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Abstract

Triple-negative breast cancer is a highly aggressive breast cancer subtype and the treatment options are mainly limited to chemotherapy, however, the patients frequently develop resistance. As endogenous regulators of gene expression, microRNAs are involved in tumor development, progression and treatment resistance. microRNA sequence variants with a shifted seed sequence are termed 5’isomiRs and extend the complexity and impact of the miRNome in cancer. A shift in the seed sequence by only one nucleotide can drastically alter the target spectrum of a 5’isomiR compared to its canonical microRNA. Hence, this study aims at identifying microRNAs and 5’isomiRs with a potential role in tumorigenesis and chemoresistance and focuses on characterizing their functional differences in triple-negative breast cancer. I selected microRNAs and 5’isomiRs that were differentially expressed between tumor and normal tissue of patients from the TCGA cohort and, thus, potentially involved in tumorigenesis and chemoresistance. Growing mammospheres from MDA-MB-231, HCC1806 and SUM-159 cells that overexpressed the selected microRNAs as pooled library enriched for cells with increased stemness and chemoresistance. Read-out of the library composition by NanoString after several sphere generations revealed strong enrichment of pre-miR-103a-1. In validation experiments, pre-miR-103a-1 overexpression did not influence stemness or chemoresistance. In the second part of the project, I focused on the functional characterization of miR-1307-3p I0 and its 5’isomiR miR-1307-3p I1. Both were selected from the list of differentially expressed microRNAs based on their similar expression levels. Phenotypic assays in triple-negative breast cancer cell lines showed that both microRNAs reduce migration, miR-1307-3p I0 in a cell line-specific manner and less pronounced than miR-1307-3p I1. miR-1307-3p I1 repressed proliferation in a cell line-dependent context. Target predictions identified genes that might contribute to these phenotypes and explain differences between cell lines. The putative targets suggested that miR-1307-3p I0 plays a role in autophagy. In summary, I showed that miR-1307-3p I0 and I1 influence different and similar phenotypes in a partially cell line-dependent manner by targeting specific as well as shared putative target subsets. This study underlines how complex and context-dependent microRNAs and their 5’isomiRs modulate gene expression and that they are of biological relevance. Consequently, diagnostic, prognostic and therapeutic approaches should discriminate between 5’isomiRs.