1-磷酸鞘氨醇信号激活对乳腺癌BT549细胞增殖的影响

Role of sphingosine-1-phosphate signaling in the proliferation of breast cancer BT549 cells

目的研究1-磷酸鞘氨醇(S1P)信号激活对乳腺癌BT549细胞增殖的影响。方法将细胞分为对照组和实验组。实验组用0.1、1.0和10.0μmol·L^(-1)S1P受体激动药SEW2871处理乳腺癌细胞72 h;对照组用含0.1%胎牛血清培养基培养。用噻唑蓝(MTT)法检测细胞增殖情况。建立乳腺癌BT549细胞过表达S1P受体的细胞模型,将转染的细胞分为空白质粒组(LUC)、野生型S1P受体组(WT)、S1P受体磷酸化位点突变组(MUT)。用MTT法检测细胞增殖情况,并计算细胞增殖率;用克隆集落形成实验检测细胞克隆形成能力。用S1P受体拮抗药W146(10μmol·L^(-1))及蛋白激酶B(AKT)信号通路抑制药MK2206(90 nmol·L^(-1))检测S1P信号在乳腺癌BT549细胞增殖中的作用,用蛋白质印迹法检测磷酸化信号传导与转录激活因子-3(p-STAT3)蛋白、原癌基因c-Myc蛋白的表达情况。结果对照组和0.1、1.0和10.0μmol·L^(-1)实验组的细胞增殖率分别为1.00±0.03、1.13±0.06、1.06±0.10和1.07±0.03,SEW2871在0.1μmol·L^(-1)时可促进细胞增殖(P<0.05)。过表达S1P受体后,WT组、MUT组与LUC组相比,细胞增殖率和克隆集落形成数量均明显增加(均P<0.05)。用W146处理后,LUC组、MUT组、WT组的细胞相对增殖率分别为1.25±0.12、1.31±0.03和1.43±0.14;用MK2206处理后LUC组、MUT组、WT组的细胞相对增殖率分别为0.87±0.15、0.77±0.03和0.88±0.02;WT组、MUT组与相应DSMO组比较,在统计学上差异均有统计学意义(均P<0.01)。用W146处理后,LUC组、MUT组、WT组的细胞克隆形成数量分别为(65.65±5.12),(141.48±5.63)和(93.64±5.14)个,WT组、MUT组与相应DSMO组比较,在统计学上差异均有统计学意义(均P<0.05)。MK2206处理后,LUC组、MUT组、WT组的p-STAT3蛋白相对表达水平分别为0.67±0.04、0.69±0.08和0.81±0.06,原癌基因c-Myc蛋白相对表达水平分别为1.69±0.03、0.70±0.10和0.67±0.07,WT组、MUT组的上述指标与DMSO组比较,在统计学上差异均有统计学意义(均P<0.05)。结论S1P信号激活可促进乳腺癌BT549细胞增殖,其机制可能与AKT及STAT3信号通路有关。

Objective To study the role of sphingosine-1-phosphate(S1P)signal on the proliferation of breast cancer BT549 cells.Methods Cells were divided into control group and experimental group,experimental group were treated with 0.1,1.0,10.0μmol·L^(-1)S1P receptor agonist SEW2871 for 72 h.Control group was cultured with 0.1%fetal bovine serum.Cell proliferation was detected by methyl thiazolyl tetrazolium(MTT)assay.Cell models of overexpressing S1P receptors in BT549 were divided into three groups:blank plasmid group(LUC),wild type S1P receptor overexpression group(WT),S1P receptor phosphorylation site mutation overexpression group(MUT);the proliferation ratio was detected by MTT,the number of cell clones was counted by colony formation experiment.S1P antagonist W146(10μmol·L^(-1))and protein kinase(AKT)signaling inhibitor MK2206(90 nmol·L^(-1))were used to detect the role of S1P signaling in the proliferation of breast cancer cells.The expression of phosphorylate signal transducer and activator of transcription 3(p-STAT3),c-Myc proteins were detected by Western blot.Results The growth ratio of BT549 cells in control group and 0.1,1.0,10.0μmol·L^(-1)experimental groups were 1.00±0.03,1.13±0.06,1.06±0.10 and 1.07±0.03,0.1μmol·L^(-1)SEW2871 promot the cell proliferation(P<0.05).Compared between WT group,MUT group and LUC group,the growth rate and the number of clonal colonies were increased after overexpression of S1P receptor(allP<0.05).The growth ratio of BT549 cells after treatment with W146 and MK2206 in the LUC group,WT group and MUT group were 1.25±0.12,1.31±0.03,1.43±0.14 and0.87±0.15,0.77±0.03,0.88±0.02.Compared between MUT group,WT group and corresponding DMSO group,the differences were statistically significant(allP<0.01).The number of cell clony formation number after treatment with W146 were 65.65±5.12,141.48±5.63 and 93.64±5.14;compared between MUT,WT group and corresponding DMSO group,the differences were statistically significant(allP<0.05).The relative protein expression levels of p-STAT3 in LUC group,WT group and MUT group were 0.67±0.04,0.69±0.08 and 0.81±0.06,the relative protein expression levels of proto-oncogene c-Myc were 1.69±0.03,0.70±0.10 and 0.67±0.07,compared between WT group,MUT group and corresponding DMSO group,the difference was statistically significant(P<0.05).Conclusion S1P signaling can promote proliferation in breast cancer BT549 cells,and the mechanism could be related to AKT and STAT3 signaling pathway.