Figure 5

Cyclin D3 and E2F3 are direct targets of miR-503. (A) the putative miR-503 binding sequence in the 3′ UTR of Cyclin D3 and E2F3 mRNA. Mutation was generated on the Cyclin D3 and E2F3 3′ UTR sequence in the complementary site for the seed region of miR-503. (B) Analysis of luciferase activity. 293 T cells were cotransfected with 50 nM of either miR-503 or NC and 100 ng pmirGLO Vector containing Wt or Mut 3′UTR of Cyclin D3 or E2F3 (indicated as WT or MUT on the X axis). The relative firefly luciferase activity normalized with renilla luciferase was measured 48 h after transfection. (C) the effect of miR-503 on the expression of endogenous Cyclin D3 and E2F3 at mRNA levels. (D and E) Endogenous Cyclin D3 or E2F3 protein levels are down-regulated in LM3 cells transfected with miR-503 mimics and up-regulated in Bel-7402 cells transfected with the miR-503 inhibitor (F) the effect of siRNA on the expression of endogenous Cyclin D3 and E2F3 at protein level. (G) knockdown of CCND3 or E2F3 induced a significant accumulation of G1-phase cells and blocked G1/S transition. *** P < 0.001.