溶血磷脂酸对乳腺癌MDA-MB-231细胞迁移和侵袭能力影响的机制探讨

摘要

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.%目的 探讨溶血磷脂酸(LPA)对乳腺癌MDA-MB-231细胞迁移和侵袭能力影响的机制.方法 ①将体外培养的MDA-MB-231细胞分为转染组、对照序列组和空白组.转染组转染(YAP)siRNA 3 h后加入浓度50 μmol/L的LPA继续培养16 h.对照序列组转染对照序列 3 h后加入浓度50 μmol/L的LPA继续培养16 h.空白组不转染、不加LPA,常规培养16 h.用Transwell小室行细胞迁移实验和细胞侵袭实验观察各组细胞迁移、侵袭能力.②用50 μmol/L的LPA培养MDA-MB-231细胞0、10、30、60、120、240、480 min,收集细胞,用Western blotting法检测磷酸化YAP(p-YAP)相对表达量.用0、1、10、20、50 μmol/L的LPA培养MDA-MB-231细胞120 min,收集细胞,用Western blotting法检测磷酸化YAP相对表达量.③将体外培养的MDA-MB-231细胞分为1、2、3、4、5、6、7组,分别加入培养基、MAPK通路阻断剂SB203580、PI3K通路阻断剂LY294002、Akt通路阻断剂MK2206、 MEK通路阻断剂PD98059、Rho抑制剂(C3转移酶)和ROCK抑制剂(Y27632),然后加入50 μmol/L的LPA继续培养120 min.采Western blotting法检测pYAP表达.结果 ①转染组、对照序列组、空白组细胞迁移实验穿膜细胞数分别为(10.01±2.05)、(12.13±2.44)、(7.06±2.54)个,细胞侵袭实验穿膜细胞数分别为(9.24±2.18)、(11.54±2.09)、(6.75±1.48)个.对照序列组细胞迁移实验、细胞侵袭实验穿膜细胞数均高于空白组(P均<0.05).转染组细胞迁移实验、细胞侵袭实验穿膜细胞数均低于对照序列组细(P均<0.05).②用10 μmol/L的LPA培养MDA-MB-231细胞0、10、30、60、120、240、480 min,MDA-MB-231细胞YAP相对表达量分别为0.11±0.023、0.08±0.002、0.05±0.027、0.04±0.012、0.01±0.002、0.03±0.004及0.05±0.007.培养时间为120 min时MDA-MB-231细胞pYAP相对表达量最低(P均<0.05).用0、1、10、20、50 μmol/L的LPA培养MDA-MB-231细胞120 min后pYAP相对表达量分别为0.23±0.024、0.18±0.020、0.09±0.022、0.06±0.017、0.02±0.012.随着LPA浓度的升高,MDA-MB-231细胞pYAP相对表达量逐渐降低(P均<0.05).③1、2、3、4、5、6、7组MDA-MB-231细胞pYAP相对表达量分别为0.34±0.027、0.32±0.022、0.35±0.019、0.33±0.021、0.34±0.025、0.74±0.029及0.65±0.022.1、2、3、4、5组MDA-MB-231细胞pYAP相对表达量相比,P均>0.05.6组MDA-MB-231细胞pYAP相对表达量高于1~5组(P均<0.05).7组MDA-MB-231细胞YAP相对表达量高于1~5组(P均<0.05),但低于6组(P<0.05).结论 LPA通过Rho和ROCK通路导致乳腺癌MDA-MB-231细胞YAP去磷酸化,进而促进乳腺癌MDA-MB-231细胞迁移和侵袭.