Figure 4.

MMP8 is a direct target of miR-204. Luciferase reporter vectors containing a single highly conserved miR-204 (A) binding site (seed nucleotide region) on the rhesus macaque MMP8 mRNA 3′ UTR or the corresponding construct with the binding sites deleted were co-transfected into HEK293 cells with 30 nM miR-204 or Negative control mimic. Firefly and Renilla luciferase activities were detected using the Dual-Glo luciferase assay system 96 h after transfection. Note significantly reduced Firefly/Renilla ratios following co-transfection of miR-204 mimic with a pmirGLO vector containing wild-type (WT) miRNA binding sties (B). Luciferase reporter assays were performed thrice in six replicate wells. Representative Immunoprecipitation/western blot (Triplicate wells) (C) and quantification (D) showing reduction in MMP8 protein expression (~52 kD) 96 h post transfection of HEK293 cells in triplicate wells with negative control (cel-miR-67) or miR-204 mimics. miR-204 overexpression followed by immunoprecipitation/western blot was performed twice in triplicate wells. Firefly/Renilla ratios were analyzed using one-way ANOVA followed by Benjamini-Hochberg post hoc test (p < 0.05). Western blot densitometry data were analyzed using unpaired “t”-test. A p-value of ≤0.05 was considered significant.