Anti-miRNA-221 sensitizes human colorectal carcinoma cells to radiation by upregulating PTEN

Figure 1 Modulation of miR-221 expression and cell proliferation in Caco2 cells. A: Expression of miR-221 was detected by real-time RT-PCR. Expression of U6 snRNA was used as internal control; B: The status of cell proliferation was determined by MTT assay. CPIR in the presence of pre-miR-221 or anti-miR-221 was compared with those of controls. n = 6, mean ± SD; ^bP < 0.01 vs control group.

Figure 2 miR-221 regulated expression of phosphatase and tensin homolog deleted on chromosome 10 in colorectal carcinoma-derived cells. A: Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) 3’-UTR site potentially targeted by miR-221 as predicted by Miranda; B: Luciferase activity assay showing direct interaction between miR-221 and PTEN 3’-UTR site. Firefly luciferase reporter activity in the presence of both pGL3-PTEN vector and pre-miR-221 or anti-miR-221 was compared with that of the controls. Luciferase activity in Caco2 cells cotransfected with pGL3-PTEN vector and pre-miR-221 was decreased markedly compared with the negative control. Luciferase activity in Caco2 cells transfected with anti-miR-221 was increased significantly compared with the negative control, ^bP < 0.01 vs control group; C: Western blotting showing PTEN protein expression in Caco2 cells transfected with pre-miR-221 or anti-miR-221. β-actin was used as a housekeeping gene to normalize PTEN protein expression. The relative PTEN protein levels normalized against β-actin are shown at the top of each panel; D: Real-time RT-PCR analysis showing PTEN mRNA expression in Caco2 cells transfected with pre-miR-221 or anti-miR-221. All the results are representative of three independent experiments.

Figure 3 Effects of X-ray dose on miR-221 and phosphatase and tensin homolog deleted on chromosome 10 expression in colorectal carcinoma-derived cells. A: Expression of miR-221 was detected by real-time RT-PCR. The miR-221 expression level improved in a dose-dependent manner with the increase in radiation dose, ^bP < 0.01 vs control group; B: Expression of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) protein was detected by Western blotting, β-actin was used as a housekeeping gene to normalize PTEN protein expression. The relative PTEN protein levels normalized against β-actin are shown at the top of each panel. The expression of PTEN protein was decreased in a dose-dependent manner with the increase in radiation dose; C: Expression of PTEN mRNA was detected by real-time RT-PCR. No significant changes in the PTEN mRNA levels were observed in the cells exposed to irradiation. The results are representative of three independent experiments.

Figure 4 miR-221 modulates colorectal carcinoma cell radiosensitivity. Caco2 cells transfected with negative control, pre-miR-221 or anti-miR-221 oligonucleotides were exposed to 0-8 Gy radiation and incubated for 14 d prior to fixation, staining and assessment of colony formation. The colony formation assays were performed in triplicate.

Figure 5 Effects of anti-miR-221 on irradiation-induced death in Caco2 cells. The percentages of necrotic and apoptotic cells were measured by calculating the ratio of the number of corresponding cells to the number of total cells. A: Blank control; B: Caco2 cells transfected with negative control; C: Caco2 cells transfected with anti-miR-221; D: Caco2 cells under irradiation; E: Caco2 cells transfected with negative control under irradiation; F: Caco2 cells transfected with anti-miR-221 under irradiation. The right upper quadrant (FITC⁺/PI⁺) shown as necrotic cells. The right lower quadrant (FITC⁺/PI^-) shown as apoptotic cells; G: The status of cell death was determined by flow cytometry. Groups A-F are described as above. n = 3, mean ± SD; ^bP < 0.01 vs blank or negative control group. ^dP < 0.01 vs sole anti-miR-221 or irradiation group.

Figure 6 Ectopic expression of miR-221 affects the radiosensitivity of colorectal carcinoma cells by targeting phosphatase and tensin homolog deleted on chromosome 10. A: miR-221 regulates the phosphatase and tensin homolog deleted on chromosome 10 (PTEN)/AKT pathway. Caco2 cells transfected with negative control or anti-miR-221 were mock treated or irradiated (4 Gy). Total cell lysates were obtained for Western blotting using the indicated antibodies. Caco2 cells were pretreated with or without anti-PTEN-siRNA (80 nmol/L) for 24 h prior to the addition of pre-miR-221 (50 nmol/L) or anti-miR-221 (50 nmol/L); B, C: Real-time RT-PCR and Western blotting showing PTEN mRNA and protein reduced markedly after transfection with anti-PTEN-siRNA (^bP < 0.01 vs blank control and negative control group); D: The status of cell radiosensitivity was determined by colony formation assay. The suppression of Caco2 cell survival fraction at 4 Gy by anti-miR-221 was partially, but not completely, abrogated by anti-PTEN-siRNA (SF₄ from 29.77% to 47.88%). n = 3, mean ± SD; ^bP < 0.01 vs negative control group; ^dP < 0.01 vs sole anti-miR-221 group.