Ex Vivo Infection of Human Placental Explants by Trypanosoma cruzi Reveals a microRNA Profile Similar to That Seen in Trophoblast Differentiation

Figures

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  Figure 1. MiR-512-3p- and miR-515-5p = mediated trophoblast differentiation. MiR-512-3p promotes trophoblast differentiation by repressing the caspase 8 inhibitor c-FLIP and, consequently, increases caspase 8 expression, promoting cellular fusion in the trophoblast. Conversely, miR-515-5p inhibits trophoblast differentiation by repressing the transcription factor hGCM-1, which mediates hCG expression.

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  Figure 2. HPE can be effectively transfected with miR-512-3p and miR-515-5p mimics and antagomirs. HPE was transfected with 100 nM of miR-512-3p (A) or miR-515-5p (B) antagomirs (A-512 or A-515), mimics (M-512 or M-515), and their respective scrambles (AS or MS) for 24 h. The miRNA expression was determined by means of real-time PCR. All values are given as the means ± SD; data were normalized to control values and analyzed via one-way ANOVA and Dunnett’s post-test. ** p ≤ 0.01; *** p ≤ 0.001.

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  Figure 3. HPE transfects are localized in the trophoblast. HPE was transfected with 100 nM of AntagomiR-Cy3 for 24 h. Samples were processed using routine fluorescence methods; nuclei were stained with DAPI. White arrows indicate the fluorescence label in the trophoblast. Scale bar: 20 μm.

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  Figure 4. T. cruzi increases and decreases miR-512-3p and miR-515-5p levels, respectively, in HPE. HPE was transfected with 100 nM of miR-512-3p (A) or miR-515-5p (B) antagomirs (A-512 or A-515), mimics (M-512 or M-515), and their respective scrambles (AS or MS) for 24 h and then challenged with 105 T. cruzi trypomastigotes for 2 h. The miRNA expression was determined by means of real-time PCR. All values are given as the means ± SD; data were normalized to control values and analyzed via one-way ANOVA and Dunnett’s post-test. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.

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  Figure 5. T. cruzi does not affect c-FLIP expression. HPE was transfected with 100 nM of miR-512-3p (A,B) antagomirs (A-512), mimics (M-512), and their respective scrambles (AS or MS) for 24 h and then challenged with 105 T. cruzi trypomastigotes for 2 h. The c-FLIP mRNA expression was determined by means of real-time PCR. All values are given as the means ± SD; data were normalized to control values and analyzed via one-way ANOVA and Dunnett’s post-test. * p ≤ 0.05.

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  Figure 6. MiR-515-5p and miR-512-3p regulate T. cruzi-induced increase in hGCM-1. HPE was transfected with 100 nM of miR-515-5p (A,B) or miR-512-3p (C,D) antagomirs (A-512 or A-515), mimics (M-512 or M-515), and their respective scrambles (AS or MS) for 24 h and then challenged with 105 T. cruzi trypomastigotes for 2 h. The hGCM-1 mRNA expression was determined by means of real-time PCR. All values are given as the means ± SD; data were normalized to control values and analyzed via one-way ANOVA and Dunnett’s post-test. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.

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  Figure 7. MiR-512-3p and miR-515-5p regulate T. cruzi-induced increase in hCG. HPE were transfected with 100 nM of miR-512-3p (A,B) or miR-515-5p (C,D) antagomirs (A-512 or A-515), mimics (M-512 or M-515), and their respective scrambles (AS or MS) for 24 h and then challenged with 105 T. cruzi trypomastigotes for 2 h. The hCG mRNA expression was determined by means of real-time PCR. All values are given as the means ± SD; data were normalized to control values and analyzed via one-way ANOVA and Dunnett’s post-test. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.

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  Figure 8. MiR-512-3p and miR-515-5p levels determine HPE susceptibility to T. cruzi infection. HPE was transfected with 100 nM of miR-512-3p (A) or miR-515-5p (B) antagomirs (A-512 or A-515), mimics (M-512 or M-515), and their respective scrambles (AS or MS) for 24 h and then challenged with 105 T. cruzi trypomastigotes for 2 h. Parasite DNA was detected using real-time qPCR. Data analysis was performed using the ΔΔCt method. All values are presented as the mean ± SD (of normalized values) and analyzed via one-way ANOVA and Dunnett’st’s post-test. ** p ≤ 0.01; **** p ≤ 0.0001.