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Comparative 5’ isomiRome analysis uncovers dysregulated 5’ isomiRs in Trypanosoma cruzi-infected macrophages

ABSTRACT: Trypanosoma cruzi (T. cruzi), the causative agent of Chagas disease, remains a major global health threat lacking effective therapies. Macrophages are key effector cells in the host response to T. cruzi infection, yet their incomplete clearance of T. cruzi contributes to Chagas disease pathogenesis. 5’ isomiRs, miRNA isoforms with shifted 5’ ends and altered seed sequences, have emerged as important regulators in diverse diseases. However, their expression alternations in T. cruzi-infected macrophages remain largely unknown. Here, we performed comparative analyses of 5’ isomiR expression in T. cruzi-infected macrophages. We found that T. cruzi infection induced a pronounced and selective increase in miRNA 5’-end heterogeneity in THP-1-derived macrophages, but not in cardiomyocytes or epithelial cells. Comparative 5’ isomiRome analyses further identified 68 differentially expressed 5’ isomiRs in THP-1 macrophages, most of which were specific to T. cruzi infection compared with other pathogens. Among these, 56 5’ isomiRs were associated with Argonaute proteins, including three originating from the miR-1246 precursor. Focusing on miR-1246|+1, a 5’ isomiR generated by a one-nucleotide downstream shift at the 5’ end, we showed that its overexpression significantly downregulated target genes involved in NF-κB signaling, cytokine responses, and cell migration. Notably, miR-1246|+1 broadly suppressed NF-κB family transcription factors and their downstream effector genes, thereby inhibiting pro-inflammatory M1 macrophage phenotype. Collectively, these findings reveal cell type- and pathogen-specific reprogramming of the 5’ isomiR landscape in T. cruzi-infected macrophages and identify miR-1246|+1 as a potential post-transcriptional regulator of macrophage inflammatory polarization.

ORGANISM(S): Homo sapiens

PROVIDER: GSE328447 | GEO | 2026/08/26

REPOSITORIES: GEO

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Project description:miRNAs are small noncoding RNA molecules that play an important role in post-transcriptional regulation of gene expression. Length and/or sequence variants of the same miRNA are termed isomiRs. While most isomiRs are functionally redundant compared to their canonical counterparts, so-called 5âisomiRs exhibit a shifted 5â end and therefore a shifted seed sequence resulting in a different target spectrum. However, not much is known about the functional relevance of these isoforms. Analysis of miRNA-seq data from breast cancer cell lines identified six pairs of highly expressed miRNAs and associated 5âisomiRs. Among them, hsa-miR-140-3p was of particular interest because its 5âisomiR showed higher expression compared to the canonical miRNA annotated in miRbase. This miRNA has previously been shown to control stemness of breast cancer cells. MiRNAseq data of breast cancer patients (TCGA dataset) showed that both the canonical hsa-miR-140-3p and its 5âisomiR-140-3p were highly expressed in patients compared to normal breast tissue. In the current work, we present the functional characterization of 5âisomiR-140-3p and the cellular phenotypes associated with its overexpression in MCF10A and MDA-MB-231 cell lines in comparison to the canonical hsa-miR-140-3p. Contrary to the effect of the canonical hsa miR 140-3p, overexpression of the 5âisomiR-140-3p led to a decrease in cell viability. The latter observation was supported by cell cycle analysis, where the 5âisomiR-140-3p but not the hsa-miR-140-3p caused cell cycle arrest in G0/G1-phase. Additionally, 5âismoiR-140-3p overexpression was found to cause a decrease in cell migration in MCF10A cells. We identified three novel direct target genes of the 5â isomiR-140-3p; COL4A1, ITGA6 and MARCKSL1. Finally, we have shown that knocking down these genes partially phenocopied the effects of the 5âisomiR-140-4p overexpression, where COL4A1 and ITGA6 knockdown led to reduced cell viability and cell cycle arrest, while MARCKSL1 knockdown resulted in a decrease in the migratory potential of cells. In summary, this work presents evidence that there is a functional synergy between the canonical hsa-miR-140-3p and the newly identified 5âisomiR-140-3p in suppressing growth and progression of breast cancer by simultaneously targeting genes related to differentiation, proliferation, and migration. With this array, we aimed to address the question which genes are regulated by either of the two forms of the miRNA. Samples were measured in one biological replicate of cells transfected with mimic-ctrl1 and mimic-ctrl2 (Dharmacon) as control samples and two biological replicates of cells transfected with hsa-miR-140-3p and 5'isomiR-140-3p (Exiqon) in 30nM concentration using Lipofectamin 2000 as transfection reagent.

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Comparative 5’ isomiRome analysis uncovers dysregulated 5’ isomiRs in Trypanosoma cruzi-infected macrophages

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