摘要
目的探讨敲低微小RNA（microRNA，miRs）-221／222表达上调p27kp1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p27kp1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p27kp1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，p27kp1是miR-221／222的靶基因。Northern印迹分析显示反义miR221／222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／ee2共转染组的p27kp1蛋白表达明显上调。结论反义miR-221／222通过上调p27kp1蛋白表达可增加U251胶质母细胞瘤细胞系的放射敏感性。
Objective To study the radiation-sensitizing effect of up regulating p27kp1 expression by knocking down miR-221/222 in the U251 human glioblastoma cell line. Methods By bioinformatic analysis, we searched the miRNA-221/222 sequence and found the relationship between p27kp1 and miRNA- 221/222. miRNA-221/222 antisense oligonucleotides were transfected into U25I human glioblastoma cells. Northern blot analysis was conducted to detect the expression of miR-221/222 in control, scrambled oligonucleotide （ODN） transfected and anti-miR 221/2220DNs transfected cell groups. The cell cycle kinetics was detected by flow cytometry. Clonogenic assay was used to measure the mitotic cell death and p27kp1 expression was examined by 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 p27kp1 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 miR- 221/222 combined with irradiation could synergistically enhance mitotic cell death. The expression of p27kip1 was up-regulated in the anti-miR-221/222 group revealed by Western blot analysis. Conclusion Anti-miR- 221/222 may enhance the radiosensitivity of U251 human glioblastoma through upregulation of p27kp1.
出处
《中华医学遗传学杂志》
CAS
CSCD
北大核心
2009年第6期634-638,共5页
Chinese Journal of Medical Genetics
基金
基金项目：教育部新世纪优秀人才支持计划（NCET-070615）
国家自然科学基金（30772231）
天津市科技计划项目（07ZCGHHZ1000）
