The role of mirna in the infection of dog spleen leukocytes by Leishmania Infantum

Author(s)

Larissa Martins Melo

• Document type: Doctoral Thesis
• Press: Araçatuba. 2019-01-28.
• Institution: Universidade Estadual Paulista (Unesp). Faculdade de Medicina Veterinária. Araçatuba
• Defense date: 2018-12-20
• Advisor: Valéria Marçal Félix de Lima

Abstract

Canine visceral leishmaniasis (VL) in Brazil represents a serious public health problem. In VL cellular immune suppression is determinant of disease progression. Studies have shown that the regulation of the immune response depends on miRNAs. The objective of this study was the characterization of the miRNAs in spleen leukocytes (SL) from dogs naturally infected by L. infantum. This study was carried out in Araçatuba, an endemic region for canine LV (LVC). A group of 4 healthy dogs and 8 dogs with VL was studied. Total RNA was extracted from SL using Mirvana Kit (Invitrogen®) according to manufacturer’s recommendations Total RNA was quantified using a fluorometer (Qubit 3.0, Invitrogen®) the degree of purity performed by capillary electrophoresis (Bioanalyser, Agilent®), the samples were then stored at -80°C. Total RNA was prepared for the microarray using the FlashTag Biotin HSR RNA Labeling Kit (Affymetrix®). The microarray was performed using the Affymetrix ™ miRNA 4.1 Strip according to manufacturer’s recommendations. cDNA production was performed using the miScript RTII kit (Qiagen®). Differentially expressed miRNAs were validated by qPCR was performed using the inventoried dog miRNAs (Qiagen®) and SYBR Green (kit miScript SYBR Green PCR, Qiagen®), according to the manufacturer's recommendation. The validated miRNAs were analyzed in the program Ingenuity Pathway Analysis (Qiagen®). After analysis of the pathways, then the LS from infected dogs were transfected with miR21 using miScript miRNA Mimics and Inhibitor (Qiagen®) according to manufacturer's recommendations. After the transfection LE the transcription factor T-bet and GATA 3, parasite load were measured by flow cytometry and IL-12 was quantified by ELISA Kit (R&D Systems®). The analyses of microarray was performed on Expression Console, Transcriptome Analysis Console (Affymetrix®); ANOVA, qPCR validation data was analyzed by Mann-Whitney test, the canonical pathway of miRNA differentially expressed in IPA with the Fisher test, the transcription factor, parasite load and expression of IL-12 was analysed by Friedman test, with the level of significance of p <0.05. It was found that 7 miRNAs had altered expression in infected dogs compared to healthy dogs: miR148a, miR21, miR7, miR615 were upregulated and miR150, miR125a and miR125b were downregulated. MiR148a, miR615 and miR21 validated the results of the microarray. The IPA showed 114 pathways regulated by these miRNAs, including pathways which regulate immunity. After transfection, the miR21 inhibitors increased T-bet and IL12 and decreased parasitic load. We conclude that miR 21 interferes in the immune response of dogs infected with L. infantum, inhibiting the cell type response. This information can assist in the identification of therapeutic targets in LVC. (AU)

• FAPESP's process: 15/16101-6 - The role of miRNAs in mononuclear cells infection of peripheral blood and spleen leukocytes the dog by Leishmania infantum
• Grantee: Larissa Martins Melo
• Support Opportunities: Scholarships in Brazil - Doctorate