EXPRESSION PROFILING OF MICRORNA IN LIPOPOLYSACCHARIDE CHALLENGED PERIPHERAL BLOOD MONONUCLEAR CELLS OF CATTLE
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Date
2023-03-23
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COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, KERALA VETERINARY AND ANIMAL SCIENCES UNIVERSITY
Abstract
MicroRNAs are endogenous, small non-coding RNAs approximately 20-22 nucleotides long which play a key role in gene regulation by affecting translation and transcription mechanisms. The present study was undertaken to analyse the
differences in the microRNA (miRNA) expression in (LPS) challenged peripheral blood mononuclear cells (PBMCs) of cattle. The current research work was carried out using the isolated blood samples from apparently healthy female crossbred cattle maintained at the University Livestock Farm and Fodder Research and Development Scheme, Mannuthy. Isolation of PBMCs were done by density gradient centrifugation using HisepTM lymphocyte separation medium 1077.Expression of miRNAs viz; bta-miR-451, bta-miR-107, bta-miR-93 and bta-miR-125a, whose expression was found to be varying in the LPS treated and untreated bovine PBMCs by miRNAome analysis of previous studies was validated in the present study by qRT-PCR assay. Insilico analysis was done using various online tools for the prediction of target genes of real time PCR validated miRNAs, gene ontology analysis and also to study the cellular pathways associated to the target genes. Targets of each miRNAs were predicted using the online target prediction programme TargetScan Human 8.0. Through the use of the online DAVID bioinformatics tool, gene ontology analysis of the real time PCR validated miRNAs was carried out. Predicted targets for each miRNAs were annotated to biological process, molecular function and cellular component categories. The pathway analysis programme (KEGG) of the DAVID database was utilized to analyze and interpret the pathways associated with the predicted targets of real time PCR verified miRNAs. The study also included the assessment of cytokines associated to the cellular pathways influenced by the target genes of real time PCR validated miRNAs in the supernatants of PBMC cultures from both treatment and control groups by ELISA. The expression of miRNAs; bta-miR-451and bta-miR-107 was found to up regulated while bta-miR-93 and bta-miR-125a showed decrease in their expression in LPS treated PBMCs when compared to control cells. The results of real time PCR validation of aforementioned miRNAs expression were in accordance with the findings of miRNAome analysis. Besides, significant enrichment of target genes of the real time validated miRNAs was noticed in many immune related GO terms as well as in critical immune associated cellular pathways. Measurement of cytokine, TNF α revealed a significantly higher level in LPS treated PBMCs when compared to LPS untreated cells. The findings of the current study will help in understanding the role of miRNAs in LPS mediated immune responses in cattle.