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5'isomiR-183-5p|+2 elicits tumor suppressor activity in a negative feedback loop with E2F1
Xiaoya Li1,2,3, Birgitta Elisabeth Michels1, Oyku Ece Tosun1,4
1Division of Molecular Genome Analysis, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 580, 69120, Heidelberg, Germany.
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|+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|0, miR-183-5p|+1 and miR-183-5p|+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|+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|+2. Knockdown of E2F1 partially phenocopied the effect of miR-183-5p|+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|+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.
Insights
This study reveals that different variants of microRNA-183-5p (isomiRs) have distinct roles in triple-negative breast cancer. One specific isomiR, miR-183-5p|+2, inhibits cancer cell proliferation by targeting E2F1, suggesting a regulatory feedback loop.
Area of Science:
- Molecular Biology
- Cancer Research
- Genetics
Background:
- MicroRNAs (miRNAs) and their variants (isomiRs) are crucial regulators of gene expression in tumorigenesis.
- 5'isomiRs possess unique seed sequences, leading to altered target specificity and broader phenotypic effects compared to canonical miRNAs.
- The specific functions of most 5'isomiRs remain largely uncharacterized.
Purpose of the Study:
- To investigate the distinct functions of microRNAs (miRNAs) and their 5'isomiRs.
- To explore the role of miR-183-5p and its variants in breast cancer, particularly in triple-negative breast cancer (TNBC).
Main Methods:
- Assessed 5'isomiR expression in The Cancer Genome Atlas (TCGA) breast cancer dataset.
- Investigated phenotypic effects (migration, proliferation, tumor growth, metastasis) of miR-183 overexpression in TNBC cell lines in vitro and in vivo.
- Validated direct targeting of E2F1 by miR-183-5p|+2 using a 3'UTR luciferase assay.
Main Results:
- Three variants of miR-183-5p (miR-183-5p|0, miR-183-5p|+1, miR-183-5p|+2) were highly expressed in TCGA breast cancer data.
- Overexpression of pre-miR-183 reduced proliferation and invasion in TNBC cell lines; miR-183-5p|+2 specifically inhibited proliferation and cell cycle progression.
- miR-183-5p|+2 directly targets E2F1, reducing E2F target gene expression and potentially forming a negative feedback loop with E2F1-mediated regulation of miR-183-5p expression.
Conclusions:
- 5'isomiRs derived from the same pre-miRNA can possess distinct functions, collectively influencing phenotypes.
- miR-183-5p|+2 counteracts pre-miRNA expression by downregulating the transcriptional activator E2F1.
- This regulatory axis may serve as a mechanism to prevent uncontrolled cell proliferation, a process often dysregulated in cancer.
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