巴马汀抑制人口腔癌KB细胞迁移、侵袭及上皮间充质转化的研究

Effect of palmatine inhibiting migration,invasion and epithelial mesenchymal transformation in human oral cancer KB cells

目的 研究巴马汀对人口腔癌KB细胞迁移、侵袭及上皮间充质转化(EMT)的影响。方法 将KB细胞分为对照组及巴马汀低、中、高浓度组,分别加入含0、4、8及16μmol·L^(-1)巴马汀的培养液。孵育48 h后,用划痕实验检测迁移能力,用Traswell实验检测侵袭能力,用酶联免疫吸附测定(ELISA)实验检测基质金属蛋白酶-2(MMP-2)、MMP-9及纤连蛋白(FN)含量,用实时定量聚合酶链反应实验检测波形蛋白(Vimentin)、N钙黏蛋白(N-cadherin)及E钙黏蛋白(E-cadherin) mRNA表达水平,用蛋白质印迹实验检测Vimentin、N-cadherin、E-cadherin、Wnt3、β连环蛋白(β-catenin)表达水平。结果 对照组和巴马汀低、中、高浓度组KB细胞迁移率分别为(69.27±8.62)%、(52.94±4.49)%、(45.22±5.05)%和(37.63±4.88)%,穿膜细胞数为(197.33±20.26)、(125.33±12.01)、(97.00±9.17)和(62.67±7.51)个,MMP-2含量为(2.93±0.21)、(1.49±0.13)、(1.16±0.15)和(0.95±0.09)ng·mL^(-1),MMP-9含量为(3.51±0.36)、(2.37±0.23)、(2.06±0.35)和(1.72±0.16)ng·mL^(-1),FN含量为(41.28±4.02)、(24.03±3.17)、(20.67±2.63)和(13.82±2.19)ng·mL^(-1);上述指标,巴马汀低、中、高浓度组与对照组比较,在统计学上差异均有统计学意义(P<0.05,P<0.01)。巴马汀低、中、高浓度组Vimentin、N-cadherin、E-cadherin mRNA和蛋白表达以及Wnt3及β-catenin蛋白表达,与对照组比较,在统计学上差异均有统计学意义(P<0.05,P<0.01)。结论 巴马汀具有抑制人口腔癌KB细胞迁移、侵袭及EMT作用,其机制与调控Wnt/β-catenin信号通路有关。

Objective To investigate the effects of palmatine on migration,invasion and epithelial mesenchymal transformation(EMT) in human oral cancer KB cells.Methods KB cells were divided into control group and palmatine-L,-M,-H groups,cultured with 0,4,8and 16 μmol·L^(-1) palmatine.After incubation for 48 h,scratch assay was used to detect migration;Traswell assay was used to detect invasion;matrix metalloproteinase 2(MMP-2),MMP-9 and fibronectin(FN)contents were detected by enzyme-linked immunosorbent assay;the expression of Vimentin,N-cadherin and E-cadherin mRNA were detected by real-time quantitative polymerase chain reaction;the expression of Vimentin,N-cadherin,E-cadherin,Wnt3and β-catenin protein were detected by Western blot.Results Cell mobility in control group and palmatine-L,-M,-H groups were(69.27±8.62) %,(52.94±4.49) %,(45.22±5.05) % and(37.63±4.88) %;the number of transmembrane cells were 197.33±20.26,125.33±12.01,97.00±9.17 and 62.67±7.51;the content of MMP-2 were(2.93±0.21),(1.49±0.13),(1.16±0.15) and(0.95±0.09) ng·mL^(-1);the content of MMP-9were(3.51±0.36),(2.37±0.23),(2.06±0.35) and(1.72±0.16) ng · mL^(-1);the content of FN were(41.28±4.02),(24.03±3.17),(20.67±2.63) and(13.82±2.19) ng·mL^(-1);the above indexes in palmatine-L,-M,-H groups were compared with the control group,and the differences were statistically significant(P <0.05,P <0.01).The mRNA and protein expressions of Vimentin,N-cadherin and E-cadherin,and the expressions of Wnt3 and β-catenin protein in palmatine-L,-M,-H groups were statistically significant compared with those in control group(P <0.05,P <0.01).Conclusion Palmatine can inhibit the migration,invasion and EMT of human oral cancer KB cells,and its mechanism is related to the regulation of Wnt/β-catenin signaling pathway.