Modulation of miR-145-5p and miR-146b-5p levels is linked to reduced parasite load in H9C2 Trypanosoma cruzi infected cardiomyoblasts

Figures

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  Figure 1. Standardization of T. cruzi infection in H9C2 cells. (A) H9C2 cells were infected with 5:1, 10:1 and 20:1 T. cruzi trypomastigotes for 4 h, washed and collected 24 h and 48 h post-infection. (B) Percentage of infected cardiomyoblasts. (C) Rate of intracellular forms/cardiomyoblasts. (D) Endocytic index. (E) Microscopic images of H9C2 cells infected with T. cruzi Colombian strain stained with panoptic. Red arrows indicate intracellular forms of T. cruzi. For all graphs, significance was determined using unpaired Student’s t test: 10:1/24 h, 20:1/24 h vs. 5:1/24 h (*p < 0.05, **p < 0.01, ***p < 0.001); 10:1/48 h, 20:1/484 h vs. 5:1/48 h (#p < 0.05, ##p < 0.01, ###p < 0.001). The experiment was repeated three times, using different cell culture flasks in each experimental condition.

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  Figure 2. T. cruzi infection modulates miR-145-5p and miR-146b-5p expression. (A) H9C2 cells were infected with 10:1 T. cruzi trypomastigotes for 4 h, washed and collected 4, 6, 8, 10, 24, 48 and 144 h post-infection. (B) Parasite load of H9C2 cells infected with T. cruzi. (C) Concentration of parasites released into the supernatant. (D) miR-145-5p levels, (E) miR-146b-5p levels. For all graphs, significance was determined using unpaired Student’s t test (*p < 0.05, **p < 0.01, ***p < 0.001). The experiment was repeated three times, using different cell culture flasks in each experimental condition.

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  Figure 3. Combined therapy with Benznidazole and Pentoxifylline reverse miR-145-5p and miR-146b-5p upregulation. (A) H9C2 cells were infected with 10:1 T. cruzi trypomastigotes for 4 h, washed, treated 48 h post-infection with 3 µM and 5 µM of Bz and collected 72 h later. (B) Parasite load of H9C2 cells infected with T. cruzi and treated with 3 µM and 5 µM of Bz. (C) miR-145-5p gene expression, (D) miR-146b-5p gene expression, (E) H9C2 cells were infected with 10:1 T. cruzi trypomastigotes for 4 h, washed, treated 48 h post-infection with 3 µM and 5 µM of Bz + PTX 0.125 mg/mL and 0.250 mg/mL and collected 72 h later. (F) Parasite load of H9C2 cells infected with T. cruzi and treated with Bz + PTX. (G) miR-145-5p levels, (H) miR-145-5p levels. For all graphs, significance was determined using unpaired Student’s t test or Mann–Whitney Rank Sum (*p < 0.05, **p < 0.01, ***p < 0.001. The experiment was repeated three times, using different cell culture flasks in each experimental condition.

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  Figure 4. Pre- and post-treatment of miR-145-5p and miR-146b-5p TaqMan® mimic/inhibitor systems alters T. cruzi infection dynamics. (A) H9C2 cells were pre-treated with miR-145-5p or miR-146b-5p TaqMan mimic/inhibitor systems for 48 h, infected with T. cruzi for 4 h, washed and collected 72 h later. (B) Parasite load of H9C2 cells pre-treated with miR-145-5p TaqMan mimic/inhibitor system. (C) Parasite load of H9C2 cells pre-treated with miR-146b-5p TaqMan mimic/inhibitor system. (D) H9C2 cells were infected with T. cruzi for 4 h, washed, post-treated with miR-145-5p or miR-146b-5p TaqMan mimic/inhibitor systems for 48 h and collected 72 h later. (E) Parasite load of H9C2 cells post-treated with miR-145-5p TaqMan mimic/inhibitor system. (F) Parasite load of H9C2 cells post-treated with miR-146b-5p TaqMan mimic/inhibitor system. All controls are added with Lipofectamine RNAiMAX. For all graphs, significance was determined using unpaired Student’s t test or Mann–Whitney Rank Sum (*p < 0.05, **p < 0.01, ***p < 0.001). The experiment was repeated three times, using different cell culture flasks in each experimental condition.