[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.

[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.