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MicroRNAs and their isomiRs function cooperatively to target common biological pathways (Illumina expression beadchip)

ABSTRACT: Variants of microRNAs, (isomiRs) are commonly reported in deep-sequencing studies; however the functional significance of these variants remains controversial. To assess their biological relevance, we have performed ultra-deep miRNA-seq on ten adult human tissues, and created an analysis pipeline (miRNA-MATE) to align, annotate, and analyze miRNAs and their isomiRs. We find that isomiRs share sequence and expression characteristics with canonical miRNAs, and are generally strongly correlated with canonical miRNA expression. We isolated polyribosome-associated mRNA, and captured the mRNA-bound miRNAs, and found that isomiRs and canonical miRNAs are equally associated with translational machinery. Finally, we transfected cells with biotinylated RNA duplexes encoding isomiRs or their canonical counterparts and directly assayed their mRNA targets. These studies showed substantial overlap in functional mRNA networks suppressed by both canonical miRNAs and their isomiRs. Together, these results find isomiRs to be biologically relevant and functionally synergistic partners of canonical miRNAs that act coordinately to target pathways of functionally related genes. This work exposes the complexity of the miRNA-transcriptome, and helps explain a major miRNA paradox: how specific regulation of biological processes can occur with predominantly non-specific miRNA-mRNA interactions.

ORGANISM(S): Homo sapiens

PROVIDER: GSE29043 | GEO | 2011/12/21

SECONDARY ACCESSION(S): PRJNA142907

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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MicroRNAs and their isomiRs function cooperatively to target common biological pathways (Illumina expression beadchip)

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