Zhang Chunzhi, Wang Guangxiu, Kang Chunsheng, Du Yue, Pu Peiyu
Department of Neurosurgery, Tianjin Medical University General Hospital and Laboratory of Neuro-Oncology, Tianjin Neurological Institute, Tianjin, 300052 P. R. China.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi. 2009 Dec;26(6):634-8. doi: 10.3760/cma.j.issn.1003-9406.2009.06.006.
To study the radiation-sensitizing effect of up-regulating p27(kip1) expression by knocking down miR-221/222 in the U251 human glioblastoma cell line.
By bioinformatic analysis, we searched the miRNA-221/222 sequence and found the relationship between p27(kip1) and miRNA-221/222. 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 oligonucleotide (ODN) transfected and anti-mi-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(kip1) expression was examined by Western blot analysis.
Based on bioinformatic analysis, we found that the seed sequences of miR-221 and miR-222 coincide with each other, and p27(kip1) 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 or 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 p27(kip1) was up regulated in the anti-miR-221/222 group revealed by Western blot analysis.
Anti-miR-221/222 may enhance the radiosensitivity of U251 human glioblastoma through upregulation of p27(kip1).
研究在U251人胶质母细胞瘤细胞系中通过敲低miR-221/222上调p27(kip1)表达的辐射增敏作用。
通过生物信息学分析，搜索miRNA-221/222序列，发现p27(kip1)与miRNA-221/222之间的关系。将miRNA-221/222反义寡核苷酸转染至U251人胶质母细胞瘤细胞中。进行Northern印迹分析以检测对照组、乱序寡核苷酸（ODN）转染组和抗mi-221/222 ODNs转染细胞组中miR-221/222的表达。通过流式细胞术检测细胞周期动力学。采用克隆形成试验测量有丝分裂细胞死亡情况，并通过蛋白质印迹分析检测p27(kip1)的表达。
基于生物信息学分析，我们发现miR-221和miR-222的种子序列相互一致，且p27(kip1)是miRNA-221/222的一个靶点。与亲本细胞或乱序ODN转染的细胞相比，用抗miR-221/222转染的细胞中miR-221/222的表达水平显著降低。抗miR-221/222组细胞周期阻滞于G0或G1期。与辐射联合时，抗miR-221/222细胞组的S期比例低于其他两个对照组。抗miR-221/222与辐射联合可协同增强有丝分裂细胞死亡。蛋白质印迹分析显示抗miR-221/222组中p27(kip1)的表达上调。
抗miR-221/222可能通过上调p27(kip1)增强U251人胶质母细胞瘤的放射敏感性。
