Figure 1

Metformin downregulated the miR-21-5p expression. (a) Quantitative PCR. Expression of the pri- and pre-miR-21-5p in SUM159PT cells treated with metformin (0.5 mM) for 24 h. Bars indicate the average of three independent experiments. Statistics (t-test): P<0.05. (b, c) Modulation of miR-21-5p happened in vivo. (b) Relative levels of miR-21-5p (log2) expression in pooled SUM159PT tumors treated intraperitoneally with vehicle (n=4) or metformin (n=4) (16 mg kg⁻¹, bi-weekly, for 10 weeks) when the average starting tumor volume was 0.2 cm³. Statistics (t-test): P<0.05. (c) Scatter plot indicating the miR-21-5p fold changes (log2) in the sera of women (n=96) with established breast cancer collected before and after 3 months of daily treatment with metformin (500 mg day⁻¹). Each circle indicates a patient with decreased (blue ones) or increased (red ones) levels of miR-21-5p as compared with the pretreatment levels. Statistics: (Wilcoxon sign-rank test, matched samples) P<0.001. (d) Luciferase assay. HEK-293 cells co-transfected with the wild-type or mutated miR-21-5p promoter/LUC reporter construct and with different doses of E2F3. Histograms show the normalized luciferase values from three independent experiments. Statistics (t-test): P<0.05. (e) E2F3 binding to the miR-21-5p promoter mediated the effect of metformin. Upper panel. Predicted E2F-binding sites in the miR-21-5p promoter. Horizontal bars indicate the three different E2F-binding sites amplified in the chromatin immunoprecipitation experiment. Lower panel. Histograms showing the amplification of the E2F-binding sites in the chromatin precipitated with anti-E2F3 and anti-acetylated Histone H4 (Acetyl-H4), from vehicle and metformin-treated (0.5 mM, 24 h) SUM159PT cells. Statistics (t-test): P<0.05. Please see additional data in Supplementary Figures S1d and e (f) Metformin induced E2F3 transcriptional expression. Quantitative PCR. Relative expression of E2F3 in SUM159PT cells treated with metformin (0.5 mM) for different times (0–720 min). Statistics (t-test): P<0.05 over untreated cell (dashed line). (g) Altering the levels of E2F3 impaired the metformin effect on the SUM159PT clonogenic ability. Histograms showing the average percentage of colonies formed by SUM159PT cells transfected with two different E2F3 siRNAs (or scrambled control) and treated with vehicle or metformin (0.5 mM, 24 h) before seeding at clonal density. Statistics (t-test): P<0.05 *Significant versus vehicle, **Significant versus control transfected cells treated with metformin.