Figure 6: miR-106b suppresses Wnt signalling pathway by targeting BRMS1L.

(a) Luciferase reporter assays for 293T cells co-transfected with pMIR-REPORT reporter vector carrying the 3′ UTR of BRMS1L mRNA with wild-type (WT) or mutated (MUT) target site with microRNA mimics or lin4. *P≪0.05, **P≪0.01, ***P≪0.001 as compared with MUT. Bars correspond to mean±s.d.. These data are representative of three independent experiments. Student’s t-test was used for the comparison of two independent groups. (b) qRT–PCR for miR-106b expression in primary breast tumour cells (PT) with or without lymph nodes metastasis (LNM; n=4 per group). Bars correspond to mean±s.d. Eight independent experiment were repeated (each patient sample was considered as one independent experiment. Five technological replications were repeated in each sample.). Student’s t-test was used for the comparison of two independent groups. (c–e) Wnt/β-catenin activity measured by TCF/LEF luciferase reporter assay (c) and β-catenin translocation (d) and wnt target gene expression using qRT–PCR (e) in T47D cells that were mock transfected (mock), or transfected with lin4 mimics (lin4), an empty vector as negative control (NC), miR-106b mimics (106b), or co-transfected with miR-106b mimics and a vector carrying BRMS1L with a mutated miR-106b-target site at its 3′ UTR (BRMS1L-MUT). Bars correspond to mean±s.d., **P≪0.01 and ^##P≪0.01 as compared with 106b. Bars correspond to mean±s.d. The data are representative of three independent experiments. Student’s t-test was used for the comparison of two independent groups. (f,g) Western blotting of E-cadherin and vimentin for T47D cells treated as in c (f) or treated with PBS or TGF-β (g). (h) Correlation between the percentages of BRMS1L⁺ and miR-106b⁺ breast cancer cells. BRL, BRMS1L. Linear regression was used to analyse the correlation. (i,j) In situ hybridization (ISH) staining for miR-106b in primary breast cancers with or without regional lymph node (LN) metastasis (i), scale bar corresponds to 20 μm, as well as metastatic lymph nodes (LNM) and brain metastasis (BM; j), scale bar corresponds to 50 μm. Sense, using sense probe; other group, using antisense probe. E-cad, E-cadherin; Vim, vimentin; β-cat, β-catenin; BRL, BRMS1L.