茯苓酸通过上调miR-133a影响子宫肌瘤细胞增殖凋亡及表皮生长因子的水平

Pacymic acid affects proliferation,apoptosis and epidermal growth factor levels in uterine fibroid cells by up-regulating miR-133a

目的:探讨茯苓酸(PA)对子宫肌瘤细胞增殖、凋亡及表皮生长因子(EGF)水平的影响。方法:选取58例子宫肌瘤患者的子宫肌瘤组织进行细胞原代培养。将子宫肌瘤细胞分为NC组、PA 10μg/ml组、PA 20μg/ml组、PA 40μg/ml组、PA 80μg/ml组。采用甲基噻唑基四唑(MTT)检测各组细胞存活率,筛选PA适宜浓度进行后续实验。采用酶联免疫吸附(ELISA)法检测PA对EGF水平的影响;蛋白免疫印迹法(Western blot)检测PA对表皮生长因子受体(EGFR)蛋白表达的影响;采用实时荧光定量聚合酶链反应(qRT-PCR)检测PA对细胞中微小RNA-133a(miR-133a)表达水平的影响。miR-133a mimics及阴性对照miR-NC转染至子宫肌瘤细胞,观察miR-133a过表达对子宫肌瘤细胞增殖、凋亡及EGF、EGFR表达水平的影响。Western blot检测细胞周期蛋白1(CyclinD1)、活化的含半胱氨酸的天冬氨酸蛋白水解酶3(C-caspase-3)蛋白表达。结果:与NC组比较,PA 10μg/ml组、PA 20μg/ml组、PA 40μg/ml组、PA 80μg/ml组细胞存活率均显著降低(P<0.05),选用PA 40μg/ml作为后续研究。与NC组比较,PA组细胞凋亡率显著升高(P<0.05),C-caspase-3蛋白表达水平显著升高(P<0.05),EGFR蛋白表达水平显著降低(P<0.05),而EGF水平显著降低(P<0.05);与NC组比较,PA组细胞中miR-133a的表达水平显著升高(P<0.05);与miR-NC组比较,miR-133a组细胞存活率显著降低(P<0.05),CyclinD1、EGFR蛋白表达水平显著降低(P<0.05),EGF水平显著降低(P<0.05),而细胞凋亡率显著升高(P<0.05),C-caspase-3蛋白表达水平显著升高(P<0.05)。抑制miR-133a的表达可逆转PA对子宫肌瘤细胞增殖、凋亡及EGF的影响。结论:PA可通过上调miR-133a的表达抑制子宫肌瘤细胞增殖并促进细胞凋亡,同时降低EGF、EGFR表达水平。

Objective:To investigate the effect of PA on the proliferation,apoptosis and epidermal growth factor(EGF) levels of uterine fibroid cells.Methods:The uterine fibroid tissues of 58 patients with uterine fibroids were selected for primary culture.Uterine fibroid cells were divided into NC group,PA 10 μg/ml group,PA 20 μg/ml group,PA 40 μg/ml group,and PA 80 μg/ml group.The cell survival rate of each group was detected by MTT,and the suitable concentration of PA was screened for subsequent experiments.The effect of PA on the level of EGF was measured by enzyme-linked immunosorbent assay(ELISA).The effect of PA on the expression of epidermal growth factor receptor(EGFR) protein was detected by Western blot.The effect of PA on the expression level of microRNA-133 a(miR-133 a) in cells was detected by qRT-PCR.miR-133 a mimics and negative control miR-NC were transfected into uterine fibroid cells,and the effect of miR-133 a overexpression on proliferation,apoptosis and expression of EGF and EGFR in uterine fibroid cells was observed.The expression of CyclinD1 and C-caspase-3 protein was detected by Western blot.Results:Compared with NC group,the cell viability of PA 10 μg/ml group,PA 20 μg/ml group,PA 40 μg/ml group,and PA 80 μg/ml group were significantly decreased(P<0.05),and PA 40 μg/ml was selected as a follow-up study.Compared with NC group,the apoptosis rate of PA group was significantly decreesed(P<0.05),the expression level of C-caspase-3 protein was significantly increased(P<0.05),and the expression level of EGFR protein was significantly decreased(P<0.05),the level of EGF was significantly increased(P<0.05).Compared with NC group,the expression of miR-133 a in PA group was significantly decreased(P<0.05).Compared with miR-NC group,the cell survival rate of miR-133 a group was significantly decreased(P<0.05),the expression of CyclinD1,EGFR protein was significantly decreased(P<0.05),and the level of EGF was significantly decreased(P<0.05),but the apoptosis rate was significantly increased(P<0.05),and the expression level of C-caspase-3 protein was significantly increased(P<0.05).Inhibition of miR-133 a expression reversed the effects of PA on proliferation,apoptosis and EGF of uterine fibroid cells.Conclusion:PA can inhibit the proliferation of uterine fibroid cells and promote apoptosis by up-regulating the expression of miR-133 a,and reduce the expression levels of EGF and EGFR.