The Role And Mechanism Of CCR7/CCL21in Lymph Node Metastasis In Non-Small Cell Lung Cancer

In the process of invasion and lymph node metastasis of lung cancer, there are at least five types of factors are involved:Sports factors, adhesion molecules, chemokines, extracellular matrix-degrading enzymes, angiogenic factors. CCR7and its ligands CCL21is a chemokine, the main destination is the secondary lymphoid tissue chemokine, VEGF-C is the most effective to promote lymphatic growth factor, MMPs are most effective extracellular matrix degrading enzymes, among these three whether interactions in the metastasis of lung cancer, worthy of exploration and research.The experiment study the expression of CCR7and VEGF-C in lung cancer tissue and adjacent tissue, and the correlation with the capillary lymphatic generated and lymph node metastasis. It also study the effect of CCR7/CCL21axis to the proliferation, metastasis, invasion and the expression of relevant metastasis genes of lung cancer. To discuss the mechanism of action of CCR7/CCL21axis in regulating the biologic characteristics and lymph node metastasis non-small cell lung cancerFirst part:The relation between CCR7and VEGF-C expression and their clinicopathological parameters in non-small cell lung cancerObjective:study CCR7and VEGF-C expression in lung cancer and lymphatic vessel density in adjacent tissue and cancer tissue adjacent tissue by the immunohistochemistry, and statistical analysis the relevant of clinicopathological parameters in patients with non-small cell lung cancer and the expression of CCR7and VEGF-C.Methods:1)60cases of pathology paraffin blocks are confirmed to be the non-small cell lung cancer by collecting clinical pathology, Statistics localization and the amount of CCR7and VEGF-C expression in cancer tissues, adjacent tissues and normal lung tissue by using immunohistochemical methods, analysis the relevant between the expression level and patient age, gender, tumor size, histological type and tumor differentiation degree off, TNM stage and lymph node metastasis.2)statistics the newborn micro lymphatic capillaries density(MLVD),in cancerous tissue, cancer tissue and normal lung tissue, by the use of immunohistochemical methods. Study the relevant between CCR7and VEGF-C expression in lung cancer, cancer tissue and new capillary lymphatic generated, and the correlation of lymphangiogenesis density and lymph node metastasis.Results:1) CCR7and VEGF-C are expressed in lung cancer cells pack slurry and envelope area, CCR7positive expression rate is66.7%, including23cases in low expression,17cases of high expression. Adjacent tissues, CCR7positive expression rate is56.7%, which is highly expressed in25cases,9cases of low expression. CCR7expression in cancer tissue is higher than in adjacent tissues. Both are statistically significant (P=0.007);2) VEGF-C in cancer tissues positive expression rate is76.7%, of which14cases in low expression and32cases in high expression. Adjacent tissues, VEGF-C positive expression rate is38.3%, of which13cases in low expression, high expression is10cases. VEGF-C expression in cancerous tissue is higher than adjacent tissues. Its expression level has statistical significance (P=0.036);3) The labeled lymphatic vessels by D2-40in cancer tissues, cancer tissue,,lymphatic micro-vessel density (15.62±9.64),(16.949.31), lymphatic micro-vessel number in cancer tissue and adjacent tissue was significantly higher than normal lung tissue’s(P=0.074). When lymph node metastasis happened, lymphatic vessel density in cancer tissue (24.38±9.01)、,and adjacent tissues (23.06±9.26) was significantly higher than the.no lymphatic vessel density in cancer tissue (9.79±3.96),and adjacent tissues (12.87±6.84), Both are statistically significant (P=0.000);4) No relevant between CCR7expression levels in lung cancer with age (P=0.357), gender (P=0.05), tumor size (P=0.361), histological type (P=0.453) and tumor differentiation degree (P=0.92), however it is correlated with lymph node metastasis and tumor stage (P=0.005);5) No relevant between the expression levels of VEGF-C in lung cancer with age (P=0.967), gender (P=0.347), tumor size (P=0.640), histological type (P=0.691) and tumor differentiation degree (P=0.103), but it is correlated with lymph node metastasis and tumor stage (P=0.025);6) It was weakly correlated in the expression level of CCR7and VEGF-C in cancer tissues and adjacent organization (r=0.29; r=0.263).Conclusion:CCR7and VEGF-C in non-small cell lung cancer and adjacent tissues are expressed, and the expression level is related with the lymphatic micro-vessel density, lymph node metastasis, suggesting that CCR7may be involved in tumor lymph-angiogenesis and lymph node metastasis, like VEGF-C. Second part:The effect of CCR7/CCL21axis to the proliferation and invasion of lung cancer cellA549Objective:In the vitro experiments, study the changes in lung cancer cell proliferation and invasion that after cellsA549was stimulated by Chemokine CCL21, and then clear the effect of CCR7/CCL21axis to the biological activity of lung cancer.Method:1) The use of MTT (MTT colorimetric assay), measured the proliferation curve of cellsA549and the cell proliferation curve with the stimulation of different concentrations of CCL21, compare the different concentrations of Chemokines on cell proliferation activity.2) study the effect of CCR7/CCL21axis on vitro migration and invasion of lung cancer cells through the migration assay and Matrigel invasion assay by the use of the transwell chamber.Results:1) Proliferation activity of cellsA549was significantly higher than ordinary cell proliferation activity after CCL21stimulate chemotaxis; when CCL21is100ng/ml, the cell proliferation is the strongest.2) the cells A549migration and invasion is higher than ordinary cells (A549) after CCL21-stimulated.Conclusion:CCR7/CCL21axis can enhanced cellA549proliferation and migration and invasion, and when CCL21concentration is100ng/ml, the proliferative activity are the strongest. Third part:The effect of CCR7/CCL21axis to theVEGF-C and MMP9gene and protein expression of cells A549Objective:To amplify the VEGF-C and MMP9mRNA by quantitative RT-PCR technology, to confirm CCR7/CCL21axis effect to the gene expression, to confirm the effect of CCL21to the VEGF-C and MMP9protein expression by the Western blot technology.Methods:Design the PCR primers of VEGF-C and MMP9, using quantitative PCR, amplify the cells A549and VEGF-C and MMP9gene in cells A549which is CCL21 chemokines, to confirm whether the activation of CCL21would impact the expression of VEGF-C mRNA and MMP9mRNA or not, to confirm the effect of CCL21to its ligand of CCR7and protein expression of VEGF-C and MMP9by Western blot technique.Results:VEGF-C and MMP9gene expression in cells A549was significantly enhanced after CCL21activation, with the time effect, on the other hand, the expression of CCR7protein was affected by CCL21,CCR7/CCL21axis can enhance VEGF-C and MMP9protein expression.Conclusion:CCR7/CCL21axis can regulate the tumor cell VEGF-C and MMP9gene and protein expression, procure the lymph nodes distant metastasis ability increased by affecting tumor invasion and lymphatic forming ability.