5 ' isomiR-183-5p vertical bar+2 elicits tumor suppressor activity in a negative feedback loop with E2F1

Background: MicroRNAs (miRNAs) and isomiRs play important roles in tumorigenesis as essential regulators of gene expression. 5'isomiRs exhibit a shifted seed sequence compared to the canonical miRNA, resulting in different target spectra and thereby extending the phenotypic impact of the respective common pre-miRNA. However, for most miRNAs, expression and function of 5'isomiRs have not been studied in detail yet. Therefore, this study aims to investigate the functions of miRNAs and their 5'isomiRs. Methods: The expression of 5'isomiRs was assessed in The Cancer Genome Atlas (TCGA) breast cancer patient dataset. Phenotypic effects of miR-183 overexpression in triple-negative breast cancer (TNBC) cell lines were investigated in vitro and in vivo by quantifying migration, proliferation, tumor growth and metastasis. Direct targeting of E2F1 by miR-183-5p vertical bar+2 was validated with a 3'UTR luciferase assay and linked to the phenotypes of isomiR overexpression. Results: TCGA breast cancer patient data indicated that three variants of miR-183-5p are highly expressed and upregulated, namely miR-183-5p vertical bar 0, miR-183-5p vertical bar+1 and miR-183-5p vertical bar+2. However,TNBC cell lines displayed reduced proliferation and invasion upon overexpression of pre-miR-183. While invasion was reduced individually by all three isomiRs, proliferation and cell cycle progression were specifically inhibited by overexpression of miR-183-5p vertical bar+2. Proteomic analysis revealed reduced expression of E2F target genes upon overexpression of this isomiR, which could be attributed to direct targeting of E2F1, specifically by miR-183-5p vertical bar+2. Knockdown of E2F1 partially phenocopied the effect of miR-183-5p vertical bar+2 overexpression on cell proliferation and cell cycle. Gene set enrichment analysis of TCGA and METABRIC patient data indicated that the activity of E2F strongly correlated with the expression of miR-183-5p, suggesting transcriptional regulation of the miRNA by a factor of the E2F family. Indeed, in vitro, expression of miR-183-5p was regulated by E2F1. Hence, miR-183-5p vertical bar+2 directly targeting E2F1 appears to be part of a negative feedback loop potentially fine-tuning its activity. Conclusions: This study demonstrates that 5'isomiRs originating from the same arm of the same pre-miRNA (i.e. pre-miR-183-5p) may exhibit different functions and thereby collectively contribute to the same phenotype. Here, one of three isomiRs was shown to counteract expression of the pre-miRNA by negatively regulating a transcriptional activator (i.e. E2F1). We speculate that this might be part of a regulatory mechanism to prevent uncontrolled cell proliferation, which is disabled during cancer progression.

Automated summary

This study investigates the functions of miRNAs and their 5'isomiRs. The results show that three variants of miR-183-5p are highly expressed and regulate cell proliferation and invasion in different ways. Proteomic analysis reveals that miR-183-5p vertical bar+2 modulates the cell cycle by directly targeting E2F1. Gene set enrichment analysis suggests that the activity of E2F correlates with the expression of miR-183-5p. This study demonstrates that different isomiRs from the same pre-miRNA may have different functions, collectively contributing to the same phenotype.