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

Effect of docetaxel combined with antisense miR-21 on proliferation and apoptosis of human pancreatic cancer cell

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Mia Pa Ca-2细胞增殖抑制最佳的IC50(15μmol/L)进行后续试验。随机将Mia Pa Ca-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。ASmiR-21组、DTX+AS-miR-21组转染AS-miR-21,无义序列组转染Lipofectamine2000,均转染24 h;DTX+AS-miR-21组转染后予15μmol/L的DTX作用48 h;空白对照组未行任何处理,DTX组加入15μmol/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 ap- optosis of human pancreatic cancer cell line MiaPaCa-2, and to discuss its possible mechanism. Methods Seven concen- trations of docetaxel （DTX） were selected to detect their proliferation inhibition rates on human pancreatic cancer MiaPaCa- 2 cells to choose the best ICs0 （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 an- tisense 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 miR- 21 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 df miR-21 in the AS-miR-21 group and DTX ＋ AS miR-21 group was significant- ly 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 ex- pression 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 pan-creatic cancer cells and induce the apoptosis, which may be associated with the decrease of CDK5 protein expression.