Tehran University of Medical Sciences Iranian Journal of Parasitology 1735-7020 18 3 2023 09 22 390 399 EN Faezeh Hamidi 1. Department of Parasitology and Mycology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran 2. Department of Laboratory Sciences and Microbiology, Faculty of Medical Sciences, Tabriz Medical Sciences, Islamic Azad University, Tabriz, Iran Samira Mohammadi -Yeganeh 1. Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran 2. Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran Mostafa Haji Molla Hoseini Department of Immunology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran Seyyed Javad Seyyed Tabaei Department of Parasitology and Mycology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran Niloofar Taghipour Department of Tissue Engineering and Applied Cell Sciences, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran Ameneh Koochaki Department of Immunology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran Vahedeh Hosseini Department of Molecular Medicine and Genetics, Faculty of Medicine, Kurdistan University of Medical Sciences, Sanandaj, Iran Ali Haghighi Department of Parasitology and Mycology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran 2022 08 02 2023 09 22 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 TM- 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. https://ijpa.tums.ac.ir/index.php/ijpa/article/view/3696 https://ijpa.tums.ac.ir/index.php/ijpa/article/download/3696/1312