反义miR-221/222上调p27~(kipl)对胶质母细胞瘤U251的放射增敏作用

目的 探讨敲低微小RNA(micro RNA,miRs)-221/222表达上调p27~(kipl)对U251胶质母细胞瘤细胞系放射敏感性的影响.方法 经生物信息学分析查询miR-221/222成熟体序列和它们与p27~(kipl)的关系.脂质体共转染反义寡聚核苷酸(反义miR-221/222)下调U251胶质母细胞瘤细胞系miR-221与miR-222的表达.使用Northern印迹方法鉴定转染后U251细胞miR-221、miR-222表达水平下调;流式细胞术检测转染后U251联合X线照射的细胞周期分布;克隆形成实验验证各组细胞增殖性死亡;Western 印迹分析p27kipl蛋白的表达变化.结果 经生物信息学分析显示miR-221/222成熟体序列的种子序列几乎一致,p27~(kipl)是miR-221/222的靶基因.Northern印迹分析显示反义miR-221/222共转染组使miR-221/miR-222的表达水平明显下降.转染无义序列组及对照组的miR-221/miR-222表达水平没有改变.流式细胞术检测可见反义miR-221/222共转染组细胞周期阻滞于G0/G1期且明显高于其它各组.经X线照射后,可明显降低S期比例.反义miR-221/222联合X线照射可增加U251细胞增殖性死亡.Western印迹分析显示反义miR-221/222共转染组的p27~(kipl)蛋白表达明显上调.结论 反义miR-221/222通过上调p27kipl蛋白表达可增加U251胶质母细胞瘤细胞系的放射敏感性.

Up-regulation of p27~(kipl) by miR-221/222 antisense oligonucleotides enhances the radiosensitivity of U251 glioblastoma

Objective To study the radiation-sensitizing effect of up-regulating p27~(kipl) expression by knocking down miR-221/222 in the U251 human glioblastoma cell line.MethodsBy bioinformatic analysis,we searched the miRNA-221/222 sequence and found the relationship between p27~(kipl) and miRNA221/222.miRNA-221/222 antisense oligonucleotides were transfected into U251 human glioblastoma cells.Northern blot analysis was conducted to detect the expression of miR-221/222 in control,scrambled o1igonucleotide(ODN)transfected and anti-miR-221/222ODNs transfected cell groups.The cell cycle kinetics was detected by flow cytometry.Clonogenic assay was used to measure the mitotic cell death and p27~(kipl) expression was examined bv Western blot analysis.Results Based on bioinformatic analysis,we found that the seed sequences of miR-221 and miR-222 coincide with each other,and p27~(kipl) is a target for miRNA-221/222.The expression level of miR-221/222 was significantly knocked down in cells transfected with antimiR-221/222 as compared to the parental cells or cells transfected with scrambled ODN.Cell cycle was arrested in G0/G1 phase in the anti-miR-221/222 group.When combined with irradiation,S phase fraction in the anti-miR-221/222 cell group is lower than that in the other two control groups.Anti-miR221/222 combined with irradiation could synergistically enhance mitotic cell death.The expression of p27~(kipl) was up-regulated in the anti-miR-221/222 group revealed by Western blot analysis.Conclusion Anti-miR221/222 may enhance the radiosensitivity of U251 human glioblastoma through upregulation of p27~(kipl).