Fig. 1
Expression pattern of miR-31 in mammary gland and tumors. a qRT-PCR for miR-31, miR-22 and miR205 in Lin-CD24+CD29high, Lin-CD24+CD29low, Lin-CD24-CD29+ and Lin-CD24-CD29- populations at 12 weeks of age. n = 3 biological replicates. b In situ hybridization for miR-31 in 12-week-old WT mammary gland ducts and tertiary branches. DTG mammary ducts, a positive control. The DTG mice have been administered with Dox at 1 week of age. miR-31 KO mammary ducts, a negative control. Scale bar, 25 μm. c qRT-PCR for miR-31 in WT mammary epithelial cells at 6, 10 weeks, P14.5 (14.5 days post pregnancy), P18.5, L1 (1 day post lactation) and Inv (10 days post involution). n = 3 at each time points. d The schematic diagram showing two potential p65 (NF-κB) binding sites (-1746 bp and -1375 bp) in the miR-31 promoter. TSS, transcription start site. e, f qRT-PCR for miR-31 e and western blotting for RANKL, p-p65, p65 f in HC11 mouse mammary epithelial cells treated with RANKL siRNA (SiRANKL), and scramble RNA (NC). g Luciferase activity in lysates of HC11 mouse mammary epithelial cells transfected with luciferase reporter plasmids of pGL3-basic plasmid, miR-31 promoter or miR-31 mutant promoter with mutation at the -1375 (p65-mut-1) or -1746 (p65-mut-2) binding site, treated with scramble RNA (negative control, NC) and RANKL siRNA. h ChIP assays carried out on HC11 mammary epithelial cells using antibodies against p65 under indicated conditions. i–k In situ hybridization and qRT-PCR analysis for miR-31 i and western blotting for p-Ikkα, p-p65 and p65 j, and immunohistochemistry for RANKL k in WT mouse mammary gland and PyVT tumors. Scale bar, 25 μm. l western blotting for p-Akt, p-Ikkα, p-p65 and p65 in MCF7 breast cancer cells treated with Pten inhibitor bpV(pic) at indicated concentrations for 12 h. m qRT-PCR for miR-31 in MCF7 breast cancer cells treated with PTEN inhibitor bpV(pic) at indicated concentrations for 12 h. Data represented as mean ± S.D. Sample size: WT (n = 3) and PyVT (n = 3) for i–k. Two tailed unpaired t-test for e, g, i, m (*P < 0.05; **P < 0.01; ***P < 0.001)
