Figure 5.

Functional analysis of miR-181c, miR-294-5p, miR-30e, and miR302d inhibition. Macrophages (5 × 10⁵) were transiently transfected with the negative control (NC, gray bars), 30 or 100 nM of miR-181c-5p, miR-294-3p, miR-30e-5p, or miR-302d-3p inhibitors (purple bars), or left non-transfected (untreated, white bars). After 36 h of incubation, the cells were infected with L. amazonensis (MOI 5:1) for 4 and 24 h. The inhibition with 100 nM of NC or miRNAs were performed to evaluated miR-181c-5p, miR-294-3p, miR-30e-5p, or miR-302d-3p expression (A) and for Nos2 (B), Tnf, (C) Mcp-1 (D), and Rantes (E) mRNA expression, by qPCR; the inhibition with 30 or 100 nM of NC or miRNAs were also performed for infectivity evaluation (F) by microscopy analysis, counting the numbers of infected macrophages and amastigotes per macrophage (n = 1,000 macrophages/treatment). The values of miRNAs were represented by Fold change using SNORD95, as a normalizing endogenous control. The values of mRNAs were normalized (100%) based on the average values of the negative control (NC) at 4 h of infection. Each bar represents the average ± SEM of the values obtained in three independent experiments (n = 4–6). Statistical significance was determined based on unpaired two-tailed Student's t-test. In infectivity analysis (F), ^*p < 0.05, compared to negative control (NC) infected macrophages.