Inhibition Of Akt Pathway By MiR-221/222Antisense Oligonucleotides Enhances The Radiosensitivity Of Glioblastoma In Vitro And In Vivo

Objective:Glioblastoma grows aggressively, which is difficult to be completely excised by surgery. Postoperative radiotherapy is an important treatment of glioblastoma. However, the therapeutic effect of radiotherapy is not good enough because of the radiation resistance of glioblastoma. People are trying to explore the mechanism and therapies of glioblastoma ceaselessly. Recently, it is demonstrated that there is a distinct difference of the expression of microRNAs (miRs) between glioblastoma and normal brain tissues. Among which the expression of miR-221and miR-222(miR-221/222)is significant different than that in normal brain tissues. Meanwhile, it is reported in some literature that miR-221/222performs its function through Akt pathway. Therefore, we plan to enhance the radiation-sensitizing effect of inhibiting of Akt pathway by knocking down miR-221/222in the human glioblastoma cells.Methods:1. In vitro experiment:U251and LN-229cells were cultured in DMEM media. These two kinds of cells were divided into three groups respectively:the control group, the antisense sequence transfected cell group and the antisense miR-221/222ODNs transfected cell group. miR-221/222antisense oligonucleotides (antisense miR-221/222) were transfected by Lipofectamine2000:The total RNA of each group cells were extracted by Trizol. The expression of miR-221and miR-222in transfected cells were measured by Northern blot analysis;Cells of each group were exposed to0Gy,2Gy,4Gy and6Gy X-ray(source skin distance was100cm, dose rate was320cGy/min, Varian600C. accelerator6MV). Each dose points of each group were repeated3times. Clonogenic assay was used to measure its radiosensitivity. The expression changes of pAkt protein in each cell group were examined by Western blot analysis.2. In vivo experiment:U251nude mice subcutaneous tumor-burdened model was established. These32subcutaneous tumor-burdened nude mice were divided into four groups:control group, irradiated group, antisense miR-221/222ODNs transfected group and antisense miR-221/222ODNs transfected plus irradiation group. Each group had8mice, and the mean tumour volume of each group was50mm3:The subcutaneously transplanted tumor of nude mice was treated by antisense miR-221/222ODNs transfected plus X-ray irradiation, then observed the growth levels of tumour; The expression levels of miR-221and miR-222after therapy were detected by Real-time PCR; The expression levels of pAkt protein in subcutaneous transplanted tumor tissue of each group were analyzed by immunohistochemistry.Results:The expression level of miR-221/222was significantly decreased in cells transfected with antisense miR-221/222as compared to the parental cells or cells transfected with scrambled ODN.AS-miR-221/222combined with irradiation could syncrgistically enhance its radiosensitivity. The expression of pAkt was down-regulated in the AS-miR-221/222group revealed by Western blot analysis. The growth of xenograft tumors treated with antisense miR-221/222and X-ray radiation were also inhibited.In antisense miR-221/222and X-ray radiation treated tumor tissue.the expression of miR-221/222and pAkt was down-regulated.Conclusions:The expression levels of miR-221and miR-222in glioblastoma cells were knocked down by antisense oligonucleotides transfected by lipofectamine. The results were checked by Northern blot analysis. Clonogenic assay showed that the cell colony formation rate was reduced significantly by antisense miR-221/222ODNs transfected plus X-ray irradiation, which means that the proliferative death risk and the radiosensitivity of glioblastoma cells can be obviously increased by combination of these two treatments. The result of Western bolt analysis indicated that the expression of pAkt protein remarkably downregulated after knocking down the expression of miR-221and miR-222in glioblastoma cells. The vitro experiment results were confirmed in vivo experiments as well. The growth of subcutaneously sransplanted tumor of nude mice was obviously suppressed after the treatment of antisense miR-221/222ODNs transfected plus X-ray irradiation. Furthermore, the expression levels of miR-221/222and pAkt protein in tumour tissue significantly decreased. All of the evidence shows that the improvement of radiation sensitivity of glioblastoma is closely related to the expression of pAkt protein after transfection of antisense miR-221/222ODNs. Consequently, we can think that AS-miR-221/222may enhance the radiosensitivity of glioblastoma by suppressing the Akt pathway. It provides new ideas for future radiotherapy combined with gene therapy of glioblastoma.