Figure 1.
The effects of the putative miR-101 binding sites in the 3′-UTR of DNA-PKcs or ATM on the luciferase activity.
(A) The putative miR-101* binding site in the 3′-UTR of DNA-PKcs (WT). (B) The putative miR-101* or miR-101 binding sites in the 3′-UTR of ATM (WT1, WT2). (C) The effects of miR-101*-binding site in the 3′-UTR of DNA-PKcs on the luciferase activity. 293T cells were transfected with the firefly luciferase reporter plasmid containing partial 3′-UTR of DNA-PKcs with the putative miR-101* binding site (WT) or without the binding site (DM). Luciferase activity was assayed 48 h after transfection with the miR-101 mimic (miR-101) or without the mimic (mock), **, p<0.01. (D) The effects of miR-101* or miR-101-binding sites in the 3′-UTR of ATM on the luciferase activity. 293T cells were transfected with the firefly luciferase reporter plasmid containing partial 3′-UTR of ATM with the putative miR-101* (WT1) or miR-101 (WT2) binding site or without the binding site (DM1, DM2). Luciferase activity was assayed 48 h after transfection with the miR-101 mimic (miR-101) or without the mimic (mock), **, p<0.01.
Figure 2.
DNA-PKs and ATM as the targets of miR-101.
(A) Up-regulating miR-101 in 95C cells and U87GMD cells. The images reflect GFP signals, which represent the infection efficiencies of the lentivirus vectors. (B) The miR-101 level (including both strands: miR-101 and miR-101*) was measured by qRT-PCR in 95 C cells. (C) The miR-101 level (including both strands: miR-101 and miR-101*) was measured by qRT-PCR in U87GMD cells. (D) The effects of up-regulation of miR-101 on DNA-PKcs and ATM expression in 95C cells. Ku70 was used as an internal loading control. (E) The effects of up-regulation of miR-101 on DNA-PKcs and ATM expression in U87GMD cells. Ku70 was used as an internal loading control.
Figure 3.
Effects of up-regulation of miR-101 on the cell radiosensitivity.
(A) The effect of up-regulation of miR-101 on 95C cell radiosensitivity. The clonogenic assay was performed as described in Materials and Methods. Data shown are the mean and SE from three independent experiments. (B) The effect of up-regulation of miR-101 on U87GMD cell radiosensitivity. At 72 h after infection with the lentivirus encoding pri-miR-101, the cells were exposed to different doses. The clonogenic assay was performed as described above. Data shown are the mean and SE from three independent experiments. (C) The effects of the miR-101 or miR-101* inhibitor on the sensitivity of the miR-101 over-expressed U87MGD cells to IR. At 48 h after infection with the lentivirus encoding pri-miR-101, the cells were transfected with the inhibitor for an additional 36 h. The cells were exposed to 4 Gy and the clonogenic assay was performed as described above. Data shown are the mean and SE from three independent experiments, **, p<0.01.
Figure 4.
Effects of up-regulation of miR-101 on the xenograft radiosensitivity.
(A) Tumor size reflected the effects of miR-101 on the subcutaneous tumor radiosensitivity. Both hind legs of each nu/nu mouse were injected with the 95C cells with or without miR-101 up-regulated (5 mice were injected with the miR-101 up-regulated 95C cells and 5 mice were injected with the vector-transfected 95C cells, 10 mice total). The right hind leg that born the developed tumor was exposed to IR (5 Gy×2, at 72 h interval) at 12 days after the tumor cell injection and the left hind leg that born the developed tumor was used as the mock-irradiated control. The mice were sacrificed at 21 days after the tumor cell injection and the tumors were removed for weight comparison. (B) Tumor weight reflected the effects of miR-101 on the subcutaneous tumor radiosensitivity. The data shown are the mean and SE: *, p<0.05; **, p<0.01; ***, p<0.001. (C) Brain tumor that developed from U87MGD glioma cells and injected with the lentiviral vector. At 72 h after the viral vector was injected, the mice were sacrificed and the brain tissues were prepared for the pathological slides. The GFP signals were detected by a fluorescence microscope from the frozen samples. H&E staining was used for distinguishing the tumor and normal brain tissue from the formalin-fixed samples. (D) MRI reflected the effects of miR-101 on the brain tumor radiosensitivity. MRI scans of individual mouse brain 18 d after intracranial inoculation of U87GMD cells. The presence of a glioma (white arrows) was detected as the bright areas with an MRI contrast agent (Gd-DTPA). (E) Survival days reflected the effects of miR-101 on the brain tumor radiosensitivity. The data shown are the mean and SE; *, p<0.05.