Figure 4

miR-29c directly targets RCC2, PPIC and CDK6. (A) Gene-expression microarray was performed at 24 h after transfection with pre-29c or pre-Neg. The Venn diagram shows the relationship between the sets of genes differentially expressed in MKN45, MKN74 and MKN7 cells. Changes in the expression of 4 genes (6 probes) were common to three cell lines. In the 3’-UTRs of PPIC, RCC2 and CDK6 mRNAs, the target sequences of miR-29c were found (according to TargetScan). (B) MKN45 cells were transfected with pre-29c or pre-Neg. The mRNA and protein levels of RCC2, PPIC and CDK6 were determined by quantitative RT-PCR and immunoblotting at 72 h. The mRNA levels relative to negative controls are indicated as mean ± SD of triplicate determinations. PPIC protein was not detectable (N.D.), presumably because the specificity of the PPIC antibody was insufficient. (C) Interaction of miR-29c with the 3’UTR of RCC2 and PPIC. At 24 h after transfection with pre-29c or pre-Neg, a reporter plasmid containing RCC2 or PPIC wt-3’UTR or mut-3’UTR and a plasmid expressing renilla luciferase (pRL-CMV) were cotransfected into MKN45 cells. Luciferase activities were measured at 48 h after transfection with the plasmids, and normalized data are shown. The data are indicated as mean ± SD of quadruplicate determinations. Three independent experiments were performed and representative data are shown. **P <0.01.