FIGURE 3.

miR-424 targets the 3′ UTR of SIAH1, SMURF1, and SMURF2, inhibiting transcript expression in uninfected and DENV2 infected cells. (A) Dual luciferase reporter plasmids were constructed containing the 3′ UTRs of SIAH1, SMURF1, or SMURF2. The 3′ UTR of SMURF1 contains two putative miR-424 binding sites, prompting the construction of two plasmids containing one binding site each. HEK293 cells were transfected with the reporter plasmid and the miR-424 mimic or control small RNA. Cells were lysed 48 h post-transfection, and luciferase expression measured by luminometer. Activity is represented as relative luciferase units (RLUs), calculated as test normalized to control. (B) HeLa cells were transfected with siRNA against SMURF1 or SMURF2, the miR-424 mimic, or a control mimic, and 48 h post-transfection total RNA was collected in Trizol. Relative expression of the SMURF1 and SMURF2 mRNAs was determined by qPCR and normalized to β-actin. (C) HeLa cells were transfected with the SIAH1 siRNA, the miR-424 mimic, or control mimic. At 48 h post-transfection, they were treated with thapsigargin (1 μM) for 6 h. Total RNA in Trizol was collected and relative mRNA levels of SIAH1 determined as described above. (D) HeLa cells were transfected as indicated and infected with DENV2 (MOI = 5 ffu/cell). Total RNA was collected in Trizol at the indicated time points and relative mRNA levels determined as described above. (* p-value < 0.05, ** p-value < 0.01, *** p-value < 0.005, **** p-value < 0.001).