miR-182对乳腺癌MCF-7细胞增殖和凋亡的影响研究

Effect of microRNA-182 on proliferation and apoptosis of breast cancer MCF-7 cells

目的探讨微小核糖核酸-182(miR-182)对乳腺癌MCF-7细胞增殖、凋亡及磷酸酶和张力蛋白同源基因(PTEN)/三磷酸磷脂酰肌醇激酶(PI3K)/蛋白激酶B(Akt)通路的影响。方法人乳腺癌MCF-7细胞分为miR-182组、anti-miR-182组、miR-NC组和对照组,分别转染miR-182拟似物、miR-182阻遏物、miR-182阴性对照物和空白试剂,每组设置6个复孔。用噻唑蓝法检测MCF-7细胞增殖情况,用流式细胞仪检测MCF-7细胞凋亡情况,用实时荧光定量逆转录聚合酶链反应测定MCF-7细胞miR-182和PTEN mRNA的表达,用蛋白质印迹法测定MCF-7细胞PTEN和p-AKT蛋白表达。结果培养24 h时,对照组、miR-182组、anti-miR-182组和miR-NC组的OD值分别0.58±0.21,0.73±0.19,0.41±0.16和0.56±0.17;培养48 h时,上述4组OD值分别为0.87±0.19,1.32±0.31,0.49±0.14和0.83±0.18;培养72 h时,上述4组OD值分别为1.33±0.38,2.08±0.41,0.58±0.19和1.28±0.31,anti-miR-182组的上述指标与其他组相比,差异均有统计学意义(均P<0.05)。对照组、miR-NC组、anti-miR-182组和miR-182组的细胞凋亡率分别为(3.59±0.54)%,(3.64±0.73)%,(8.23±0.82)%和(2.12±0.47)%,miR-182组和arti-miR-182组与其他组相比,差异均有统计学意义(均P<0.05)。anti-miR-182组、对照组、miR-NC组、miR-182组的miR-182 mRNA相对表达量分别为0.24±0.11,0.62±0.16,0.65±0.13,0.87±0.09,PTEN mRNA相对表达量分别为0.72±0.16,0.35±0.08,0.34±0.09,0.12±0.06,PTEN蛋白相对表达量分别为0.73±0.11,0.55±0.09,0.52±0.09,0.35±0.06,p-AKT蛋白相对表达量分别为0.28±0.09,0.48±0.10,0.49±0.08,0.69±0.06,miR-182组和anti-miR-182组与其他组别比较,差异均有统计学意义(均P<0.05)。结论miR-182可能通过降低抑癌基因PTEN表达,激活PI3P/AKT信号通路促进乳腺癌细胞增殖和抑制其凋亡,在乳腺癌发生发展中发挥重要作用,提示抑制miR-182进而靶向PTEN/PI3K/AKT信号通路可能是乳腺癌治疗的有效途径。

Objective To investigate the effects of microRNA-182(miR-182)on proliferation,apoptosis and phosphatase and tensinhomolog deleted on chromosome 10(PTEN)/phosphatidylinositol triphosphate kinase(PI3K)/protein kinase B(Akt)pathway of breast cancer MCF-7 cells.Methods Human breast cancer MCF-7 cells were divided into microRNA-182 group,anti-microRNA-182 group,microRNA-NC group and control group,microR-NA-182 mimimetic,microRNA-182 repressor,microRNA-182 negative control substance and blank reagent were transfected respectively.Six replicates were set in each group.Thiazolyl blue tetrazolium bromide method was used to detect the proliferation of MCF-7 cells,flow cytometry was used to detect the apoptosis of MCF-7 cells,quantitative real-time polymerase chain reaction was used to detect the expression of miR-182 and PTEN mRNA in MCF-7cells,and Western blot was used to detect the expression of PTEN and p-AKT protein in MCF-7 cells.Results At24 h,the OD values of control group,miR-182 group,anti miR-182 group and miR-NC group were 0.58±0.21,0.73±0.19,0.41±0.16 and 0.56±0.17,at 48 h,the OD values were 0.87±0.19,1.32±0.31,0.49±0.14and 0.83±0.18,at 72 h,the OD values were 1.33±0.38,2.08±0.41,0.58±0.19 and 1.28±0.31,all with significant difference(all P<0.05).The apoptotic rates of control group,miR-NC group,anti-miR-182 group and miR-182 group were(3.59±0.54)%,(3.64±0.73)%,(8.23±0.82)%and(2.12±0.47)%,with significant difference(all P<0.05).The relative expressions of miR-182 mRNA in anti-miR-182 group,control group,miR-NC group and miR-182 group were 0.24±0.11,0.62±0.16,0.65±0.13,0.87±0.09,the relative expression levels of PTEN mRNA were 0.72±0.16,0.35±0.08,0.34±0.09 and 0.12±0.06,the relative expression levels of PTEN protein were 0.73±0.11,0.55±0.09,0.52±0.09,0.35±0.06,the relative expression of pAKT protein were 0.28±0.09,0.48±0.10,0.49±0.08 and 0.69±0.06.Compared with other groups,there were significant diffe-rences in microRNA-182 and anti-microRNA-182(all P<0.05).Conclusion MiR-182 may play an important role in the development of breast cancer by reducing the expression of PTEN and activating PI3P/AKT signaling pathway,suggesting that inhibiting the expression of miR-182 and targeting PTEN/PI3K/AKT signaling pathway may be an effective way to treat breast cancer.