Systems Studies Uncover miR-146a as a Target in Leishmania major Infection Model

Abstract

Leishmaniasis, the second most neglected tropical disease, has been reported to affect approximately 12 million people worldwide. The causative protozoan parasite Leishmania has shown drug resistance to available chemotherapies, owing to which we need to look for better approaches to deal with the clinical situations. As per recent reports, several miRNAs have been found to be differentially expressed during Leishmania major infection in host macrophages. We aim to evaluate the impact of miRNA-mediated gene regulation on the key players of inflammation and macrophage dysfunction. The origin of Leishmania miRNAs and their processing is a questionable phenomenon as of yet. Through our study, we aim to provide a framework of their characterization. We amalgamate chemical systems biology and synthetic biology approaches to identify putative miRNA targets and unravel the complexity of host-pathogen gene regulatory networks.

Figure 1

During L. major infection in macrophages, miRNAs targeting the mRNA of proteins involved in the pro-inflammatory response gets upregulated and miRNAs targeting mRNA of proteins involved in the anti-inflammatory response gets downregulated, aiding in the survival of the parasite inside the macrophages.

Figure 2

(a) Robust miRNA regulatory network generated after simulated annealing. (b) Generated network in a circular layout after applying out- and in-degree filters; miR-146a-3p, miR-146a-5p, miR-155-3p, miR-155-5p, miR-122, miR-9-3p, miR-9-5p, miR-188, and miR-147 were filtered out.

Figure 3

Heatmap and hierarchical clustering of infection-specific miRNAs. Heatmap and hierarchical clustering of 48 infection-specific differentially expressed key miRNAs in THP-1 cells in response to Leishmania infection at 3, 6, 12, and 24 h as compared to the infected 0 h control. The heatmap shows several members of key families of miRNAs (miR-146, let-7, miR-30, miR-155, miR-21, and miR-9) differentially modulated across different time points. The miRNA expression values are presented using a green (upregulated), yellow (median value), and red (downregulated) color scheme.

Figure 4

Heatmap generated and Pathway cluster dendrogram generated by DIANA Tools representing involvement of key miRNAs in inflammatory response such as Fc-gamma R-mediated phagocytosis, mTOR signaling pathway, TGF-β signaling pathway, B-cell receptor signaling pathways, T-cell receptor signaling pathways and MAPK signaling pathways.

Figure 5

Ten motifs generated against 24 human miRNAs with their respective E values and length of the motifs and predicted 3D structures of discovered motifs generated by RNAComposer.

Figure 6

Graph depicting the frequency of occurrence of human miRNA motifs in the L. major genome. It is observed that motifs 6 (AGCAGCA), 5 (CTCAGC), and 9 (CCCTTT) are frequently occurring in the L. major genome.

Figure 7

Human Argonaute 2 proteins were blind-docked with each motif from which motifs 5 and 7 had more stable ligand receptors, considering their binding energies (kCal/mol).

Figure 8

TGF-β signaling cascade in relation to the miRNA of interest (miR-146a).

Figure 9

(a) Synthetic circuit design for miR-146a, SMAD 7 with their respective plasmid maps and (b) Transfection study on THP1 cells transfected with miR-146a and SMAD7 designed synthetic circuit via Lipofectamine 3000.