Background: Immune cells and their secreted cytokines are known as the first barrier against pathogens. Leishmania major as an intracellular protozoan produces anti-inflammatory cytokines that lead to proliferation and survival of the parasite in the macrophages. miRNAs are small non-coding RNA molecules that regulate mRNAs expression. We aimed to investigate the relationship between the expression of TGF-β and a bioinformatically candidate miRNA, in leishmaniasis as a model of TGF-β overexpression.
Methods: The miRNAs that target TGF-β -3'UTR were predicted and scored by bioinformatic tools. After cloning of TGF-β-3'UTR in psi-CHECK ™- 2 vector, targeting validation was confirmed using Luciferase assay. After miRNA mimic transfection, the expression of miR-27a, TGF-β, as well as Nitric Oxide concentration was evaluated.
Results: miR-27a received the highest score for targeting TGF-β in bioinformatic predictions. Luciferase assay confirmed that miR-27a is targeting TGF-β-3'UTR, since miR-27a transfection decreased the luciferase activity. After miRNA transfection, TGF-β expression and Nitric Oxide concentration were declined in L. major infected macrophages.
Conclusion: Bioinformatic prediction, luciferase assay, and miRNA transfection results showed that miR-27a targets TGF-β. Since miRNA and cytokine-base therapies are developing in infectious diseases, finding and validating miRNAs targeting regulatory cytokines can be a novel strategy for controlling and treating leishmaniasis.
Keywords: Immunity; Leishmania major; Luciferase assay.
© 2023 Hamidi et al. Published by Tehran University of Medical Sciences.