Figure 2

Calcium-binding protein 39-like (CAB39L) and Sestrin-1 (SESN1) are targets of metformin-miR-21-5p. (a, b) Quantitative PCR. Histograms showing the relative levels of CAB39L (a) and SESN1 (b) mRNAs in SUM159PT, BT-474, MCF-7 and BT-549 cells, treated with vehicle or metformin (0.5 mM) for 24 h. Statistics (t-test): P<0.05. (c) Representative western blotting with anti-CAB39L or SESN1 antibodies of whole-cell lysates from SUM159PT cells treated with vehicle or metformin (0.5 mM) for 24 h. Actin staining was used as a loading control. (d–i) Modulation of CAB39L and SESN1 by metformin required miR-21-5p. (d–f) Quantitative PCR. Histograms showing the relative levels of miR-21-5p (d), CAB39L (e) and SESN1 (f) mRNAs in SUM159PT transfected with different doses of miR-21-5p inhibitor. Statistics (t-test): P<0.05. (g) Representative western blotting of whole-cell lysates from SUM159PT cells treated as from panel (d–f), stained with the indicated antibodies. Actin staining was used as a loading control. (h, i) Colony-forming assay (h) and invasion assay (i). Histograms showing average colony counts (h) or the number of migrated cells (i) of SUM159PT cells treated as from panel (g). Statistics (t-test): P<0.05. (j) Quantitative PCR. mRNA levels of SESN1 or CAB39L in SUM159PT cells expressing either a control vector or a miR-21-5p mimic vector. Bars indicate the average of three independent experiments. Statistics (t-test): P<0.05. (k, l) Dual luciferase assay. SUM159PT cells co-transfected with the wild-type or mutated CAB39L (k) or SESN1 (l) 3′-untranslated region luciferase reporter and, subsequently, with either a scrambled- or a specific miR-21-5p agonist. Histograms show the normalized luciferase values from three independent experiments. Statistics (t-test): P<0.05.