摘要
目的观察Wnt3a对子宫内膜癌细胞转移的影响,并探讨其是否通过Dishevelled-2(Dvl-2)激活PI3K-Akt-GSK3β通路起作用。方法取子宫内膜癌细胞Ishikaw,MTT法确定Wnt3a作用于细胞的安全浓度<500 ng/mL;以0、50、100、200、400 ng/mL Wnt3a处理后,用细胞划痕实验测算细胞迁移距离;400 ng/mL Wnt3a处理细胞0、12、24、48 h后,Western blotting法检测细胞中Dvl-2、PI3K、Akt和GSK3β的磷酸化水平;转染Dvl-2 siRNA质粒敲低Dvl-2表达后,观察Wnt3a对细胞迁移的影响;分别用LY294002(PI3K抑制剂)、CCT128930(Akt抑制剂)预处理细胞后,用Western blotting法检测Wnt3a诱导的Akt、GSK3β磷酸化水平变化;敲低Dvl-2表达后,用Western blotting法检测Wnt3a诱导的PI3K磷酸化水平变化;分别用LY294002、CCT128930、AR-A014418(GSK3β抑制剂)预处理细胞,或敲低Dvl-2表达、IGF-1(PI3K-Akt激活剂)预处理细胞后,用细胞划痕实验测算Wnt3a对细胞迁移的影响。结果与未经处理(0 ng/mL)的细胞相比,细胞迁移距离随Wnt3a浓度增加而增加(P均<0.01);用400 ng/mL Wnt3a处理细胞后,Dvl-2、PI3K、Akt、GSK3β蛋白磷酸化水平随作用时间延长而不断升高(P均<0.01);敲低Dvl-2表达后,Wnt3a诱导的PI3K磷酸化水平降低,细胞迁移距离变小(P均<0.01);用LY294002预处理细胞后,Wnt3a诱导的Akt磷酸化水平下降,细胞迁移距离变小(P均<0.01);用CCT128930预处理细胞后,Wnt3a诱导的GSK3β磷酸化水平降低,细胞迁移距离变小(P均<0.01);用AR-A014418预处理细胞后,Wnt3a诱导的细胞迁移距离变小(P<0.001);且敲低Dvl-2表达后,Wnt3a和IGF-1诱导的细胞迁移距离变小(P均<0.01)。结论Wnt3a通过Dvl-2激活了PI3K-Akt-GSK3β信号通路,从而促进子宫内膜癌细胞的转移。
Objective To observe the effect of Wnt3a on the metastasis of endometrial cancer cells and to investigate whether it plays a role in activating the PI3K-Akt-GSK3βpathway through Dishevelled-2(Dvl-2).Methods We took the endometrial cancer cells Ishikaw.MTT was used to determine the safe concentration of Wnt3a acting on endometrial cells(<500 ng/mL).After treatment with 0,50,100,200 and 400 ng/mL Wnt3a,the cell migration distance was measured by cell scratch test and phosphorylation levels of Dvl-2,PI3K,Akt,and GSK3βwere detected by Western blotting after 400 ng/mL Wnt3a treatment for 0,12,24,and 48 h.The effect of Wnt3a on cell migration was observed after transfection of Dvl-2 siRNA plasmid by knocking down Dvl-2 expression.After pretreatment of cells with LY294002(PI3K inhibitor)and CCT128930(Akt inhibitor),phosphorylation levels of Akt and GSK3βinduced by Wnt3a were detected by Western blotting.After the expression of Dvl-2 was knocked down,the phosphorylation level of PI3K induced by Wnt3a was detected by Western blotting.After pretreatment of cells with LY294002,CCT128930 and AR-A014418(GSK3βinhibitor),or with knockdown of Dvl-2 expression and IGF-1(PI3K-Akt activator),the effect of Wnt3a on cell migration was measured by cell scratch test.Results Compared with the untreated cells(0 ng/mL),Wnt3a-induced cell migration increased in a concentration-dependent manner(P<0.01).After the cells were treated with 400 ng/mL Wnt3a,Wnt3a promoted phosphorylation of Dvl-2,PI3K,Akt and GSK3βin a time-dependent manner(all P<0.01).After knocking down Dvl-2 expression,the PI3K phosphorylation level induced by Wnt3a decreased and the cell migration distance decreased(both P<0.01).After pretreatment with LY294002,Wnt3a-induced Akt phosphorylation level decreased and cell migration distance decreased(both P<0.01).After pretreatment with CCT128930,the phosphorylation level of GSK3βinduced by Wnt3a decreased and the cell migration distance decreased(both P<0.01).After pretreatment with AR-A014418,the cell migration distance induced by Wnt3a decreased(P<0.001).In addition,the cell migration distance induced by Wnt3a and IGF-1 decreased after the expression of Dvl-2 was knocked down(both P<0.01).Conclusion Wnt3a activates the PI3K-Akt-GSK3βsignaling pathway through Dvl-2,and thus promotes the metastasis of endometrial cancer cells.
作者
刘晶晶
刘海娟
王荣
LIU Jingjing;LIU Haijuan;WANG Rong(Xi′an Fourth Hospital,Xi′an 710075,China;不详)
出处
《山东医药》
CAS
2020年第29期23-27,共5页
Shandong Medical Journal
基金
陕西省重点研发计划项目(2019SF-210)。
