Objective: 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.
Methods: 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.
Results: 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.
Conclusion: Anti-miR-221/222 may enhance the radiosensitivity of U251 human glioblastoma through upregulation of p27(kip1).