miR-370通过FoxM1相关通路调控人视网膜母细胞瘤细胞凋亡机制研究

Mechanism of miR-370 regulating apoptosis of human retinoblastoma cell via FoxM1-related pathway

目的探讨miR-370对人视网膜母细胞瘤细胞Y79增殖和凋亡的影响及其可能的作用机制。方法通过Lipofectamine TM 2000将miR-370-mimics、miR-370-NC及miR-370-inhibitor转染至Y79细胞内,分别建立miR-370过表达组(mimics组)、对照组(NC组)和抑制组(inhibitor组),三组转染24 h、48 h、72 h和96 h时,均使用CCK-8检测各组细胞增殖活性;转染48 h后,RT-PCR检测各组细胞内miR-370表达;Annexin V-FITC/PI联合流式细胞仪检测各组细胞凋亡率,Western Blot检测各组细胞PI3K、P-AKT、FoxM1蛋白表达。结果mimics组细胞miR-370 mRNA表达远高于其他两组(均为P<0.05),inhibitor组细胞miR-370 mRNA表达显著低于其他两组(均为P<0.05)。自转染48 h起,inhibitor组细胞增殖活性显著高于其他两组(均为P<0.05),而mimics组细胞增殖活性显著低于其他两组(均为P<0.05),这种趋势一直持续至转染后96 h。mimics组细胞早期凋亡率显著高于其它两组(均为P<0.05),而inhibitor组细胞早期凋亡率在三组中最低(P<0.05)。mimics组细胞内PI3K、P-AKT、FoxM1蛋白表达均显著低于其他两组(均为P<0.05),而inhibitor组细胞内PI3K、P-AKT、FoxM1蛋白表达显著高于其他两组(均为P<0.05)。结论miR-370能够抑制人视网膜母细胞瘤细胞Y79的增殖,促进其凋亡,这种影响可能是通过降低PI3K/AKT/FoxM1信号通路的活性实现的。

Objective To investigate the effect of miR-370 on proliferation and apoptosis of human retinoblastoma cell line Y79,and its possible mechanism.Methods miR-370-mimics,miR-370-NC and miR-370-inhibitor were transfected into Y79 cells by Lipofectamine TM 2000,to establish miR-370 over-expression group(mimics group),normal control group(NC group)and inhibitor group.CCK-8 was used to detect the proliferative activity in each group at the 24 h,48 h,72 h and 96 h after transfection.RT-PCR was used to detect the expression of miR-370 in the cells at the 48 h after transfection.The apoptosis rate in each group was detected by Annexin V-FITC/PI with flow cytometry.The protein expression of PI3K,P-AKT and FoxM1 were detected by Western Blot.Results The expression of miR-370 mRNA was much higher in the mimics group than that in the other two groups(both P<0.05),and significantly lower in the inhibitor group than that in the other two groups(both P<0.05).Since 48 hours after transfection,proliferative activity was significantly higher in the inhibitor group than that in the other two groups(both P<0.05),while significantly lower in the mimics group than that in the other two groups(all P<0.05).This trend continued until 96 hours after transfection.The early apoptosis rate was the highest in the mimics group,and the lowest in the inhibitor group(all P<0.05).The protein expression of PI3K,P-AKT and FoxM1 were significantly lower in the mimics group than those in the other two groups(all P<0.05),while were significantly higher in the inhibitor group than those in the other two groups(all P<0.05).Conclusion miR-370 can inhibit the proliferation of human retinoblastoma cell line Y79 and promote its apoptosis,which may be achieved by lowering the activity of PI3K/AKT/FoxM1 signaling pathway.