Figure 1
CDK5R1 is the target gene of miR-152. (a) The binding site of CDK5R1 and miR-152 is shown. (b) The sequence of miR-152 and the sequence of miR-152 mutant with mutation in the seeded region. After transfection of SKES-1 cells, miR-152 was detected by quantitative PCR from RNA extracted from the cells. miR-152 was appropriately introduced into the cells, and the miR-152 mutant did not differ from the control group. (c) Intracellular expression of CDK5R1 after transfection with miR-152 or miR-152 mutant as analyzed by reverse transcriptase-quantitative polymerase chain reaction (qRT-PCR). (d) Predicted binding sites of miR-152 in the 3′-untranslated region (UTR) of CDK5R1 binding sites 1 and 2. Sites mutated as controls for the luciferase assay are underlined. WT, wild-type; M1, mutation of CDK5R1 3′-UTR binding site 1; M2, mutation of CDK5R1 3′-UTR binding site 2. (e) Luciferase assay identified CDK5R1 as two targets of miR-152. Luciferase reporter assay for the direct and specific interaction of miR-152 with predicted two target sites in the 3′-UTR of CDK5R1. Reporter activity was significantly reduced in miR-152 mimic transfected cells. Control-miR cells were used as control here for normalization. The mutations at the two target sites did not reduce luciferase activity in response to the miR-152 mimic; it remained equivalent to the Control-miR group. Images (f) and plots (g) from the immunoblot analysis of CDK5R1 protein expression following changes in miR-152 expression. Images (h) and plots (i) of protein expression of CDK5R1 following changes in the concentration of CDK5R1 siRNA. GAPDH level was used as loading control.
