Figure 7

Identification of novel miR-324-5p targets using 3′UTR luciferase assays. (a) C3H10T1/2 murine cells were transfected with 3′UTR-pmiRGLO constructs and subsequently also with either a negative control miRNA mimic (miCon) or a mimic of miR-324-5p or miR-324-3p. Luciferase activity was measured after cells were incubated at 37 °C for 24 h. Relative luciferase activity was calculated as a ratio of renilla activity, an internal transfection control of pmiRGLO, and values were plotted as a percentage of mean miCon luciferase activity for each construct. Suox and Cd300lf, but not Sp6, were identified as direct miR-324-5p targets in vitro. (b) When 2nts of the miR-324 binding sites of the Cd300lf and Suox 3′UTRs were mutated, shown as Cd300lf(mut) and Suox(mut), the miR-324-5p-mediated repression was inhibited in both cases. The predicted miR-324-5p binding sites of each 3′UTR are shown above the panel corresponding to that 3′UTR, with mutations highlighted in black. For panels a and b, the means of at least 3 independent experiments were used to test statistical significance in two-tailed Student’s t-tests. A negative control was included for each independent experiment (pmiRGLO) in addition to miCon and, after miR-324 targets were identified, one of these was used as a positive control for each independent experiment. (c) Western Blotting was used to confirm that the changes to the quantity of target gene mRNAs affected levels of the corresponding proteins in female hippocampal tissue aged 7 months. SUOX levels were significantly increased in miR-324-null samples relative to WT controls and although levels of CD300lf were not statistically significantly increased at a 5% level (p value = 0.077), there was a trend towards increased expression in miR-324-null samples. SUOX and CD300lf band intensities were normalised against GAPDH. In all panels, ** indicates p values ≤ 0.01.