The Autism-Related lncRNA MSNP1AS Regulates Moesin Protein to Influence the RhoA, Rac1, and PI3K/Akt Pathways and Regulate the Structure and Survival of Neurons.

Autism spectrum disorder (ASD) is a complex disease involving multiple genes and multiple sites, and it is closely related to environmental factors. It has been gradually revealed that long noncoding RNAs (lncRNAs) may regulate the pathogenesis of ASD at the epigenetic level. In neuronal cells, the lncRNA moesin pseudogene 1 antisense (MSNP1AS) forms a double-stranded RNA with moesin (MSN) to suppress moesin protein expression. MSNP1AS overexpression can activate the RhoA pathway and inhibit the Rac1 and PI3K/Akt pathways; however, the regulation of Rac1 by MSNP1AS is not associated with MSN, and the effect on the RhoA pathway may also be associated with other factors. MSNP1AS can decrease the number and length of neurites, inhibit neuronal cell viability and migration, and promote apoptosis. Downregulation of MSN expression functions similarly to MSNP1AS, and its overexpression can block the above functions of MSNP1AS. In addition, in vivo experiments show that MSN improves social interactions and reduces repetitive behaviors in BTBR mice, decreases the activity of RhoA and restores the activity of PI3K/Akt pathway. Therefore, the abnormal expression of MSNP1AS in ASD patients might influence the structure and survival of neuronal cells through the regulation of moesin protein expression to facilitate the development and progression of ASD. These findings provide new evidence for studying the mechanisms of lncRNAs in ASD. LAY SUMMARY: Autism spectrum disorder (ASD) is a common neurodevelopmental disease and its neurodevelopmental mechanisms have not been elucidated. More and more studies have found that long noncoding RNAs (lncRNAs) can regulate the development of central nervous system in many ways and affect the pathogenic process of ASD. Moesin pseudogene 1 antisense (MSNP1AS) is an up-regulated lncRNA in ASD patients. In-depth functional experiments showed that MSNP1AS inhibited moesin protein expression and regulated the activation of multiple signaling pathways, thus decreasing the number and length of neurites, inhibiting neuronal cell viability and migration, and promoting apoptosis. Therefore, MSNP1AS is an important lncRNA related to ASD and can regulate the biological function of neurons.

Downregulation of iASPP Expression Suppresses Proliferation, Invasion and Increases Chemosensitivity to Paclitaxel of Head and Neck Squamous Cell Carcinoma In Vitro.

Objective Our previous study has revealed that iASPP is elevated in human head and neck squamous cell carcinoma (HNSCC) and iASPP overexpression signifcantly correlates with tumor malignant progression and poor survival of HNSCC. This study investigated the function of iASPP playing in proliferation and invasion of HNSCC in vitro. Methods HNSCC cell line Tu686 transfected with Lentiviral vector-mediated iASPP-specific shRNA and control shRNA were named the shRNA-iASPP group and shRNA-NC group, respectively. The non-infected Tu686 cells were named the CON group. CCK-8 assay, flow cytometry, transwell invasion assay were performed to detect the effects of iASPP inhibition in vitro. Results Our results demonstrated that the proliferation of shRNA-iASPP cells at the time of 72 h (F=32.459, P=0.000), 96 h (F=51.407, P=0.000), 120 h (F=35.125, P=0.000) post-transfection, was significantly lower than that of shRNA-NC cells and CON cells. The apoptosis ratio of shRNA-iASPP cells was 9.42% ± 0.39% (F=299.490, P=0.000), which was significantly higher than that of CON cells (2.80% ± 0.42%) and shRNA-NC cells (3.18% ± 0.28%). The percentage of shRNA-iASPP cells in G0/G1 phase was 74.65% ± 1.09% (F=388.901, P=0.000), which was strikingly increased, compared with that of CON cells (55.19% ± 1.02%) and shRNA-NC cells (54.62% ± 0.88%). The number of invading cells was 56 ± 4 in the shRNA-iASPP group (F=84.965, P=0.000), which decreased significantly, compared with the CON group (111 ± 3) and the shRNA-NC group (105 ± 8). The survival rate of shRNA-iASPP cells administrated with paclitaxel was highly decreased, compared with CON cells and shRNA-NC cells (F=634.841, P=0.000). Conclusion These results suggest iASPP may play an important role in progression and aggressive behavior of HNSCC and may be an efficient chemotherapeutic target for the treatment of HNSCC.

Knockdown of Annexin A1 Enhances Radioresistance and Inhibits Apoptosis in Nasopharyngeal Carcinoma.

Radiotherapy is the primary treatment for nasopharyngeal carcinoma while radioresistance can hinder efficient treatment. To explore the role of annexin A1 and its potential mechanisms in radioresistance of nasopharyngeal carcinoma, human nasopharyngeal carcinoma cell line CNE2-sh annexin A1 (knockdown of annexin A1) and the control cell line CNE2-pLKO.1 were constituted and CNE2-sh annexin A1 xenograft mouse model was generated. The effect of annexin A1 knockdown on the growth of xenograft tumor after irradiation and radiation-induced DNA damage and repair was analyzed. The results of immunohistochemistry assays and Western blotting showed that the level of annexin A1 was significantly downregulated in the radioresistant nasopharyngeal carcinoma tissues or cell line compared to the radiosensitive nasopharyngeal carcinoma tissues or cell line. Knockdown of annexin A1 significantly promoted CNE2-sh annexin A1 xenograft tumor growth compared to the control groups after irradiation. Moreover, the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assays revealed that knockdown of annexin A1 significantly inhibited apoptosis in vivo compared to the control groups. We assessed the intracellular reactive oxygen species levels and the extent of radiation-induced DNA damage and repair using reactive oxygen species assay, comet assays, and immunohistochemistry assay. The results showed that knockdown of annexin A1 remarkedly reduced the intracellular reactive oxygen species levels, level of DNA double-strand breaks, and the phosphorylation level of H2AX and increased the accumulation of DNA-dependent protein kinase in nasopharyngeal carcinoma cells after irradiation. The findings suggest that knockdown of annexin A1 inhibits DNA damage via decreasing the generation of intracellular reactive oxygen species and the formation of γ-H2AX and promotes DNA repair via increasing DNA-dependent protein kinase activity and therefore improves the radioresistance in nasopharyngeal carcinoma cells. Together, our findings suggest that knockdown of annexin A1 promotes radioresistance in nasopharyngeal carcinoma and provides insights into therapeutic targets for nasopharyngeal carcinoma radiotherapy.

Prevalence survey of nosocomial infections in the Inner Mongolia Autonomous Region of China [2012-2014].

BACKGROUND: To investigate the prevalence of nosocomial infections, the distribution of nosocomial infection sites, the use of antibiotic and the situation of detected nosocomial infection pathogens in the Inner Mongolia Autonomous Region of China from 2012 to 2014, to grasp the current conditions of regional nosocomial infections in timely, for the development of infection prevention and control measures to provide a basis for effective hospital. METHODS: A survey of the prevalence of nosocomial infections was conducted in target hospitals using the combination of a bedside survey and medical record review. RESULTS: In total, 101,907 inpatients were surveyed from 2012 to 2014. There were 1,997 cases of nosocomial infections, accounting for an average prevalence of 1.96%. The infection site was mainly the lower respiratory tract. Higher prevalence of nosocomial infections occurred in the comprehensive intensive care unit (ICU), Neurosurgery Department, and Hematology Department. The average rate of antibiotic use was 33.72%, and the average submission rate for bacterial cultures for patients who received therapeutic treatment with antibiotics was 28.26%. The most common pathogens associated with nosocomial infections were Gram-negative (G(-)) bacteria, and frequently detected bacterial pathogens included Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii, and Staphylococcus aureus. CONCLUSIONS: The survey of the prevalence of nosocomial infections helped to identify problems in the control process of nosocomial infections and to develop targeted measures for the prevention and control of these infections accordingly.

Metadherin regulation of vascular endothelial growth factor expression is dependent upon the PI3K/Akt pathway in squamous cell carcinoma of the head and neck.

Our previous study indicated overexpression of metadherin (MTDH) is an adverse prognostic factor in squamous cell carcinoma of the head and neck (SCCHN) and promotes SCCHN cell proliferation and invasion. However, its mechanism remains unclear. Recent studies have indicated that MTDH is a cancer-metastasis-associated molecule that participates in the process of angiogenesis. Therefore, the study is aimed to investigate that whether vascular endothelial growth factor (VEGF), as one of the most potent proangiogenic cytokines, is regulated by MTDH and the role of the phosphatidylinositide 3-kinases/Protein Kinase B (PI3K/Akt) pathway in this process of regulation and the clinical significance of both MTDH and VEGF in SCCHN.Immunohistochemistry was used to assay the expression of MTDH and VEGF in a cohort of 189 SCCHN patients with intact follow-up information. The expression of MTDH was then upregulated or inhibited by lentivirus-mediated MTDH Complementary deoxyribonucleic acid or MTDH short hairpin ribonucleic acid (shRNA) to observe the resulting alterations in VEGF expression and the PI3K/Akt signaling pathway in SCCHN cell lines. In addition, the PI3K/Akt pathway was modulated to observe the resulting changes in the MTDH-mediated expression of VEGF.The immunohistochemistry data showed that MTDH expression is positively correlated with VEGF expression in SCCHN tissues. Moreover, the overexpression of MTDH in SCCHN Tu686 and 5-8F cells led to increases in the expression of VEGF, and this effect was accompanied by activation of the PI3K/Akt pathway. Conversely, shRNA-mediated knockdown of MTDH led to decreased VEGF expression. In addition, inhibition of the Akt signaling pathway reversed the upregulation of VEGF resulting from MTDH overexpression. Moreover, the survival analysis revealed that VEGF is an independent prognostic factor, and a combined survival analysis based on both MTDH and VEGF showed synergistic effects in the prognosis evaluation of SCCHN patients.The findings of the present study demonstrate that MTDH regulates the expression of VEGF via the PI3K/Akt signaling pathway, indicating the potential role of the MTDH-mediated activation of VEGF signaling pathway in SCCHN angiogenesis and metastasis.

Meta-analysis of the aldehyde dehydrogenases-2 (ALDH2) Glu487Lys polymorphism and colorectal cancer risk.

A number of studies have explored the association of the aldehyde dehydrogenases-2 (ALDH2) Glu487Lys polymorphism and risk of colorectal cancer; however, the results are inconsistent. We performed this meta-analysis to clarify this issue using all the available evidence. Relevant studies were retrieved by searching PubMed. Eleven case-control studies were included in the meta-analysis, representing 2909 cases and 4903 controls. The pooled results based on all included studies showed a decreased colorectal cancer risk in the analysis of the GA genotype vs. the GG genotype (OR = 0.81, 95%CI = 0.68-0.98, p = 0.03) and in the dominant genetic model analysis (OR = 0.81, 95%CI = 0.67-0.98, p = 0.03). However, there was no statistical difference in the AA vs. GG analysis (OR = 0.74, 95%CI = 0.52-1.06,p = 0.11) and the recessive genetic model analysis (OR = 0.86, 95%CI = 0.69-1.07, p = 0.17). Cumulative meta-analysis based on publication time confirmed these findings. Patients with colorectal cancer had a higher frequency of the GG genotype (OR = 1.10, 95% CI = 1.02-1.20, p = 0.02) and a lower frequency of the GA genotype (OR = 0.89, 95%CI = 0.81-0.98, p = 0.02) comparing with control population. Our results suggested that the ALDH2 Glu487Lys polymorphism may be associated with a decreased risk of colorectal cancer.

[Irradiation induced epithelial-mesenchymal transition in nasopharyngeal carcinoma in vitro].

OBJECTIVE: To investigate the irradiation induced epithelial-mesenchymal transition (EMT) in nasopharyngeal carcinoma (NPC) in vitro. METHODS: NPC CNE-2 cells with radioresistance (CNE-2-Rs) were established by exposure to gradiently increased dose of irradiation. CCK-8 cell viability kits, colony formation assay and fluorescence-activated cell sorting analysis were used to confirm the capacity of radioresistance of CNE-2-Rs cells. Invert microscope was used to monitor the morphological changes and western blot was applied to detect the expression of epithelial cell marker E-cadherin and mesenchymal cell marker Vimentin during the phase of CNE-2 exposure to irradiation. RESULTS: Irradiation exposure successfully induced the radioresistance of CNE-2 cells. After exposed to irradiation, the survival rate in CNE-2-Rs was higher than that in CNE-2 by CCK-8 assays. No significant difference of proliferation ability was observed between the CNE-2 and CNE-2-Rs pre-radiotherapy, but a higher proliferation ability in the CNE-2-Rs post-radiotherapy. By using the colony forming assay, the parameters of CNE-2 and CNE-2-Rs in multi-target single-hit and linear quadratic model were obtained and the data demonstrated that parameters mean lethal dose (D0) , quasi-thres hold dose (Dq) , surrival fraction in 2Qy (SF2) and mean inctivation dose (MID) value increased, α and α/ß value decreased (P < 0.05) . At the same time, the CNE-2-Rs cells showed higher percentage of cells in S and G2 phase (P < 0.05) . In terms of biomorphology, CNE-2-Rs cells were more narrow, long strips or fusiform shapes, stretched out tentacles, and the contacts between them were loosened. When radiation dose accumulated to 24 Gy, an over-expression of Vimentin was observed in treated cells, while E-cadherin was down-regulated (P < 0.01) . CONCLUSIONS: NPC cells present with typical morphorlogical and biomolecular changes of EMT during exposure to irradiation, indicating the potential critical roles of EMT in the malignant behavior of radioresistance in NPC.

[EphA2 mediated vascular endothelial growth factor expression via the p38 MAPK signaling pathway in squamous cell carcinoma of the head and neck].

OBJECTIVE: To investigate the regulatory effect of erythropoietin-producing hepatocellular receptor (EphA2) on the expression of VEGF protein, a pro-angiogenic factor, via p38 mitogen-activated protein kinase (p38 MAPK) signaling pathway in squamous cell carcinoma of the head and neck(SCCHN) in vitro. METHODS: SCCHN Tu686 cells were transfected with EphA2 overexpression vector pEGFP-N1-EphA2. Western blot was used to detect the expression of p38 MAPK and enzyme-linked immunosorbent assay (ELISA) was applied to assay of VEGF. SB203580 as a inhibitor of p38 MAPK signaling pathway was used. RESULTS: The expression of VEGF protein was significantly up-regulated in Tu686 cells transfected with EphA2 overexpression vector (535.31 ± 45.71) pg/ml, when compared with Tu686 cells transfected with empty vector (400.99 ± 33.50) pg/ml and Tu686 cells with no transfection (385.30 ± 33.50) pg/ml (F = 17.091, P < 0.01). The expression of phosphorylated p38 MAPK was obviously increased in Tu686 cells with EphA2 overexpression. SB203580 inhibited the expressions of VEGF and phosphorylated p38 MAPK proteins in Tu686 cells with EphA2 overexpression. CONCLUSION: EphA2 can regulate the expression of VEGF protein and stimulate p38 MAPK signaling pathway.

[Expression of astrocyte elevated gene-1 protein and its clinical significance in laryngeal squamous cell carcinoma].

OBJECTIVE: To assess the protein expression of astrocyte elevated gene-1 (AEG-1) in tissue specimens of laryngeal squamous cell carcinoma (LSCC), and to correlate its expression with clinicopathological parameters and prognosis in patients with LSCC. METHODS: RT-PCR was used to assay the expression of AEG-1 mRNA in 13 pairs of LSCC tissues and their corresponding noncarcinoma epithelia. Immunohistochemistry was performed on paraffin-embedded tissue specimens to investigate the protein expression of AEG-1 in 88 cases of LSCC specimens and 15 cases of adjacent epithelial samples. RESULTS: The expression of AEG-1 mRNA was significantly increased in LSCC tissues compared to adjacent noncarcinoma epithelial tissues (0.81 ± 0.17 vs. 0.23 ± 0.10;t = 10.337, P < 0.001). Meantime, the positive rate of AEG-1 protein in 88 cases of LSCC was 87.5% (77/88). However, 15 cases of adjacent noncarcinoma epithelial merely demonstrated negative or mild expression of AEG-1 protein. AEG-1 overexpression was closely correlated with T stage (χ(2) = 6.289, P = 0.018), clinical stage (χ(2) = 11.049, P < 0.01), metastasis (χ(2) = 20.859, P < 0.01) and recurrence(χ(2) = 13.459, P < 0.01). The overall survival rates of patients with AEG-1 overexpression and low expression were 35.9% and 86.4%, respectively (χ(2) = 23.409, P < 0.01). Multivariate Cox regression analysis revealed that AEG-1 expression was an independent prognostic factor (P = 0.016). CONCLUSION: AEG-1 protein may play a critical role in the initiation and progression of LSCC, implicating its predictive value in prognosis.

[EGCG regulates TGF-ß1-induced epithelial mesenchymal transition in squamous cell carcinoma of head and neck].

OBJECTIVE: To study the effect and molecular mechanism of epigallocatechin-3-gallate (EGCG) on epithelial-mesenchymal transition (EMT) in vitro induced by human recombinant TGF-ß1 protein in squamous cell carcinoma of the head and neck. METHODS: EMT morphological changes of Tu686 cells were observed after sequential treatment of 5 ng/ml TGF-ß1 and 20 µmol/L EGCG. Tu686 cells were collected after the treatment of 5 ng/ml TGF-ß1 for 24 h and EGCG with different concentrations (0, 10, 20, 30 µmol/L) for another 24 h or 20 µmol/L EGCG treatment for different time phase (6, 12, 24 h). Then RT-PCR and Western-blot were applied to detect mRNA and protein expression level of epithelial cell marker E-cadherin, mesenchymal cell marker Vimentin and Smad7, an inhibit molecule of TGF-ß1 mediated pathway in Tu686 cells. RESULTS: TGF-ß1 successfully induced characterized EMT morphological and molecular changes in Tu686 cells, in which expression of E-cadherin decreased, Vimentin increased and Smad7 declined. However, EGCG could reverse the TGF-ß1 mediated process of EMT by downregulating the expression of Vimentin and upregulating the expression of E-cadherin and Smad7. CONCLUSION: EGCG significantly inhibits TGF-ß1-mediated EMT inTu686 cell lines of SCCHN, which maybe associated with the upregulated-expression of Smad7, an inhibitor in TGF-ß1 signaling pathway.

[Expressions and clinical significance of high mobility group box-1 mRNA and protein in laryngeal squamous cell carcinoma tissues and serum].

OBJECTIVE: To investigate the mRNA and protein expressions of high mobility group box-1 (HMGB1) in the tumor tissues and sera of patients with laryngeal squamous cell carcinoma (LSCC) and their clinical significance. METHODS: Semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot were used to detected the expressions of HMGB1 mRNA and protein in the tumors and adjacent normal epithelial tissues in 30 patients with LSCC. Serum HMGB1 protein levels in the patients with LSCC and in 10 healthy volunteers were detected with enzyme-linked immunosorbent adsorption experiment (ELISA). RESULTS: RT-PCR demonstrated that the mean relative mRNA expression levels of HMGB1 (HMGB1/GAPDH) in LSCC tissues and in adjacent normal epithelial tissues were 1.25 ± 0.12 and 0.32 ± 0.04, respectively (t = 40.27, P < 0.05). Western blot revealed that the mean relative protein expression levels of HMGB1 (HMGB1/ß-actin) were 1.29 ± 0.10 and 0.34 ± 0.03 (t = 49.84, P < 0.05), respectively. Both mRNA and protein expression levels of HMGB1 were associated with T stage, clinical stage, lymph node metastasis status and smoking (all P < 0.05), but no significant correlation with age, alcohol consumption and primary tumor grade and location (all P > 0.05). Mean serum HMGB1 protein levels in patients with LSCC and healthy volunteers were (24.80 ± 14.08) ng/ml and (23.58 ± 14.69) ng/ml (t = 0.37, P > 0.05). CONCLUSIONS: Both mRNA and protein expressions of HMGB1 were obviously elevated in LSCC, which were associated closely with T stage, clinical stage and lymph node metastasis.

[Expression of HMGB1 protein in laryngeal squamous cell carcinoma and its clinical significance].

OBJECTIVE: To evaluate the expression of HMGB1 protein in tissue specimens of laryngeal squamous cell carcinoma (LSCC) and adjacent normal mucosa, and explore the correlation of HMGB1 protein expression with clinicopathologic features and prognosis in LSCC. METHODS: Ninty-three cases of LSCC and 5 cases of adjcent mucosal tissue samples were included in this study. Immunohistochemical staining was performed on paraffin-embedded tissue specimens to examine the HMGB1 protein expression. The data were futher correlated with the clinicopathological features and prognosis of the LSCC patients. RESULTS: The positive rates of HMGB1 expression in LSCC specimens was 87.1%, significantly higher than that in the adjcent normal mucosa samples (46.7%, P = 0.001), and its overexpresion was closely correlated with T stage (Chi2 = 10.878, P = 0.004), clinical stage (Chi2 = 21.115, P < 0.01), metastasis (Chi2 = 28.298, P < 0.01) and recurrence (Chi2 = 14. 923, P = 0.001) in patients with LSCC. Patients with HMGB1 overexpression had both poorer disease-free survival and poorer overall survival compared with that in patients with low HMGB1 expression (Chi2 = 13.815, Chi2 = 11.912; Both P < 0.01). Univariate and multivariate Cox regression analyses revealed that HMGBI expression is an independent prognostic factor for patients with LSCC. CONCLUSIONS: The results of this study demonstrate that HMGB1 protein expression is significantly increased in LSCC tissues, and HMGB1 protein overexpression is associated with a poorer prognosis in patients with LSCC. These results suggest that HMGB1 may play a critical role in the initiation and progression of LSCC, implicating HMGB1 may become a valuable marker for the prediction of prognosis in patients with LSCC.

[EphA2 promotes angiogenesis and metastasis of head and neck squamous cell carcinoma in vivo].

OBJECTIVE: To investigate the effects of EphA2 on the angiogenesis and cervical lymph node metastasis of squamous cell carcinoma of the head and neck (SCCHN) in vivo. METHODS: EphA2 short hairpin (shRNA) lentiviral particles were used to knockdown the expression of EphA2 in SCCHN cell line M2 with high lymph nodes metastasis rate. Stable clones, obtained by puromycin screening, were assayed by RT-PCR and Western blot to validate the gene silencing efficiency and were used to establish SCCHN metastatic xenograft mouse model. Hematoxylin-eosin staining was applied to identify cervical lymph node metastasis of SCCHN in xenografted tumors. Immunohistochemistry was used to observe microvessel density. Western blot was used to investigate the protein expressions of EphA2 and vascular endothelial, growth factor (VEGF). RESULTS: EphA2 shRNA lentiviral particles efficiently decreased the mRNA and protein expressions of EphA2 in SCCHN cell line M2, which were further successfully utilized to establish SCCHN metastatic xenograft mouse model. Compared with xenografted tumors in control group, xenografted tumors in M2EphA2RNAi(+) group decreased significantly tumor volume [(430.7 ± 190.0) mm(3) (x(-) ± s) vs (1179.0 ± 289.4) mm(3)] and weight [(0.26 ± 0.10) g vs (0.54 ± 0.12) g] (both P < 0.05). More importantly, bilateral cervical lymph node metastasis rate in M2EphA2RNAi(+) was also greatly declined (Mann-Whitney U = 10.0, P < 0.05). Decreased protein expressions of EphA2 and VEGF and microvessel density were observed in M2EphA2RNAi(+) group (t = 26.751, P < 0.01). CONCLUSIONS: Knockdown of EphA2 expression led to the inhibition of tumor growth and metastasis in SCCHN nude mouse model. More importantly, SCCHN angiogenesis was also impeded, which might be associated with the decreased expression of VEGF.

Analysis of transcriptional factors and regulation networks in laryngeal squamous cell carcinoma patients with lymph node metastasis.

The present study was to identify and quantitate differentially expressed proteins in laryngeal squamous cell carcinoma (LSCC) tissues with or without lymph node metastasis and to explore transcriptional factors and regulation networks associated with the process. Tissue specimens were taken from 20 patients with LSCC, including 10 cases of LSCC without metastasis LSCC (N0) and 10 cases of LSCC with metastasis LSCC (Nx). Among the 643 unique proteins identified by using iTRAQ labeling and quantitative proteomic technology, 389 proteins showed an abundance change in LSCC (Nx) as compared to LSCC (N0). Cytoskeleton remodeling, cell adhesion, and immune response activation were found to be the main processes in LSCC metastasis. The construction of transcription regulation networks identified key transcription regulators for lymph node metastasis of LSCC, including Sp1, c-myc, and p53, which may affect LSCC metastasis through the epithelial-mesenchymal transition. Furthermore, our results suggest that ubiquitination may be a critical factor in the networks. The present study provides insights into transcriptional factors and regulation networks involved in LSCC metastasis, which may lead to new strategies for treatment of LSCC metastasis.

Bone morphogenetic protein-4-induced epithelial-mesenchymal transition and invasiveness through Smad1-mediated signal pathway in squamous cell carcinoma of the head and neck.

BACKGROUND AND AIMS: Bone morphogenetic proteins (BMPs) have recently been shown to be involved in the genesis and progression of a wide variety of carcinomas. The present study was undertaken to estimate the effect of BMP-4 on squamous cell carcinoma of the head and neck (SCCHN) in tissue and cell levels. METHODS: In this study, immunohistochemistry, Western blotting and RT-PCR were utilized to detect the expression of BMP-4, Smad1 and phosphorylated Smad1 in SCCHN tissues or SCCHN cell lines. Those three proteins in tissues were further correlated with prognosis of SCCHN by Kaplan-Meier analysis. The epithelial-mesenchymal transition (EMT)-associated changes in SCCHN cells were detected after stimulation by human BMP-4 recombinant protein and knockdown of Smad1 gene. Meanwhile, the effect on invasiveness and migration was evaluated by invasion and scratch assays, respectively. RESULTS: BMP-4 and p-Smad1 protein were overexpressed in SCCHN tissues with cervical lymph node metastasis, which was significantly higher than those without metastasis. The expression of BMP-4 and p-Smad1 protein was negatively correlated with the prognosis of SCCHN. BMP-4 promoted the invasiveness and migration through EMT, which was demonstrated by morphological alterations, loss of E-cadherin, increase of vimentin and activation of the Smad1 signal pathway. Knockdown of Smad1 expression suppressed BMP-4 induced EMT in both cell lines and weakened the invasiveness and migration of Tu686 and Tu212 in vitro. CONCLUSIONS: Our results demonstrate that BMP-4 protein may contribute to the malignant metastasis of SCCHN, which presents as a novel prognostic marker and a potential therapeutic target for patients with SCCHN.

[Correlation of EphA2 protein expression with clinicopathological characteristics and prognosis in laryngeal squamous cell carcinoma].

OBJECTIVE: To evaluate the expression of EphA2 protein in tissue specimens and cell lines of laryngeal squamous cell carcinoma (LSCC), and to further study the correlation of EphA2 protein expression with clinicopathological characteristics and prognosis in LSCC. METHODS: Western blot was applied to assess the EphA2 protein expression in LSCC cell line Hep-2 cells and the head and neck immortalized epithelial cell line NP-69 cells. Immunohistochemical staining was performed on paraffin sections of 88 cases of LSCC specimens and 16 cases of adjcent normal tissue samples to investigate the EphA2 protein expression, and to futher elucidate its correlation with clinicopathological characteristics. RESULTS: Compared with the NP-69 cells, EphA2 expression in LSCC cell line Hep-2 cells was upregulated. The positive rates of EphA2 expression in LSCC and adjcent normal tissues samples were 80.7% and 43.8%, respectively, with a significant difference between the two groups (P < 0.001). EphA2 overexpresion was closely correlated with clinical stage (I + II/III + IV, P = 0.005), metastasis (P = 0.025) and recurrence (P = 0.021) in LSCC. Furthermore, patients with EphA2 overexpression had poorer tumor-free survival and 5-year overall survival compared with that in patients with low EphA2 expression (33.3% vs. 63.2%, P = 0.003; 46.7% vs. 81.6%, P = 0.002). EphA2 expression combined with clinical stage provided a better predictive value in prognosis. Univariate and multivariate Cox regression analysis revealed that EphA2 expression is an independent prognostic factor for patients with LSCC (P = 0.019). CONCLUSIONS: The results of this study demonstrate that EphA2 protein expression is significantly increased in LSCC tissues and cell lines, and EphA2 protein overexpression is associated with tumor recurrence, metastasis and poorer prognosis in LSCC patients. These results suggest that EphA2 may play a critical role in the initiation and progression of LSCC, implicating EphA2 as a valuable marker for the prediction of recurrence, metastasis and prognosis in LSCC.

[Inhibition of cell growth by rapamycin through targeting mammalian target of rapamycin signaling pathway in nasopharyngeal carcinoma].

OBJECTIVE: To study the effects of rapamycin on cell growth and cell cycle in CNE-1 and CNE-2 cells. METHODS: Growth inhibition effect of rapamycin on CNE-1 and CNE-2 cells were assessed by cell counting kit-8 (CCK-8) assay. Morphological alterations of the cells were observed by microscope. Cell cycle and cell apoptosis were analyzed by FCM. The expression of mammalian target of rapamycin (mTOR) was analyzed by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: The growth of CNE-1 and CNE-2 cells was inhibited significantly by rapamycin dose-dependently. FCM showed that CNE-1 and CNE-2 cells at 48 hours after rapamycin (150 nmol/L) treatment were arrested in the G0/G1 phase of cell cycle. However rapamycin treatment did not significantly induce apoptosis of CNE-1 and CNE-2 cells (P > 0.05). RT-PCR showed that rapamycin significantly inhibited mRNA expression of mTOR in CNE-2 cells (t = 10.625, P < 0.01). CONCLUSIONS: Rapamycin inhibits the growth of CNE-1 and CNE-2 cells by inhibiting the progression of cell cycle, which could be achieved through decreasing the expression of mTOR.

Alteration of gene expression during nasopharyngeal carcinogenesis revealed by oligonucleotide microarray after microdissection of tumor tissue and normal epithelia from nasopharynx.

BACKGROUND: Microarray and microdissection techniques were being used for many applications to study the carcinogenesis of some human tumors. But seldom studies had hitherto combined these two techniques to study carcinogenesis mechanism of nasopharyngeal carcinoma (NPC). To identify a set of genes involved in the carcinogenesis and development of NPC, we used the microdissected homogeneous NPC tissue cells and the pure normal epithelium pillar cells to construct the whole human genome expression profiles. METHODS: We preserved the tissue samples from nasopharynx of 18 patients (including 13 samples of NPC and 5 samples of normal or inflammatory mucous tissue samples from nasopharynx) in RNAlater Stabilization Reagent. The tissue samples were microdissected to harvest the homogeneous tissue cells, then total RNA was isolated from them. The sufficient antisense RNA (aRNA) was amplified from these total RNA. HG-U133.Plus.2.0 GeneChip was used to construct the human whole genome expression profiling of each sample. Differential patterns of expression of genes correlated with the carcinogenesis, classification and progression of NPC were identified with comparing the expression profiling data respectively in leave one out cross-validation analysis. Correlation between aRNA expression measured by the microarrays and semi-quantitative reverse transcription polymerase chain reaction (sqRT-PCR) were also ascertained, and found that hybridization results were validated in all of the 18 patients. RESULTS: Differential patterns of expression of 127 genes correlated with the carcinogenesis (A P value less than 0.001 with the 2-fold differentiated expression between case group and control group) of the NPC were filtered. The top most up-regulated and down-regulated 8 genes by the way of permutation test were also selected and listed in the paper. Expression of genes E2F6 and TSPAN-1 was identified using aRNA by sqRT-PCR and showed that there was significant difference between the average value of case groups and that of control group respectively (t = 2.170, df = 16, P = 0.045 and t = -2.946, df = 16, P = 0.009). CONCLUSIONS: We had identified some genes which could be the molecular marker during the carcinogenesis and the development of the NPC. The genes which selected from the different subgroups seemed to be implicated for the diagnosis,classification, and progression of NPC, and provided important insights into their underlying biology.

Differential gene expression profiling of laryngeal squamous cell carcinoma by laser capture microdissection and complementary DNA microarrays.

BACKGROUND AND AIMS: Genetic alteration associated with initiation and progression of laryngeal squamous cell carcinoma (LSCC) is largely unknown. The aim of this study was to identify genetic changes associated with the disease pathogenesis and pinpoint genes whose expression is impacted by these genetic alterations. METHODS: Tumor cells were collected from eight matched pairs of specimens of glottic carcinoma of the larynx and histologically normal epithelium tissues adjacent to the carcinoma by laser capture microdissection (LCM). RNAs prepared from these cells were used for genome-wide transcriptome analysis by probing 16 cDNA microarrays. Real-time quantitative RT-PCR and immunohistochemistry of tissue microarrays were used to validate a group of the differentially expressed genes identified by the cDNA microarrays. RESULTS: Hierarchical cluster analysis of the expressed genes showed that 2351 genes were differentially expressed and could distinguish cancerous and noncancerous samples. We also found 761 differentially expressed genes that were consistently different between early stage and later stage specimens. Furthermore, abnormal expression of some relevant genes such as MMP12, HMGA2, and TIMP4 were validated by real-time quantitative RT-PCR and immunohistochemistry. Analysis of gene ontology and pathway distributions then highlighted genes that may be critically important to laryngeal carcinogenesis. CONCLUSIONS: Our results suggest that using LCM plus DNA microarray analysis may facilitate the identification of clinical molecular markers for disease and novel potential therapeutic targets for LSCC.

[Global gene expression profiling of laryngeal squamous cell carcinoma by laser capture microdissection and complementary DNA microarrays].

OBJECTIVE: To examine the gene expression profile of laryngeal squamous cell carcinoma (LSCC) by combination of laser capture microdissection (LCM) and microarray and to identify genetic changes in disease pathogenesis. METHODS: The study analysed 8 matched pairs of specimens of glottic carcinoma of larynx and histologically normal epithelium tissues adjacent to the carcinoma preserved in the RNA later reagent. A genome-wide transcriptome analysis was performed by probing 16 cDNA microarrays with fluorescent-labeled amplified RNA derived from laser capture microdissected cells. Real-time quantitative (RT-PCR) of tissue microarray was used to validate the reliability of cDNA microarrays. RESULTS: Significant analysis of microarray (SAM) software and hierarchical cluster analysis of the expressed genes showed that 2351 genes was significantly expressed respectively according to different analysis method (false discover rate = 0.63%). A selected set of MMP12, KRT16, RARB, PRB1 genes was identified to be consistent with array data by RT-PCR. CONCLUSIONS: The analysis of gene ontology and pathway distributions futher highlighted genes that may be critically important to laryngeal carcinogenesis. The results strongly suggest that this new approach may facilitate the identification of clinical molecular markers of disease and novel potential therapeutic targets for LSCC.