Fig. 2

The miR-942-5p targets the IFI27 3′-UTR, causing translational suppression. A The IFI27 mRNA schematic showed one potential binding site for miR-942-5p in its 3′-UTR. The IFI27 3′-UTR sequence covering the potential binding site was inserted into the pmirGLO-REPORT luciferase plasmid. Control plasmids with the mutant 3′-UTR sequence were also generated as a control. B HCT-8 cells were exposed to C. parvum sporozoites for as long as 24 h, followed by western blot to evaluate IFI27 protein expression and real-time PCR analysis for IFI27 mRNA expression. Representative western blots from three independent experiments are shown. The densitometric levels of protein signals and mRNA levels were quantified and expressed as the ratio to actin. C Targeting the IFI27 3′-UTR resulted in translational suppression. Cells were transfected with a pMIR-REPORT luciferase construct containing the miR-942-5p binding site in the IFI27 3′-UTR and treated with an miR-942-5p mimic for 24 h, followed by a luciferase analysis. D Manipulation of miR-942-5p function can result in reciprocal alterations in IFI27 protein expression in HCT-8 cells. Cells were treated with an miR-942-5p mimic or miR-942-5p inhibitor for 6 h and exposed to C. parvum sporozoites, followed by a western blot for IFI27 protein and real-time PCR for IFI27 mRNA 12 hpi. Representative western blot images and quantification of IFI27 mRNA levels from three independent experiments are shown. Densitometric levels of IFI27 signals were quantified and expressed as the ratio to β-actin. Densitometric levels of protein signals and mRNA levels were quantified and expressed as the ratio to β-actin. All data represent the combined mean ± SD of three independent experiments, with two to three technical replicates per experiment, and were analyzed with a t-test vs. the controls. ^*P < 0.05; ^**P ≤ 0.01