miR-21拮抗剂、左炔诺孕酮对子宫内膜癌Ishikawa细胞增殖、凋亡的影响

Effects of miR-21 Antagonist and Levonorgestrel on Proliferation and Apoptosis of Endometrial Carcinoma Ishikawa Cells

目的探讨miR-21拮抗剂联合左炔诺孕酮(LNG)对子宫内膜癌Ishikawa细胞增殖、凋亡的影响。方法采用流式细胞仪检测在不同浓度、不同作用时间Ishikawa细胞的凋亡率,确定左炔诺酮最佳作用时间和浓度;将体外培养的Ishikawa细胞分为:Ishikawa组(Control组);Ishikawa+LNG处理组(LNG组);Ishikawa+LNG+miR-21拮抗剂处理组(LNG+miR-21组);Ishikawa+LNG+miR-21无关序列处理组(LNG+NC组),采用流式细胞法、细胞计数法(CCK-8)、Transwell实验及划痕实验检测各组Ishikawa细胞的凋亡、增殖、侵袭及迁移能力;采用Western blot检测各组Ishikawa细胞中PTEN、AKT、p-AKT、PI3K、p-PI3K蛋白表达水平。结果不同剂量左炔诺孕酮(LNG)对子宫内膜癌Ishikawa细胞作用24 h后,细胞凋亡率随着浓度增加而升高(P均<0.05),尤其100μmol·L^(-1)浓度存活率最低;100μmol·L^(-1)的LNG作用下,转染miR-21拮抗剂/转染miR-21无关序列,发现子宫内膜癌细胞Ishikawa的增殖、迁移、侵袭能力及活性均降低(P均<0.05),同时使PTEN、AKT、PI3K等蛋白表达升高,使p-AKT,p-PI3K蛋白表达降低(P均<0.05)。结论miR-21拮抗剂联合左炔诺孕酮可以促进子宫内膜癌细胞Ishikawa凋亡,抑制子宫内膜癌细胞Ishikawa增殖。

Objective To investigate the effects of miR-21 antagonist combined with levonorgestrel(LNG)on proliferation and apoptosis of endometrial cancer Ishikawa cells.Methods The apoptotic rate of Ishikawa cells at different LNG concentrations and different time was detected via flow cytometry to determine the optimal time and concentration of LNG;Ishikawa cells cultured in vitro were divided into Ishikawa group(Control group),Ishikawa+LNG treatment group(LNG group),Ishikawa+LNG+miR-21 antagonist treatment group(LNG+miR-21 group)and Ishikawa+LNG+miR-21 unrelated sequence treatment group(LNG+NC group).Flow cytometry,cell counting method(CCK-8),Transwell experiment and scratch experiment were adopted to detect the apoptosis,proliferation,invasion and migration ability of Ishikawa cells in each group.Western blot was used to detect PTEN,AKT,p-AKT,PI3K,p-PI3K gene expression levels in each group.Results After different doses of LNG were added to endometrial cancer Ishikawa cells for 24 h in each group,the apoptotic rate increased significantly with the increase in concentration(P all<0.05).Especially for 100μmol·L^(-1) LNG,the lowest survival rate was obtained.In 100μmol·L^(-1) LNG subgroup,the miR-21 antagonist/miR-21 unrelated sequence was transfected and showed the proliferation,migration,invasion ability and activity of endometrial cancer cell Ishikawa reduced(P all<0.05).Meanwhile,the expression of PTEN,p-AKT,p-PI3K and other genes were increased(P all<0.05).Conclusion MiR-21 antagonist combined with levonorgestrel can promote the apoptosis of endometrial cancer cell Ishikawa cells,and inhibit the proliferation of endometrial cancer Ishikawa cells.