FIGURE 5:

miR-222 directly interacts with CDK4 mRNA through its CR. (A) Schematic representation of CDK4 mRNA depicting two predicted target sites for miR-222 in the CDK4 CR. Alignment of the CDK4 mRNA sequences with miR-222: top strand, CDK4 mRNA; bottom strand, miR-222. (B) Levels of biotinylated miR-222 after transfection for 48 h: (a) schematic representation of biotinylated miR-222; (b) miR-222 levels as measured by Q-PCR analysis; (c) U6 RNA levels. Values are means ± SE from three separate experiments. *p < 0.05 compared with cells transfected with control scrambled RNA. (C) Binding of biotinylated miR-222 to CDK4 mRNA in pull-down materials. *p < 0.05 compared with input control. (D) Changes in the levels of CDK4 and CUGBP1 proteins after ectopic overexpression of miR-222: (a) levels of miR-222 as measured by Q-PCR analysis 48 h after transfection; (b) representative immunoblots of CDK4. (E) Changes in CDK4 translation efficiency as measured by analysis of CDK4-CR (a) and CDK4 3′-UTR (b) luciferase reporter. (F) Polyamine depletion enhances CDK4 mRNA association with miR-222: (a) binding of CDK4 mRNA to miR-222; (b) total CDK4 mRNA levels. (G) Effect of miR-222 silencing on CDK4 expression in polyamine-deficient cells: (a) binding of miR-222 to CDK4 mRNA; (b) CDK4 protein levels.