survival in a large sample of patients with NPC. The level of plasma EBV-DNA in 637 NPC patients prior and subsequent to treatment was determined by quantitative polymerase chain reaction. The value of pre- and post-treatment plasma EBV-DNA in predicting the survival of NPC patients was then analyzed. The results revealed that pre-treatment plasma EBV-DNA loads were significantly higher in patients with NPC than those in healthy controls (P<0.001). The percentage of patients with positive plasma EBV-DNA was markedly higher prior to treatment (70.64%; median, 1150 copies/ml; range, 0-9.75×106 copies/ml) than following treatment (25.99%; median, 0 copies/ml; range, 0-3.83×106 copies/ml) (P<0.001). Patients with a high plasma EBV-DNA load presented with a higher clinical tumor classification, lymph node status, metastatic status and overall cancer stage. The risk of NPC relapse and mortality was higher in patients with pre-treatment plasma EBV-DNA levels of ≥1,500 copies/ml than that in patients with <1,500 copies/ml. Furthermore, the risk of relapse and mortality was higher in patients with positive post-treatment plasma EBV-DNA than in patients with negative post-treatment plasma EBV-DNA. Detectable post-treatment plasma EBV-DNA was the most significant prognostic factor to affect relapse-free survival, whilst metastasis was the prognostic factor with the greatest effect on overall survival. These data indicated that pre- and post-treatment levels of plasma EBV-DNA were able to predict the prognosis of NPC. This finding may provide novel references for research and clinical practice.

[Salvage surgery for recurrent laryngocarcinoma].

OBJECTIVE: To study the curative effect and prognosis of salvage surgery performed for recurrent laryngocarcinoma. METHOD: The clinical data of 43 patients with salvage surgery for recurrent laryngocarcinoma hospitalized in Nanfang Hospital between 2003 and 2011 were analyzed retrospectively. Survival analysis was performed by using Kaplan-Meier method and prognosis factors were analyzed by Log-rank test. RESULT: After salvage surgery, 10 (23.3%) patients developed postoperative complications. Pharyngocutaneous fistula and infection occurred in 8 (18.6%) patients. Kaplan-Meier analysis showed that patients survived for 3-year (n = 32) and 5-year 25) after salvage surgery were 56.3% and 32.0% respectively. Univariate analysis indicated that clinical stage in the first diagnosis was significantly correlated with 3 year overall survival (P < 10.05). Local recurrence of laryngeal cavity group and preservation of laryngeal function group had a good prognosis respectively, but no sig nificant difference between them. CONCLUSION: Salvage surgery is a good choice for recurrent laryngeal carcinoma. Outcomes of these patients were correlated with clinical stages. Laryngeal function preserving surgery for local recurrent laryngeal carcinoma can achieve a expected curative effect and prognosis.

[Clinical application of heterogeneous acellular dermal matrix with autologous bone meal in open tympanoplasty].

OBJECTIVE: To evaluate the clinical value of heterogeneous acellular dermalmatrix with autologous bone meal in open tympanoplasty. METHOD: Twenty-eight cases (30 ears) with middle ear cholesteatoma were trea- ted by open tympanoplasty and repaired by heterogeneous acellular dermalmatrix autologous bone meal on study team. Twenty-two cases (22 ears) with middle ear cholesteatoma were treated by open tympanoplasty on control team. All patients were followed up for 12 to 18 months and assessed the fuction postoperatively. RESULT: The re- construction of external auditory canal structure is close to normal, and no narrow happens on study team. The rate of dry ear was about 90%. All cases had no recurrence of cholesteatoma. CONCLUSION: Application of decellu- larized dermal matrix with autologous bone meal can rise early to cover the wound, promote wound healing and to reduce the external auditory canal, reduce the effect of granulation and scar formation. It is a kind of method of repair to be promoted.

EZH2 promotes angiogenesis through inhibition of miR-1/Endothelin-1 axis in nasopharyngeal carcinoma.

Emerging evidence clearly indicates that EZH2 plays a crucial role in tumor angiogenesis. However, the role of EZH2 in angiogenesis is still unknown in nasopharyngeal carcinoma (NPC). We here showed that the elevated EZH2 level was closely associated with an aggressive and poor prognostic phenotype, and was positively correlated with microvessel density (MVD) in NPC tissues. Functional studies showed that EZH2 upregulation promoted cell proliferation, migration and tubule formation of endothelial cells, and knockdown of EZH2 suppressed tumor growth, metastasis and angiogenesis in vivo. Mechanistic investigations revealed that EZH2 inhibited miR-1 transcription via promoter binding activity, leading to enhanced expression of Endothelin-1 (ET-1) which is suppressed by miR-1 targeting of ET-1 3'UTR. Furthermore, knockdown of EZH2 or overexpression of miR-1 exerted anti-angiogenic effect on NPC cells. More importantly, the neutralizing antibody against ET-1 significantly abrogated the pro-angiogenic effect of EZH2, and forced expression of ET-1 rescued the anti-angiogenic effect induced by EZH2 knockdown. In clinical specimens, ET-1 was widely overexpressed and associated with clinical stage and MVD. Taken together, our results identify a novel signaling pathway involved in NPC angiogenesis, and also suggest that EZH2-miR-1-ET-1 axis represents multiple potential therapeutic targets for NPC.

[Screening and mass spectrometry analysis of differentially expressed proteins of plasm between laryngocarcinoma and healthy individuals].

OBJECTIVE: To compare and analyze the expression difference of proteins between laryngocarcinoma and healthy individuals to search for protein biomarkers that may be detected in plasm of laryngocarcinoma patients. METHOD: Pooled plasm from 6 laryngocarcinoma patients and 6 healthy individuals as controls were collected. Two-dimensional gel electrophoresis (2-DE) was used to isolate the total proteins, and the differential protein spots were identified by MALDI-TOF/TOF mass spectrometer, and then the biological information of the proteins was analyzed. RESULT: Twenty differential expressed protein spots with more than 1.5-fold between the laryngocarcinoma and healthy individuals were selected, and there were 8 proteins upregulated and 12 proteins downregulated among these proteins. After identifying by MALDI-TOF/TOF, compared with healthy controls, the spots that were L1, C-reactive protein, haptoglobin, ceruloplasmin; the spots that were downregulated were: Serotransferrin, alpha-2-HS-glycoprotein, fibrinogen gamma chain, haptoglobin related protein, Ig lambda-1 chain C regions, Ig kappa chain C region, apolipoprotein A-I, transthyretin, apolipoprotein C-III. CONCLUSION: Compared with laryngocarcinoma and healthy individuals, the plasm proteins profile showed differently. The proteins of differential expressed are expected to be the specific plasm biomarkers for laryngocarcinoma patients.

[Screening of plasma tumor biomarkers for hypopharyngeal carcinoma by proteomics method].

OBJECTIVE: To screen tumor biomarkers in the plasma close related with hypopharyngeal carcinoma. METHODS: Pooled plasma from 6 patients with hypopharyngeal carcinoma and 6 healthy individuals were collected. After removal of high-abundance plasma proteins, two-dimensional gel electrophoresis (2-DE) was performed to isolate the total proteins, and the protein spots with more than 2-fold differential expressions were detected by 2D analysis software followed by identification by MALDI-TOF/TOF mass spectrometer. Western blotting was performed to validate the expression level of α2-HS-glycoprotein. RESULTS: A total of 11 differentially expressed protein spots were selected, including 5 upregulated proteins and 6 downregulated proteins. MALDI-TOF/TOF identified the upregulated proteins in hypopharyngeal carcinoma patients as alpha-2-HS-glycoprotein and haptoglobin and downregulated ones as Ig kappa chain C region and apolipoprotein A-I. Western blotting demonstrated that α-2-HS- glycoprotein expression level was consistent with that detected by 2-DE. CONCLUSION: Patients with hypopharyngeal carcinoma show different plasma protein profiles from healthy individuals. These differentially expressed proteins may serve as potential specific tumor biomarkers for hypopharyngeal carcinoma.

miR-26a inhibits invasion and metastasis of nasopharyngeal cancer by targeting EZH2.

Nasopharyngeal carcinoma (NPC) is a highly invasive and metastatic type of cancer that is widely prevalent in Southern China. Studies have shown that several microRNAs (miRNAs) are implicated in NPC metastasis. Our previous studies have demonstrated that miRNA miR-26a inhibits cell growth and tumorigenesis of NPC through the repression of enhancer of zeste homolog 2 (EZH2). However, the role of miR-26a in NPC metastasis remains unknown. In this study, we showed that ectopic expression of miR-26a inhibited the migratory and invasive capacities of NPC cells in vitro. Additionally, we used a murine model to investigate the role of miR-26a in NPC metastasis and results showed that miR-26a overexpression suppresses the metastatic behavior of NPC cells in vivo. Furthermore, the data demonstrated that miR-26a decreased the expression levels of EZH2 in vitro and in vivo, suggesting that the antimetastatic effect of miR-26a in NPC was mediated by regulating EZH2. Therefore, these findings indicate that miR-26a functions as an antimetastatic miRNA in NPC and that its antimetastatic effects are mediated mainly by repressing EZH2 expression.

Diagnostic and prognostic value of plasma microRNA deregulation in nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is uncommon worldwide but often highly invasive in late stages. Due to its special location and lack of specific symptoms, NPC is hardly detected in regular medical examination at the beginning. Development of sensitive and specific biomarkers should help to save lives against this type of disease. In the present report, we investigated the value of plasma miRNAs for diagnosis and prognosis of NPC. Using candidate approach, we selected 21 miRNAs from literature to compare their expression levels in the plasma of NPC patients and controls. As a result, 5 miRNAs showed diagnostic potentials (P<0.01). Among them, miR-16, -21, -24, and -155 had increased levels in NPC patients, whereas the level of miR-378 was decreased. There was a negative correlation between plasma miRNA expression and cancer progression, where miR-21 was statistically significant in T and N staging and miR-16 and 24 were significant in N staging only. Combination of miR-16, -21, -24, -155, and -378 gives 87.7% of sensitivity and 82.0% of specificity for NPC diagnosis. Without miR-16, combination of the rest 4 miRNAs gives the same sensitivity but a slightly reduced specificity. After treatment, all 5 miRNAs were somewhat back to normal levels in patients without cancer recurrence but the prognostic value was not statistically significant. In conclusion, plasma miRNA expression is a useful biomarker for NPC diagnosis but not for its prognosis. More importantly, it is simple, effective, and non-invasive. Combination of several plasma miRNAs can increase both NPC diagnostic sensitivity and specificity.

[Effects of enhancer of zeste homolog (EZH2) downregulation on the proliferation and invasion of nasopharyngeal carcinoma cell and the possible mechanisms].

OBJECTIVE: To investigate the effects of the enhancer of zeste homolog 2 (EZH2) gene on cell growth and invasion of the nasopharyngeal carcinoma (NPC). METHODS: Recombinant lentivirus vector for shRNA delivery of EZH2 was constructed and transfected into 293FT cells. After collecting the viral particles, the NPC cell line 5-8F cells were transfected. The effects of EZH2 silence on cell proliferation and cell cycle were detected using MTT assay, plate colony formation assay and flow cytometry. The migration and invasion of 5-8F cells were determined by wound healing assay and matrigel invasion assay, respectively. The expressions of EZH2 and epithelial-mesenchymal transition (EMT)-related markers at mRNA and protein levels were examined by real-time PCR and Western blot respectively. RESULTS: The expressions of EZH2 mRNA and protein in the transfected 5-8F cells were obviously reduced. MTT assay showed that EZH2 downregulation significantly inhibited the growth of 5-8F/shEZH2 cells (P < 0.001). Colony formation rate (84.44%) of 5-8F/shEZH2 cells was lower than control (31.56%, P = 0.001). Cell cycle analysis showed that most 5-8F/shEZH2 cells were arrested in G0/G1 phase, with a very low ratio of cells in S phase. Wound healing assay indicated that the migration ability of cells silencing EZH2 decreased significantly, and the 48-hour relative migration distance of 5-8F/ShEZH2 cells and control cells was 0.58 ± 0.05, and 0.81 ± 0.02, respectively (P < 0.000). Matrigel invasion assay, showed the invasive capacity of cells silencing EZH2 was significantly inhibited, with less penetrating cells (72.23 ± 4.08) compared to control (150.95 ± 16.27), P < 0.000. The mRNA expressions of epithelial markers E-cadherin and Keratin 18 in the cells silencing EZH2 increased by 177% and 158% respectively, and the mRNA expressions of mesenchymal markers ß-catenin and N-cadherin decreased by 18.04% and 41.18% respectively. Similar results also were obtained with Western blot analysis. CONCLUSION: EZH2 significantly enhanced the proliferation and invasion of nasopharyngeal carcinoma cells in vitro, which might be mediated by inducing EMT.

[Enhancement of target gene expression by recombinant adeno-associated virus combined with recombinant adenovirus in vivo].

OBJECTIVE: To study the changes in the intensity and temporal pattern of target gene expression in the tumor tissue of nude mice bearing human nasopharyngeal carcinoma (NPC) following injection of recombinant adeno-associated virus (rAAV) and recombinant adenovirus (AdV) in vivo. METHODS: EBV-positive human NPC cell line C666-1 was inoculated subcutaneouly in nude mice. After the tumor mass reached 3 mm in diameter, 1.5 × 10(11) v.g (virus genome) rAAV-EGFP, 2.5 × 10(8) pfu rAdV-EGFP or their balanced mixture was injected intratumorally. At 5 and 10 days after the injection, the tumor tissues were harvested for immunohistochemical staining of GFP, and the ratio of the GFP-positive cells and the intensity of GFP expression was determined. RESULTS: Immunohistochemistry for GFP showed that 5 days after the injection, GFP expression was detected (1.70 ∓ 0.48) in the tumor tissue in rAAV group, and the peak expression levels was seen in rAdV group (6.00∓1.94); the expression level was comparable between the combination group (6.90 ∓ 1.92) and rAdV group. At 10 days, GFP expression was considerably lowered to 2.00 ∓ 0.67 in rAdV group but increased to 8.00∓1.15 in rAAV group. The expression in the combination group maintained a high level at 10 days (10.10∓1.63), which was significantly higher than that in rAAV group (P%0.001). CONCLUSION: Transfection with rAAV combined with rAdV allows instant, sustained and significantly enhanced expression of the target gene in the tumor tissue. This approach takes advantages of the two viruses and can be ideal for exogenous gene delivery into the tumor tissues.

[Imageology features and transoral approach of benign parapharyngeal space tumors].

OBJECTIVE: To analyze the imageology features of benign parapharyngeal space (PPS) tumors, and also to summarize our experience in removing PPS benign tumors through transoral approach. METHOD: A retrospective review was conducted to 48 patients with benign tumors in PPS during a 10-year period. CT were performed in all patients, and only a few required MRI. Transoral approach (33.3%) and transcervical (39.6%) were the most commonly performed surgical procedures followed by the transcervical-transparotid approach (27.1%). RESULT: CT scan and MRI often provided complementary information to help the surgeons delineate the size, precise location and likely cause of these tumors. After a follow-up of three years, only 2 of 48 patients had disease recurrence. The transoral approach described herein safely allowed for en bloc resection of most benign neoplasms. No significant complications attributed to the approach itself. CONCLUSION: CT or MRI scan can distinguish prestyloid from poststyloid lesions, and to assess the extension of the tumor as well as its relationship with adjacent structures. The transoral approach safely provides access to some benign PPS tumors with a low rate of complications and recurrence as well as traditional transcervical approaches.