Figure 3

miRNA (miR)-34b suppressed cell proliferation by directly regulating JAG1 and cyclin D1. (A) Cell viability after transfection of negative control (Mock) (200 pM), precursor miR-34b (200 pM) or antagomir-34b (200 pM) was detected by using 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide (n = 3). (B) Predicted binding structures of miR-34b with targets JAG1 and cyclin D1. RNAhybrid software was used for nucleotide-binding sequence prediction. (C) Western blots showing JAG1 and cyclin D1 after 24-hour treatment with precursor miR-34b or (200 pM) in MCF-7 cells. β-actin was probed as internal control. The numbers under the blots represent the semiquantitative values of the Western blot analysis. (D) and (E) Top: Diagrams depicting the pMIR-REPORT vector luciferase reporter constructs containing a cytomegalovirus (CMV) promoter (P_CMV), which was used to verify the putative miR-34b binding sites. Bottom bar graphs: 293T cells were cotransfected with negative control (Mock) (200 pM) or miR-34b (200 pM), Luc-JAG1-3'UTR (0.5 μg) or Luc-CyclinD1-3'UTR (0.5 μg), along with a pRL-SV40 reporter plasmid (0.05 μg). After 24 hours, luciferase activity was measured. Values are presented as relative luciferase activity after normalization to Renilla luciferase activity. Data are expressed as means ± SD of results derived from triplicate experiments. *P < 0.05.