title: The highly expressed 5'isomiR of hsa-miR-140-3p contributes to the tumor-suppressive effects of miR-140 by reducing breast cancer proliferation and migration.
abstract: 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, the so-called 5'isomiRs exhibit a shifted 5' 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 5'isomiRs. Among them, hsa-miR-140-3p was of particular interest because its 5'isomiR 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 5'isomiR-140-3p were highly expressed in patients' tumors compared to normal breast tissue. In the current work, we present the functional characterization of 5'isomiR-140-3p and the cellular phenotypes associated with its overexpression in MCF10A, MDA-MB-468 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 5'isomiR-140-3p led to a decrease in cell viability. The latter observation was supported by cell cycle analysis, where the 5'isomiR-140-3p but not the hsa-miR-140-3p caused cell cycle arrest in G0/G1-phase. Additionally, 5'ismoiR-140-3p overexpression was found to cause a decrease in cell migration in the three cell lines. We identified three novel direct target genes of the 5'isomiR-140-3p; COL4A1, ITGA6 and MARCKSL1. Finally, we have shown that knocking down these genes partially phenocopied the effects of the 5'isomiR-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 functional synergy between the canonical hsa-miR-140-3p and the newly identified 5'isomiR-140-3p in suppressing growth and progression of breast cancer by simultaneously targeting genes related to differentiation, proliferation, and migration.
authors: Division of Molecular Genome Analysis, German Cancer Research Center (DKFZ), INF580, Heidelberg, 69120, Germany.
authors: Omar
authors: O
authors: Salem
authors: Division of Molecular Genome Analysis, German Cancer Research Center (DKFZ), INF580, Heidelberg, 69120, Germany.
authors: Nese
authors: N
authors: Erdem
authors: Division of Molecular Genome Analysis, German Cancer Research Center (DKFZ), INF580, Heidelberg, 69120, Germany.
authors: Janine
authors: J
authors: Jung
authors: Division of Molecular Genome Analysis, German Cancer Research Center (DKFZ), INF580, Heidelberg, 69120, Germany.
authors: Ewald
authors: E
authors: Münstermann
authors: Division of Molecular Genome Analysis, German Cancer Research Center (DKFZ), INF580, Heidelberg, 69120, Germany.
authors: Angelika
authors: A
authors: Wörner
authors: Division of Molecular Genome Analysis, German Cancer Research Center (DKFZ), INF580, Heidelberg, 69120, Germany.
authors: Heike
authors: H
authors: Wilhelm
authors: Division of Molecular Genome Analysis, German Cancer Research Center (DKFZ), INF580, Heidelberg, 69120, Germany.
authors: Stefan
authors: S
authors: Wiemann
authors: Division of Molecular Genome Analysis, German Cancer Research Center (DKFZ), INF580, Heidelberg, 69120, Germany. c.koerner@dkfz.de.
authors: Cindy
authors: 0000-0003-1150-9462
authors: C
authors: Körner
mesh_terms: D001943:Breast Neoplasms / Q000235:genetics*; D002453:Cell Cycle / Q000235:genetics; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000235:genetics; D049109:Cell Proliferation; D018899:CpG Islands; D019175:DNA Methylation; D005260:Female; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D055785:Gene Knockdown Techniques; D016147:Genes, Tumor Suppressor; D006801:Humans; D035683:MicroRNAs / Q000235:genetics*; D008954:Models, Biological; D034622:RNA Interference; D059368:RNA Isoforms
chemical_list: D035683:MicroRNAs; C544302:Mirn140 microRNA, human; D059368:RNA Isoforms
keywords: Breast cancer; IsomiRs; MiR-140; MiRNA; Migration; Proliferation; Seed sequence