Expression Of NGX6 Protein In NPC And Its Correlation To Expression Of VEGF And LMVD

Objectives: To study the expression of NGX6(a kind of tumor suppressor gene)at protein level, in human nasopharyngeal carcinoma. To investigate the relationship between the expression of NGX6 and the clinical characteristics, such as age, sex lymph node metastasis,clinical stage and T,N-stages, in nasopharyngeal carcinoma.Methods: In Experiment one, immunohistochemical technique (EnVision) was used to detect the expression of NGX6 in 20 cases of non-tumor nasopharyngeal epithelial tissue(group A), 32 cases of NPC tissues without lymph node metastasis(group B) and 48 cases of NPC tissues with lymph node metastasis (group C). Analysis the difference of expreesion level among three groups. Analysis the relationship between expressive level of NGX6 and clinicopathological features of the patients, including age, sex, lymph node metastasis, clinical stage and T,N-stages, respectively.In Experiment two, Western Blot technique was used to detect the expression of NGX6 in 4 cases of non-tumor nasopharyngeal epithelial tissue(group A), 6 cases of NPC tissues without lymph node metastasis(group B) and 10 cases of NPC tissues with lymph node metastasis (group C). Analysis the relationship between expression of NGX6 and lymph node metastasis in NPC.Results: In Experiment one, the immunoreactive products of NGX6 protein were stained into Buffy granules, located in cytoplasm and cell membrane of nasopharyngeal epithelial cells and tumor cells. The positive expression of NGX6 protein showed a downward trend in turn A, B, C group. The pos-expressive rate of NGX6 protein in non-tumor tissue(85%, 17/20) was significantly higher than that in NPC tissues (52.5%, 42/80)(Χ2=6.9864,P<0.01). The pos-expressive rate of NGX6 protein in NPC tissue without lymph node metastasis(68.75%, 22/32) was significantly higher than that in NPC tissues with lymph node metastasis(41.67%, 20/48)(Χ2=5.6475,P<0.025).The pos-expressive rate expression of NGX6 protein in NPC tissue of T2 stage (45.2%) and NPC tissue of T3/4 stage (18.18%) were all significantly lower than that in NPC tissues of T1 stage (77.78%) (P<0.01,P<0.005). No significant diference was found between the expression of NGX6 protein in NPC tissue of T2 stage and of T3/4 stage(P>0.05). The pos-expressive rate of NGX6 protein in NPC tissue of N2/3 stage (26.92%) was significantly lower than that in both NPC tissues of N0 stage (68.75%) and NPC tissue of N1 stage (59.09%) (P<0.005,P<0.025). No significant diference was found between the expression of NGX6 protein in NPC tissue of N0 stage and of N1 stage (P>0.05). The pos-expressive rate of NGX6 protein in NPC tissue ofⅢ/Ⅳstage (36.37%) was significantly lower than that in NPC tissues ofⅠ/Ⅱstage (72.22%) (P<0.005). Besides, no significant diference was found in the expression of NGX6 protein in different age and sex (P>0.05,respectively).In Experiment two, the opposing integral optical density value (ODV) of NGX6 protein showed downward trend in turn A, B, C group and significant diference in each other(F=38.02,Pr<0.0001). The opposing integral ODV of NGX6 protein in non-tumor tissue(0.9098±0.2673) was significantly higher than that in NPC tissues without lymph node metastasis (0.3218±0.2003) (P<0.05). The opposing integral ODV of NGX6 protein in NPC tissue without lymph node metastasis(0.3218±0.2003) was significantly higher than that in NPC tissues with lymph node metastasis(0.0875±0.0432) (P<0.05).Conclusions: The expression of NGX6 was down regulated at protein level in nasopharyngeal carcinoma. Furthermore, the down regulation of NGX6 in NPC significantly correlated with lymph node metastasis. Lower expression of NGX6 in NPC indicated a higher incidence of lymph node metastasis. In NPC, the expression of NGX6 protein was significantly correlated to the clinicopathological characteristics, such as lymph node metastasis, T stages, N stages and clinical stages. Therefore, detection of the NGX6 protein might be used to predict metastasis of nasopharyngeal carcinoma and assess prognosis. But no significant correlation was found in the expression of NGX6 protein to both age and sex of patients.Part two Correlation of NGX6 protein expression to LMVDObjectives: To study the correlation of NGX6 protein expression to LMVD in nasopharyngeal epithelial tissue,then presume the possible function of NGX6 in regulating the formation of lymphatic vessel in tumor.Methods: Immunohistochemical technique (EnVision) was used to detect the expression of D2-40 in each group (same to Part one-Experiment one). Calculate the LMVD of each sample. Analysis the difference of LMVD among three groups. Analysis the correlation of NGX6 protein expression to LMVD.Results: The immunoreactive products of D2-40 were stained into Buffy granules, located in cytoplasm of lymphatic vessel endothelial cells. The LMVD showed a upward trend in turn A, B, C group and significant diference in each othe(rF=29.98,Pr<0.0001). The LMVD in non-tumor tissue(3.45±1.94) was significantly lower than that in NPC tissues without lymph node metastasis (6.02±2.96) (P<0.05). The LMVD in NPC tissue without lymph node metastasis(6.02±2.96) was significantly lower than that in NPC tissues with lymph node metastasis(10.99±5.10) (P<0.05).The LMVD showed a negative correlation to the expressive level of NGX6 (rs=-0.73221,P<0.0001). The more expression of NGX6, the less of LMVD.Conclusions: The LMVD was high in nasopharyngeal carcinoma. Furthermore, the LMVD in NPC significantly correlated with lymph node metastasis. Higher LMVD in NPC indicated a higher incidence of lymph node metastasis. In NPC, the LMVD was significantly correlated to the expression of NGX6 protein. Then we suppose that, the lower expression of NGX6 and higher LMVD have a synergistic effect in advancing of NPC. The NGX6 protein might has depressant effect on the formation of lymphatic vessel.Part three Correlation of NGX6 Expression to VEGF ExpressionObjectives: To study the correlation of NGX6 protein expression to VEGF protein expression in nasopharyngeal epithelial tissue,then presume the possible function of NGX6 in regulating the expression of VEGF in NPC cell.Methods: In Experiment one, immunohistochemical technique (EnVision) was used to detect the expression of VEGF in each group (same to Part one-Experiment one). Analysis the difference of expreesion level among three groups. Analysis the correlation of VEGF protein expression to LMVD. Analysis the correlation of NGX6 protein expression to VEGF protein expression.In Experiment two, Western Blot technique was used to detect the expression of VEGF in each group (same to Part one-Experiment two). Analysis the difference of expreesion level among three groups. Analysis the correlation of NGX6 protein expression to VEGF protein expression.Results: In Experiment one, the immunoreactive products of VEGF protein were stained into Buffy granules, located in cytoplasm of nasopharyngeal epithelial cells and tumor cells. The positive expression of VEGF protein showed a upward trend in turn A, B, C group. The pos-expressive rate of VEGF protein in non-tumor tissue(55%, 11/20) was significantly lower than that in NPC tissues (86.25%, 69/80)(Χ2=9.7656,P<0.005). The pos-expressive rate of VEGF protein in NPC tissue without lymph node metastasis(78.13%, 25/32) was significantly lower than that in NPC tissues with lymph node metastasis(91.67%, 44/48), but the difference was not significant(Χ2=2.9688,P>0.05). The LMVD showed a positive correlation to the expressive level of VEGF(rs=0.73332,P<0.0001). The more expression of VEGF, the more of LMVD. The expressive level of VEGF showed a negative correlation to the expressive level of NGX6(rs=-0.59988,P<0.0001). The less expression of NGX6, The more expression of VEGF.In Experiment two, the opposing integral optical density value (ODV) of VEGF protein showed upward trend in turn A, B, C group and significant diference in each other(F=20.13,Pr<0.0001). The opposing integral ODV of VEGF protein in non-tumor tissue(0.5419±0.1993) was significantly lower than that in NPC tissues without lymph node metastasis (1.3748±0.3665) (P<0.05). The opposing integral ODV of VEGF protein in NPC tissue without lymph node metastasis(1.3748±0.3665) was significantly lower than that in NPC tissues with lymph node metastasis(1.9939±0.4506) (P<0.05). The expressive level of VEGF showed a negative correlation to the expressive level of NGX6(rs=-0.80902,P<0.0001). The less opposing integral ODV of NGX6, The more opposing integral ODV of VEGF.Conclusions: The expression of VEGF protein was up regulated in nasopharyngeal carcinoma. Furthermore, the overexpression of VEGF protein in NPC significantly correlated with lymph node metastasis. Higher expression of VEGF in NPC indicated a higher incidence of lymph node metastasis. In NPC, the expression of VEGF protein was significantly negatively correlated to the expression of NGX6 protein. Then we suppose that, the lower expression of NGX6 and overexpression of VEGF have a synergistic effect in proceeding of NPC. VEGF expression might be negatively regulated by NGX6. The expression of VEGF protein was significantly correlated to the LMVD, which verify the promoting effect of VEGF on the formation of lymphatic vessel. Therefore, com-detection of the NGX6 protein , VEGF protein and LMVD might be more helpful to predict metastasis of nasopharyngeal carcinoma and assess prognosis.