[Silencing of endothelin-1 suppresses growth, migration, and invasion of nasopharyngeal carcinoma cells in vitro].

OBJECTIVE: To explore the role of endothelin-1 (ET-1) gene in regulating the proliferation, migration and invasion of nasopharyngeal carcinoma cells. METHODS: A lentivirus-mediated shRNA-ET-1 vector was infected into 5-8F cells, and the interference efficiency was examined with Western blotting. MTT assay, cell cycle analysis, plate colony formation assay, Transwell assay, Boyden chamber assay and tumor growth assay were carried out to analyze the changes in cell proliferation, migration and invasion. The expressions of genes related with epithelial-mesenchymal transition (EMT) were examined using Western blotting. RESULTS: shRNA-ET-1 transfection significantly inhibited the expression of ET-1, and suppressed the growth, migration and invasion of 5-8F cells. ET-1 knockdown enhanced the expression of E-cadherin and CK18 and inhibited the expression of N-cadherin and vimentin. CONCLUSION: ET-1 promotes cell growth, migration and invasion by modulating the genes associated with epithelial-mesenchymal transition in nasopharyngeal carcinoma cells.

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.

MiR-124 suppresses tumor growth and metastasis by targeting Foxq1 in nasopharyngeal carcinoma.

BACKGROUND: The molecular mechanisms underlying dysregulation of microRNAs have been documented in nasopharyngeal carcinoma (NPC). Our previous study demonstrated that plasma miR-124 was down-regulated in NPC using microarray analysis and quantitative PCR validation. Though growing studies showed that down-regulated miR-124 was closely related to tumourigenesis in various types of cancers, the role of miR-124 in NPC remains largely unknown. METHODS: The expression level of miR-124 was evaluated in NPC cell lines and patient specimens using quantitative reverse transcription-PCR (Real-time qPCR). The clinicopathological significance of the resultant data was later analyzed. Then, we explored the role of miR-124 in NPC tumorigenesis by in vitro and in vivo experiments. Homo sapiens forkhead box Q1 (Foxq1) was confirmed as a novel direct target gene of miR-124 by the dual-luciferase assay and western bolt. RESULTS: We found that miR-124 was commonly down-regulated in NPC specimens and NPC cell lines. The expression of miR-124 was inversely correlation with clinical stages and marked on T stages. Then, the ectopic expression of miR-124 dramatically inhibited cell proliferation, colony formation, migration and invasion in vitro, as well as tumor growth and metastasis in vivo. Furthermore, we identified Foxq1 as a novel direct target of miR-124. Functional studies showed that knockdown of Foxq1 inhibited cell growth, migration and invasion, whereas Foxq1 overexpression partially rescued the suppressive effect of miR-124 in NPC. In clinical specimens, Foxq1 was commonly up-regulated in NPC, and the level increased with clinical stages and T stages. Additionally, the level of Foxq1 was inversely correlated with miR-124. CONCLUSIONS: Our results demonstrate that miR-124 functions as a tumor-suppressive microRNA in NPC, and that its suppressive effects are mediated chiefly by repressing Foxq1 expression. MiR-124 could serve as an independent biomarker to identify patients with different clinical characteristics. Therefore, our findings provide valuable clues toward the understanding the of mechanisms of NPC pathogenesis and provide an opportunity to develop new effective clinical therapies in the future.

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.

Stunting and soil-transmitted-helminth infections among school-age pupils in rural areas of southern China.

BACKGROUND: Stunting and soil-transmitted helminth (STH) infections including ascariasis, trichuriasis and hookworm remain major public health problems in school-age pupils in developing countries. The objectives of this study were to determine the prevalence of stunting for children and its association with three major soil-transmitted helminths (STH) in rural areas of southern China. The study also aims to determine risk factors for stunting and to provide guidance on the prevention and control of stunting and STH infections for future studies in this field. RESULTS: A cross-sectional survey was carried out in the poor rural areas in Guangxi Autonomous Regional and Hainan Province where STH prevalence was higher between September and November 2009. Pupils were from 15 primary schools. All the school-age pupils aged between 9 and 12 years old (mean age 11.2 ± 3.2 years), from grades three to six took part in this study. Study contents include questionnaire surveys, physical examination and laboratory methods (stool checking for eggs of three major STH infections and haemoglobin determination was performed for the anaemia test). Finally 1031 school-age pupils took part in survey. The results showed that the overall prevalence of stunting (HAZ < 2SD) was 25.6%, based on the WHO Child Growth Standards (2007). Risk factors for stunting based on logistic regression analyses were: (1) STH moderate-to-heavy intensity infections (OR = 1.93;95%CI:1.19,3.11); (2) anaemia (OR = 3.26;95%CI: 2.02,5.27); (3) education level of mother (OR = 2.13; 95%CI: 1.39,3.25). The overall prevalence of major STH infections was 36.7%, STH moderate-to-heavy intensity infections was 16.7%. The overall prevalence of ascariasis, trichuriasis, hookworm and co-infection were 18.5%, 11.2%, 14.7% and 9.1% respectively. The prevalence of anaemic children (HB < 12 g/dl) was 13.1%. CONCLUSION: The present study showed that stunting was highly prevalent among the study population and STH infection is one of the important risk factors for stunting, with moderate-to-heavy intensity infections being the main predictor of stunting. Hence, additional interventions measures such as to promote de-worming treatment, to enhance health education and to improve hygiene and sanitation in order to reduce stunting in this population, are needed throughout the primary school age group.

[Epidemiology of soil-transmitted nematode infections in central mountain area of Hainan province].

The epidemiology of soil-transmitted intestinal nematodes were observed in the central mountain area without anti-helminthic therapy from 1986 to 2008. The results showed that the overall prevalence decreased from 96.4% in 1986 to 35.7% in 2008. The prevalence of Ancylostoma duodenale, Ascaris lumbricoides and Trichuris trichiura decreased from 84.7%. 80.9%, 31.8% in 1986 to 32.5%, 0.3%, 4.2% in 2008, respectively. The proportion of light infection with Ancylostoma duodenale, Ascaris lumbricoides and Trichuris trichiura increased from 56.6%, 41.2%, and 66.9% in 1986 to 97.9%, 100%, and 83.7% in 2008, respectively. While that of heavy infection decreased from 6.8%, 11.9%, and 3.8% in 1986 all to zero in 2008. Water and toilet renovation, rural income increase and the improvement of sanitation and living conditions made the prevalence of soil-transmitted intestinal nematode decreased.