Fig. 7
MiR-31 activates Wnt and represses TGFβ signaling pathways. a Wnt signals were evaluated by Axin2-LacZ reporter activity in mammary ducts from Control (n = 3) and DTG (n = 3) mice, as well as Control (n = 3) and miR-31 KO (n = 3) mice. Blue, LacZ signals. Scale bar, 100 μm. b Immunohistochemistry for β-Catenin in Control (n = 3) and DTG (n = 3) mammary glands at 12 weeks of age and 14.5 d.p.c. Arrows, nuclear localized β-Catenin. Scale bar, 25 μm. c Western blotting for β-Catenin in Control (n = 3) and miR-31 KO (n = 3) mice. GAPDH was used as a loading control. d Immunofluorescence for β-Catenin in HC11 mouse mammary epithelial cells in the presence of Dox (miR-31 overexpression). Control, without Dox. Red, β-Catenin; Blue, DAPI. n = 3 technical replicates. e Western blotting for Wnt target LBH in miR-31 overexpressing (Dox) or miR-31 inhibited (anti-miR-31) HC11 mammary epithelial cells. The Control HC11 cells were treated with Scramble RNA. n = 3 technical replicates. f TOP/FOPflash luciferase assays demonstrate that Dox induced miR-31 overexpression activates Wnt reporter gene expression and that miR-31 inhibitor (anti-miR-31) markedly represses Wnt reporter gene expression. Scramble RNA was used as negative control (NC). n = 3 technical replicates. g Western blotting for Smad3, p-Smad2/3 and p21 in mammary epithelial cells of Control (n = 3) and miR-31 KO (n = 3) mice at 10 weeks of age. h qRT-PCR analysis for TGFβ components and downstream target genes, Cdkn2b (encoding p15), Cdkn1a (encoding p21), Cdkn1c (encoding p57) and Tgfbr1 in mammary epithelial cells of Control (n = 3) and miR-31 KO (n = 3) mice at 10 weeks of age. i HC11 mammary epithelial cells were transfected with CAGA luciferase reporter vector, combined with scrambled RNA (negative control, NC), miR-31 mimics or miR-31 inhibitor (anti-miR-31) for 24 h and then harvested for luciferase activity determination. n = 3 biological replicates. Data represented as mean ± S.D. n = 3. Two tailed unpaired t-test for f, h, i (*P < 0.05; **P < 0.01; ***P < 0.001)
