Research On The Correlation Between PAR-1 And Metastasis Of Human Lung Cancer

Identifying the crucial genes controlling metastasis of human cancer will aid in the development of new therapeutic approaches to this disease. Recent evidence suggests that the major receptor for thrombin, protease-activated receptor-1 (PAR-1), might be a crucial gene involved in metastasis. Though thrombin has long been known to contribute to metastasis of various human cancers, the function and mechanism of its major receptor, PAR-1, in metastasis of human lung cancer have not previously been studied.To explore the correlation between the expression of PAR-1 and the metastasis of human lung cancer, we first investigated the expression of PAR-1 in the lung carcinoma specimens. The mRNA and protein expression levels of the PAR-1 gene in surgically resected lung carcinoma and corresponding normal lung tissue and lymph node were examined by RT-PCR and irnmunohistochemistry. Results showed that there were significant differences of PAR-1 expression level between the groups of metastatic and non-metastatic carcinoma, primary and metastatic carcinoma, primary carcinoma and the lung tissues. There was no significant correlation between PAR-1 expression and the tumor size, histological type and grade. The results implied a significant contribution of PAR-1 in progression and metastasis of lung carcinoma.To further confirm the function of PAR-1 in metastasis, two human lung giant cell carcinoma cell lines PLA801C (lowly metastasis potential) and PLA801D(highly metastasis potential) were chosen as research materials on the PAR-1 differential expression. The sense and anti-sense constructs "pC/PARls" and "pC/PARlas" were transfected into PLA-801C and PLA-801D cells respectively by the way of lipofection. The pCDNA3 vector was also tranfected into the same cells as a negative control. The cell lines were selected from each transfection by antibiotic reagent G418. PAR-1 expression of cell clones were confirmed by RT-PCR and western blot analysis in comparison with the vector control. It was obvious that PLA801C/PAR-ls and PLA80ID/PAR-las cells showed up-regulation and down-regulation of expression level of PAR-1 than its negative control respectively.MTT growth, flow cytometry analysis, fibronectin adhesion, and Matrigel invasion assays were used in vitro to study the effect of PAR-1 expression on transfected cellsABSTRACTproliferation, adhesion, and invasion. The results showed that transfected with sense PAR-1 could remarkably facilitate the proliferation, adhesion and invasion properties of the PLA-801C cells. And transfected with anti-sense PAR-1 could significantly inhibite growth, adhesion, invasion capabilities, and caused the cell arrested at GO/Gl phase in the cell cycle progression of the PLA-801D cells. The results suggested that PAR-1 might improve metastasis by influence on proliferation, adhesion and invasion of the lung cancer cells.The free intracellular Ca2+ ([Ca2+]i)of all transfected cells were dyed by Fluo-3-Am and then examined by confocal microscope under the laser of 464nm. The result showed that PAR-1 could regulate the concentration of ([Ca2+]i) in cytoplasm. The expression proteins such as pi6, VEGF, CD44V6, MMP2 and P53 known to play important roles in metastasis were also measured by western blot in the transfected cells. The results suggested that the stable expression of PAR-1 could up-regulate VEGF, CD44v6 or down-regulated p53 expression of in transfectant PLA-801C, and vice versa in transfectant PLA-801D cells.In summary, over expression of PAR-1 may play a definite role in the formation of tumor invasion and metastatic phenotype in human lung carcinoma. PAR-1 genes had impact on the metastasis characters of human lung giant cell carcinoma cell line PLA-801(C/D) in several steps including proliferation, adhesion, invasion etc. PAR-1 might impose on tumor metastasis by the mechanism of interacting with Ca2+ and some metastasis related molecules such as VEGF, CD44v6 and p53. PAR-1 may be one of the most important factors in the processes of metastasis of human lung cancer.