Effects Of LPA On Lung Adenocarcinoma Cell Proliferation, Migration And CFTR Ion Channel

Lung cancer ranks the top of cancer deaths, of which the incidence across the globe shows a persistent rising trend in recent years, which is particularly evident in China. Therefore, it is significant to clarify its occurrence, development mechanism and also to explore effective therapies for medical development and human health. In this study, in order to figure out the influence of LPA on lung cancer morbidity, the plasma, the concentration of LPA and the lung adenocarcinoma cell of clinical patients have been investigated. In addition, the research object is lung malignancy cell Calu-3 and how LPA affect the proliferation and migration of Calu-3 has been observed. The relevant molecular mechanism has also been investigated in the meantime. Therefore, the study may provide a new theoretical and experimental statistics basis for the treatment of lung cancer.Firstly, making use of LPA lit to inspect the plasma and the concentration of LPA in pleural effusion of lung cancer patients and pneumonia, tuberculous pleurisy patients. The results reveal that the concentration of LPA in lung cancer patients’ plasma and pleural effusion shows a remarkable increase compared to pneumonia, tuberculous pleurisy patients’, of which the difference is significant in statistical scale.(P<0.05)Secondly, exploring the influence of LPA on Calu-3 cells through CCK8 experiment and then inspecting how LPA affects the migration of Calu-3 cells and lastly detecting the variation of cell cycles with the use of flow cytometry. The experimental results are that LPA could promote the proliferation and migration of Calu-3 cells. In addition, LPA could also give rise to the quantity of S cells during the cell cycles. Besides, to further investigate how LPA make effects on proliferation and cycle of Calu-3 cells, the study employ quantitative PCR to observe the genes which play important roles in cell proliferation, migration and the cell cycle: VEGF、HIF-1α、PCNA、c-myc、Cyclin D1、CDK2、p53. The result shows that with the increase of LPA concentration, the expression of VEGF、HIF-1α、 PCNA、c-myc m RNA also rise significantly,but the expression of p53 m RNA shows a great decrease. Moreover, LPA functions by combining with the G-protein coupled receptors in cell surface. Many G-protein exist in our body and they could be approximately classified into the family of Gs, Gi and Gq. PTX is a specific blocker of Gi-protein. The research adding PTX into Calu-3 cells finds out that the expression of VEGF、HIF-1α、PCNA、c-myc and Cyclin D1、CDK2 m RNA have not increased. In the meantime, the expression of p53 m RNA has not fallen yet. In addition, the study adopted Western blot technique in protein levels to observe the change of the key proteins in cell proliferation and cell cycle: VEGF、HIF-1ɑ、PCNA、c-myc、Cyclin D1、CDK2、p53. The results indicate that LPA can make the expression of VEGF、HIF-1α、 PCNA、c-myc、Cyclin D1 and CDK2 protein increase, down-regulate p53 protein expression, which further prove LPA could promote the proliferation and migration of Calu-3 cells.It has been reported that CFTR ion channel has some effects on inhibiting tumor cell proliferation. Through immunofluorescence and confocal microscopy experiments, it has been observed that there is CFTR protein expression in lung adenocarcinoma Calu-3 cell membrane. Therefore the whole-cell patch-clamp technique has been used to observe how different concentrations of LPA affect Calu-3 cell membrane CFTR ion channel currents. The results show that after 5、10、20μmol/L LPA for Calu-3 cells, CFTR ion channel current reduce with different degree compared to untreated groups of LPA. All of the above results suggest that LPA could reduce CFTR channel currents of Calu-3 cells, which may be one of the mechanisms of LPA promoting the Calu-3. To further investigate the impact of LPA on CFTR ion channel Calu-3 on the cell membrane, methods of immunofluorescence, Western blot has been adopted to detect the effect of LPA on CFTR protein expression of cell membrane. The result is that exogenous LPA could reduce cell membrane expression of CFTR protein, which is accordance with the LPA could reduce the Calu-3 cell membrane CFTR channel currents.It has also been investigated that PTX can inhibit the current decrease initiated by LPA and justifies that Gi-protein mediates the effect of LPA on Calu-3, LPA2 is the membrane receptor of LPA, we therefore speculate that LPA could affect CFTR channel current by LPA2-receptor in the cell membrane. Thus, we adopt the methods of Immunofluorescence and western blot to detect the expression of LPA2-receptor in the cell membrane. The results indicate that the expression of LPA2-receptor exists in Calu-3, justifying that LPA could promote the migration and invasion of lung adenocarcinoma, Calu-3, through affecting LPA2-receptor in the cell membrane.The study initially proves the connection between LPA and lung cancer through the experimental result that the concentration of LPA in lung cancer patients’ plasma and pleural effusion shows a remarkable increase compared to pneumonia, tuberculous pleurisy patients’. Furthermore, LPA could promote the proliferation and migration of Calu-3 cells as well as increase the quantity of cells in S stage during the cell cycles. In addition, the research demonstrates that LPA could promote the expression of cell proliferation and migration of relevant genes and proteins. Also LPA could reduce the expression of apoptosis-related genes. Besides, with the use of patch-clamp technique and Western blot, it has been confirmed that LPA could reduce the CFTR channel currents of Calu-3 cells and the expression of CFTR protein on the cell membrane. All of the results suggest that LPA plays a crucial role in the occurrence and development of lung adenocarcinoma, which provides new ideas and theoretical basis for lung cancer targeted therapy.