微小RNA-96靶向叉头框转录因子O1促进肺癌细胞增殖和迁移

MicroRNA-96 promotes the proliferation and migration of lung cancer cells by targeting forkhead box O1

目的探讨微小RNA(miRNA,miR)-96通过靶向叉头框转录因子O1(FOXO1)促进肺癌细胞增殖和迁移的作用。方法应用茎环反转录荧光定量聚合酶链反应(RT-qPCR)法检测miR-96在肺癌细胞株中的表达;干扰慢病毒感染抑制miR-96表达后,分别利用细胞增殖试验(CCK-8)和划痕试验检测其对肺癌细胞增殖和迁移的影响;蛋白质印迹法(Western blot)和双荧光素酶活性试验验证miR-96靶基因FOXO1。应用GraphPad Prism 5统计学软件进行分析,计量资料比较采用t检验。结果CCK-8实验显示抑制H1299细胞miR-96表达后,实验组24、48、72 h吸光度值分别为1.293±0.019、1.684±0.038、2.180±0.034,明显低于对照组的1.655±0.056、1.880±0.020、2.493±0.060,差异有统计学意义(t=5.646、4.426、14.670,P<0.01);划痕试验显示对照组4 h和10 h细胞迁移率为(29.21±3.96)%、(47.49±3.33)%,明显高于实验组[(5.80±2.94)%、(13.63±3.18)%],差异有统计学意义(t=4.748、7.352,P<0.01);筛选miR-96靶基因FOXO1,将miR-96过表达后,肺癌细胞FOXO1表达明显降低;反之,抑制miR-96表达后,FOXO1表达明显增高。荧光素酶活性试验显示miR-96和FOXO13’非翻译区(3’UTR)野生型质粒共转染后,野生型为(22.5±7.6)%,与对照组的(97.2±13.2)%及突变型的(115.6±13.5)%比较,荧火虫/海肾荧光素酶比值明显降低(t=14.750、12.040,P值均<0.01)。结论miR-96通过靶向调控FOXO1促进肺癌细胞增殖和迁移。

Objective To investigate the role of microRNA(miRNA,miR)-96 in promoting the proliferation and migration of lung cancer cells by targeting forkhead box O1(FOXO1).Methods The miR-96 expression in lung cancer cell lines was detected by stem-loop reverse transcription-quantitative polymerase chain reaction(RT-qPCR).Silencing lentiviral miR-96 was used to infect with lung cancer cells and then the proliferation and migration ability of lung cancer cells was evaluated by cell counting kit(CCK)-8 assay and scratch test,respectively.Western blotting and dual luciferase activity assays was utilized to validate the target gene FOXO1 of miR-96.GraphPad Prism 5 statistical software was used,and the t-test was adopted to compare the difference of the measurement data.Results CCK-8 assay showed that after inhibiting the expression of miR-96 in H1299 cells,the optical density(OD)at 24,48 and 72 h was 1.293±0.019,1.684±0.038,and 2.180±0.034,which was lower than the controls of 1.655±0.056,1.880±0.020,and 2.493±0.060(t=5.646,4.426,and 14.670,P<0.01,0.01,and 0.01,respectively).Moreover,the scratch test showed that the cell migration rate of controls was(29.21±3.96)%and(47.49±3.33)%,higher than in the experimental group of(5.80±2.94)%and(13.63±3.18)%(t=4.748,7.352,P<0.01,0.01,respectively).We screened the target gene of miR-96 and found that upregulation of miR-96 significantly downregulated the FOXO1 expression in lung cancer cells.In contrast,FOXO1 expression was significantly increased after inhibition of miR-96 expression.Luciferase activity assay revealed that co-transfection of miR-96 and FOXO13’untranslated region(3’UTR)wild type plasmid had lower ratio of firefly/renilla luciferase(22.5±7.6)%than co-trasfection with the mutant type plasmid(115.6±13.5)%and controls(97.2±13.2)%(t=14.750,12.040,P all<0.01).Conclusion miR-96 promotes the proliferation and migration of lung cancer cells by targeting FOXO1,which might serve as potential diagnosis and therapy target,however,the detailed mechanism should be studied in future.