多烯紫杉醇联合反义miR-21对人胰腺癌细胞增殖和凋亡的影响

摘要

目的 探讨多烯紫杉醇(DTX)联合反义miR-21(AS-miR-21)对胰腺癌细胞增殖及凋亡的影响及其可能的作用机制.方法 采用7种浓度DTX筛选对人胰腺癌MiaPaCa-2细胞增殖抑制最佳的IC50(15 μmol/L)进行后续试验.随机将MiaPaCa-2细胞分为空白对照组、无义序列组、DTX组、AS-miR-21组、DTX+ AS-miR-21组.AS-miR-21组、DTX+ AS-miR-21组转染AS-miR-21,无义序列组转染Lipofectamine2000,均转染24h;DTX+ AS-miR-21组转染后予15 μmol/L的DTX作用48 h;空白对照组未行任何处理,DTX组加入15 μmol/L的DTX作用48 h.收集各组细胞,RT-PCR法检测miR-21的相对表达量,克隆形成实验检测细胞增殖能力,流式细胞仪检测细胞早期凋亡率,Western blotting法检测细胞周期依赖性蛋白激酶5(CDK5)的相对表达量.结果 与无义序列组、空白对照组比较,AS-miR-21组、AS-miR-21+ DTX组miR-21的相对表达量均明显降低(P均＜0.05).与空白对照组、无义序列组比较,DTX组、AS-miR-21组、DTX+ AS-miR-21组克隆数目明显降低、早期凋亡率明显升高、CDK5的相对表达量明显降低,以DTX+ AS-miR-21组变化最明显(P均＜0.05).结论 DTX联合AS-miR-21可明显抑制胰腺癌细胞增殖并促进其早期凋亡,其机制可能与CDK5表达下降有关.%Objective To investigate the effect of docetaxel combined with antisense miR-21 on proliferation and apoptosis of human pancreatic cancer cell line MiaPaCa-2,and to discuss its possible mechanism.Methods Seven concentrations of docetaxel (DTX) were selected to detect their proliferation inhibition rates on human pancreatic cancer MiaPaCa2 cells to choose the best IC5o (15 μmol/L) which was then used to do the following experiment.Human pancreatic cancer MiaPaCa-2 cells were divided into the control group,antisense miR-21 group,DTX group,AS-miR-21 group,and DTX + AS-miR-21 group.Cells in the AS-miR-21 group and DTX + AS-miR-21 group were transfected by AS-miR-21,and the antisense miR-21 group was transfected by Lipofectamine2000 for 24 h.Cells in the DTX + AS-miR-21 group were treated with DTX,and DTX group was treated with 15 μmol/L DTX for 48 h.RT-PCR was used to detect the expression of miR21 in each group.The proliferation capacity of cells was detected by the cloning formation assay.The apoptosis rate was measured by flow cytometry.The expression of cyclindependent kinase 5 (CDK5) in each group was evaluated by Western blotting.Results The relative expression of miR-21 in the AS-miR-21 group and DTX + AS miR-21 group was significantly lower than that in the control group,and antisense miR-21 group,which indicated that the transfection was successful.Compared with the control group and antisense miR-21 group,the number of clones in the DTX group,AS-miR-21 group and DTX + AS-miR-21 group was significantly decreased,the early apoptosis rate was significantly increased,and the relative expression of CDK5 protein was significantly decreased,and the most significant changes were found in the DTX + AS-miR-21 group (all P ＜0.05).Conclusion DTX combined with AS-miR-21 can significantly inhibit the proliferation of human pancreatic cancer cells and induce the apoptosis,which may be associated with the decrease of CDK5 protein expression.