miR-16-1 regulates CCND1 through its 3′ UTR. (A) Three GFP reporter constructs were made as previously described (“Methods”). GFP-L contains the distal CCND1 3′ UTR including both miR-16-1 binding sites. GFP-T contains the truncated CCND1 3′ UTR. GFP-M is a double mutant where both miR-16-1 binding sites were mutated. Notice the mutation changes the T from the third base pair to an A and the A in the seventh base pair to a T. (B) H157 cells were transfected with GFP-L and cotransfected with a mimic of miR-16-1 or negative control random miRNA. Shown is the GFP expression measured by flow cytometry. RFP-expressing plasmids were cotransfected to control for transfection efficiency. Error bars represent SD.(C) H157 cells were transfected with either GFP-L, GFP-T, or GFP-M. They were also cotransfected with either a mimic of miR-16-1 or negative control random miRNA. GFP expression of cells transfected by a mimic of miR-16-1 is first normalized to the RFP expression and then normalized to cells transfected with negative control random miRNA. This shows that miR-16-1 mimic cannot regulate a truncated or mutated CCND1 3′ UTR.