Figure 5

Functional characterization of miR-1254 and CCAR1 5′ UTR in breast cancer cells. (A-C) MCF-7 cells were transfected with vector or miR-1254 sponge and deprived of estrogen for 8 days. Cell viability was measured by MTT assay (A), foci formation in soft agar (B) and monolayer culture (C). Scale bars represent 5 mm. Error bars indicate SEM. ^***P< 0.001. (D-F) MCF-7 cells were transfected with vector or miR-1254 sponge and treated with different doses of tamoxifen. Cell viability was measured by MTT assay (D), foci formation on soft agar (E) and monolayer culture (F). Scale bars represent 5 mm. Error bars indicate SEM. ^***P< 0.001. (G) Tumors (red arrows) form after orthotopic injection of MCF-7 pcDNA3.1 cells or MCF-7 miR-1254 sponge cells in nude mice without estrogen supplement for 40 days. (H-I) Growth curves (H) and final sizes (I) of tumors derived from MCF-7 pcDNA3.1 cells or MCF-7 miR-1254 sponge cells orthotopically injected in nude mice with tamoxifen treatment for 40 days after the average tumor volume has reached 100 mm3 with estrogen supplementation. (J-L) MCF-7 TAM-R cells were transfected with NC or miR-1254 and further treated with different doses of tamoxifen for another 8 days. Cell viability was measured by MTT assay (J), foci formation on soft agar (K) and monolayer culture (L). Scale bars represent 5 mm. Error bars indicate SEM. ^*P< 0.05, ^**P< 0.01, ^***P< 0.001. (M-N) Transwell assay (M) measuring cell migration and FACS analysis (N) measuring CD44 and CD24 expression in MCF-7 TAM-R cells transfected with NC or miR-1254. Scale bars represent 100 μm. (O-P) Quantification of relative migrating cell number (O) and average of CD44⁺/CD24^-/low population (P) in three independent experiments. Error bars indicate SEM. ^***P< 0.001. (Q-R) MTT assay measuring viability of MCF-7 TAM-R cells transfected with vector or CEG (Q), or vector or mutCEG (R) and treated with different doses of tamoxifen for 8 days. Error bars indicate SEM. ^**P< 0.01, ^***P< 0.001. (S) Tumors derived from MCF-7 TAM-R CEG or MCF-7 TAM-R mutCEG in nude mice. (T-V) Tumor weight (T), cell proliferation measured by Ki67 staining (U), and cell apoptosis measured by TUNEL staining (V) of tumors derived from MCF-7 TAM-R CEG or MCF-7 TAM-R mutCEG cells. Error bars indicate SEM. P = 0.007 in T; ^***P < 0.001 in U and V. Student's t-test except in T, where Wilcoxon test is used. See also Supplementary information, Figure S4-S6.