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Fig. 6 | Journal of Experimental & Clinical Cancer Research

Fig. 6

From: 5’isomiR-183-5p|+2 elicits tumor suppressor activity in a negative feedback loop with E2F1

Fig. 6

Targeting E2F1 partially causes the effect of overexpression of miR-183-5p|+2. a Effect of E2F1 knockdown on cell proliferation. MDA-MB-231 and BT-549 were transfected with siE2F1_9, siE2F1_11 or siRNA negative control for 48 h. After transfection, the cells were starved for 24 h and reseeded into clear-bottom 96-well black plates. Cells were stained with Hoechst 33342 for 30 min and imaged with Molecular Devices Microscope IXM XLS after attachment and 144 h later. The cell number was obtained by counting cell nuclei with Molecular Devices Software and normalized to the cell number at 0 h time point. Data are presented as mean ± SD, n = 3 (each derived from the median of 6 technical replicates). Statistical analysis: ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test. b Effect of E2F1 knockdown on cell cycle. After transfection, MDA-MB-231 and BT-549 cells were starved with 0% FBS medium for 24 h and released with full growth medium for 24 h. Afterwards, the cells were exposed to BrdU for 2 h and stained with FITC conjugated anti-BrdU antibody and 7-AAD. Cells cycle was analyzed using a FACSCalibur device and the CellQuest Pro software. Statistical analysis: two-way ANOVA followed by Dunnett’s multiple comparisons test. c Ectopic overexpression of E2F1 ORF rescues the effect of miR-183-5p|+2 overexpression on cell cycle. MDA-MB-231 and BT-549 cells were seeded in 6-well plates and E2F1 expression was induced with the doxycycline. Cells were transfected with miRNA mimics (miR-183-5p|0, miR-183-5p|+1 and miR-183-5p|+2) or siAllstar for 48 h. After transfection, the cells were starved with 0% FBS medium for 24 h and released with full growth medium for 24 h. Cells were cultured in presence of doxycycline in each step (0.2 ng/mL for MDA-MB-231, 5 ng/mL for BT-549). Cells cycle was analyzed using a FACSCalibur device and the CellQuest Pro software. Data are presented as mean ± SD, n = 3 (each derived from the median of 3 technical replicates). *p < 0.05, **p < 0.01 compared to control, unpaired t-test

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