Figure 3.

miR‐33a directly targets Mouse double minute 4 (MDM4). (a) The predicted miR‐33a binding site within MDM4 3′ UTR and miR‐33a mutated version by site mutagenesis; (b) Caki‐1 cells were transfected with reporter constructs containing either wild type (WT) MDM4, or MDM4 3′ UTR with mutation (MUT), along with miR‐33a mimics, or negative control, respectively. Relative luciferase activity was measured. (c) qPCR analysis of MDM4 expression in renal cell cancer (RCC) Caki‐1 cells after overexpression or knockdown of miR‐33a. (d, e) Western blotting analysis of MDM4 protein expression in RCC Caki‐1 cells after overexpression or knockdown of miR‐33a. (f) Western blot analysis revealed that transfection of MDM4 siRNA into Caki‐1 cells resulted in decreased MDM4 expression compared to the cells transfected with scrambled siRNA. These effects of siRNA were attenuated by anti‐miR‐33a inhibitor transfection. NC represents normal control, *p < 0.05, **p < 0.01 versus NC