FIGURE 4.

miR-378 reduces endogenous IGF1R expression by direct targeting of 3′ UTR. A, Northern analysis showing increased expression of mature miR-378 48 h following transfection of the 378-mimic in cardiac myocytes, U6 was used as a loading control. B, IGF1R expression by Western blot in triplicate (50 μg of protein/lane) in the presence of mimic control or increasing amounts of 378-mimic, GAPDH used as a loading control. Right graph is derived from 3 additional experiments. C, real-time PCR analysis of IGF1R mRNA levels in mimic control and 378-mimic transfected cells (n = 2). D, functional assay of the IGF1R 3′ UTR in H9C2 cells using a dual luciferase reporter system following transfection of various DNA constructs in the presence of 378-mimic or mimic control. The sequence shown is the predicted target sequence of IGF1R 3′ UTR, three repeats of this sequence (WtIGF1R3X-luc) or mutated sequence (underlined nucleotide mutIGF1R3X-luc) were cloned downstream of the luciferase reporter, Renilla luciferase activity was used for normalizing data. Data are derived from triplicate transfectants of 3 independent experiments. *, p < 0.05.