土槿皮乙酸通过调控PAX2对宫颈癌细胞的影响

Effects of Pseudolaric acid B on cervical cancer cells via PAX2 regulation

目的探讨土槿皮乙酸通过调控PAX2对宫颈癌细胞的影响及其分子机制。方法MTT法检测细胞增殖情况;shRNA干扰技术构建PAX2低表达Hela细胞株,分为空白组、阴性对照组、sh-PAX2组、土槿皮乙酸组、土槿皮乙酸+sh-PAX2组;流式细胞术检测各组细胞凋亡情况;Transwell小室迁移实验检测各组细胞迁移能力;String预测PAX2潜在作用蛋白;Western blot检测蛋白表达;免疫荧光实验观察各组细胞中β-catenin的定位。结果土槿皮乙酸可时间-浓度依赖地抑制宫颈癌细胞增殖。各宫颈癌细胞中PAX2蛋白表达与土槿皮乙酸对各细胞的IC50均呈负相关(P<0.05)。土槿皮乙酸可抑制Hela细胞中PAX2蛋白表达;土槿皮乙酸和低表达PAX2均可诱导Hela细胞凋亡并抑制其迁移,可降低Bcl-2、Cyclin D1、Survivin、MMP2、MMP9、β-catenin和p-β-catenin蛋白和升高BAX蛋白表达(P<0.05),可使Hela细胞中β-catenin的进核量减少。结论土槿皮乙酸在宫颈癌细胞中抑制PAX2表达和Wnt/β-catenin信号通路进而诱导细胞凋亡并抑制其转移。

AIM To investigate the effects of Pseudolaric acid B(PAB)on cervical cancer(CC)cells and its molecular mechanism via PAX2 regulation.METHODS Both MTT assay in the determinations of cells proliferation,and shRNA interference technology in the construction of weak PAX2 expression in Hela cells were performed.CC cells divided into blank group,negative control group,sh-PAX2 group,PAB group,PAB+sh-PAX2 group were subjected to determinations of cellular apoptosis by flow cytometry,determinations of cells migration ability by transwell chamber migration experiment,and had their potential protein of PAX2 predicted by String,their proteins expression identified by Western blot.And the localization ofβ-catenin was observed by immunofluorescence assay.RESULTS PAB inhibited CC cells proliferation in a time-and concentration-dependent manner.The expression of PAX2 protein in CC cells was negatively correlated with the IC50 of PAB on each cell(P<0.05).Inhibiting PAX2 protein expression in Hela cells,PAB worked in the way of weak PAX2 expression in inducing Hela cells apoptosis,inhibiting their migration,reducing BCL-2,Cyclin D1,Survivin,MMP2,MMP9,β-catenin and p-β-catenin protein and increasing BAX protein expression(P<0.05),and decreasing the amount ofβ-catenin in Hela cells nucleus.CONCLUSION PAB inhibits PAX2 expression and Wnt/β-catenin signaling pathway in CC cells,and therefore induces cells apoptosis and inhibits their metastasis.