Abstract
Objective To investigate the effects of lysophosphatidic acid (LPA) on the migration and invasion of breast cancer MDA-MB-231 cells and the related mechanisms.Methods ① MDA-MB-231 cells were divided into three groups: transfection group, control sequence group, and blank group.Cells in the transfection group were cultured with 50 μmol/L LPA for 16 h after they were transfected with YAP-siRNA for 3 h.Cells in the control sequence group were cultured with 50 μmol/L LPA for 16 h after they were transfected with control sequence for 3 h.Cells in the blank group were normally cultured without transfection or LPA for 16 h.Transwell was performed to evaluate the cell migration and invasion.② MDA-MB-231 cells were cultured with 50 μmol/L LPA for 0, 10, 30, 60, 120, 240, and 480 min, and then we collected the cells and used Western blotting to evaluate the relative expression level of phosphorylated YAP (pYAP).MDA-MB-231 cells were cultured with 0, 1, 10, 20, and 50 μmol/L LPA for 120 min and then we collected the cells for detecting the pYAP expression level by using Western blotting.③ MDA-MB-231 cells were divided into seven groups: groups 1, 2, 3, 4, 5, 6, and 7, which were then added with cell culture medium only, SB203580 (MAPK inhibitor), LY294002 (PI3K inhibitor), MK2206 (Akt inhibitor), PD98059 (MEK inhibitor), C3 transferase (Rho inhibitor), and Y27632 (ROCK inhibitor), followed by 50 μmol/L LPA culture for 120 min.The pYAP expression was measured by Western blotting.Results ① The transwell cell migration test showed that the transmembrane cells in the transfection group, control sequence group, and blank group were 10.01±2.05, 12.13±2.44, and 7.06±2.54, respectively;the cell invasion test indicated that the transmembrane cells were 9.24±2.18, 11.54±2.09, and 6.75±1.48, respectively.The cell invasion or migration test showed that transmembrane cells were more in the control sequence group than those in the blank group (all P<0.05);both invasion and migration test showed that cell count in the transfection group was lower than that in the control sequence group (P<0.05).② The pYAP expression levels in MDA-MB-231 cells cultured with 10 μmol/L LPA for 0, 10, 30, 60, 120, 240, and 480 min were 0.11±0.023, 0.08±0.002, 0.05±0.027, 0.04±0.012, 0.01±0.002, 0.03±0.004, and 0.05±0.007, respectively.The pYAP expression level in MDA-MB-231 cells cultured with 10 μmol/L LPA for 120 min was the lowest (all P<0.05).The pYAP expression levels in MDA-MB-231 cells cultured with 0, 1, 10, 20, and 50 μmol/L LPA were 0.23±0.024, 0.18±0.020, 0.09±0.022, 0.06±0.017, and 0.02±0.012, respectively.With the increase of LPA concentration, the relative expression of pYAP in MDA-MB-231 cells decreased gradually (all P<0.05).③ The expression levels of pYAP in groups 1, 2, 3, 4, 5, 6, and 7 were 0.34±0.027, 0.32±0.022, 0.35±0.019, 0.33±0.021, 0.34±0.025, 0.74±0.029, and 0.65±0.022, respectively.No statistically significant difference in pYAP expression was detected among groups 1-5 (all P>0.05).However, the pYAP expression in groups 6 and 7 was respectively higher than that in groups 1-5 (all P<0.05).Conclusion LPA induces YAP dephosphorylation in MDA-MB-231 cell line through Rho and ROCK pathway, and thus promotes migration and invasion of MDA-MB-231 cells.关键词
乳腺癌/溶血磷脂酶A/细胞迁移/细胞侵袭/YAP蛋白/去磷酸化/Rho通路/ROCK通路Key words
breast carcinoma/lysophospholipase A/cell migration/cell invasion/YAP protein/dephosphorylation/Rho pathway/ROCK pathway分类
医药卫生
