FIG. 2.

MiR-34a suppresses cell proliferation in MCF-7 cells in vitro and in vivo and inhibition of miR-34a promotes cell proliferation. (A) Growth curves of MCF-7 cells were conducted by CCK-8 assay in the presence of 10 nM E2 as indicated. The OD value at 450 nm represented the viable cell numbers. Mock: parental MCF-7 cells without infection or transfection; E2-: without E2 treatment; E2+: treatment with E2; EV and MiR-34a: MCF-7 cells stably expressing pLL3.7 vector or miR-34a; I.C: miR-34a Inhibitor Control; MiR-34a In: MiR-34a Inhibitor. The transfection was performed with Lipofectamine 2000 and 50 nM of miR-34a inhibitor or inhibitor control were used for transfection. All experiments were carried out thrice independently. *p<0.05, **p<0.01. (B) Colony formation was performed with MCF-7 cells as indicated; Cells were cultured with or without E2 treatment for 2 weeks. The experiment was repeated thrice independently. (C) Quantification of colony formation assay. The colonies consisting of more than 50 cells were counted in 10 random visual fields. The experiment was repeated thrice independently. *p<0.05. (D) Cell viability was performed with Trypan blue staining. After treatment as indicated, cells were incubated with a final concentration of 0.04% (w/v) Trypan blue solution at room temperature for 3 min and then directly observed using a microscope (Olympus). Cell viability (%)=(viable counts/total counts)×100%. Each experiment was repeated thrice independently. *p<0.05. (E) Cell cycle analysis using propidium iodide staining and flow cytometry. The ratio of G1, S, and G2/M phase was analyzed. MiR-34a groups significantly reduced the ratio of S phase. Each experiment was repeated thrice independently. *p<0.05. (F) Tumor size observation in nude mice after the inoculation. 5×10⁶ either miR-34a (left) or EV group (right) infected MCF-7 cells were injected subcutaneously in nude mice (n=5). The tumor volume was measured every 3 days with calipers after tumor appeared. The average size of the tumors was measured every 3 days and shown in the curves. The error bars show SD (standard deviation). *p<0.05, **p<0.01. (G) One representative nude mouse was shown. Arrows indicate tumors that resulted from either EV or miR-34a stably expressing MCF-7 cells.