Proteomic Signature Analysis Reveals the Impact of miR-379 Overexpression in a model of Triple Negative Breast Cancer

[Introduction]: Triple-negative breast cancer (TNBC) remains a challenging subtype with limited targeted treatment options, necessitating a deeper understanding of its molecular mechanisms for improved therapeutic strategies. Our group delves into the intricate interplay between miR-379 and the proteome landscape of TNBC, aiming to elucidate its potential as a therapeutic target. Previously, we transduced TNBC 4T1 murine breast cancer cells expressing luciferase (4T1-Luc) with miR-379 (4T1-379). Balb/c immunocompetent mice then received an orthotopic injection of 4T1-Luc or 4T1-379 cells. The promising data generated highlighted a suppressive role for miR-379 in regulation of tumour growth. The aim of this study was to elucidate the mechanism of action of miR-379. To achieve this, we analysed the proteomes of 4T1-Luc and 4T1-379 tumour tissues using liquid chromatography tandem mass spectrometry (LC-MS/MS) and identified the differentially expressed proteins (DEPs). The key DEPs were screened and functionally annotated by Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses, and Gene Set Enrichment Analysis (GSEA). This study is the first to profile the proteome of TNBC enriched with miR379-5p and will contribute to unravelling the molecular mechanisms of this tumour suppressing microRNA.

[Method]: Tissue samples of three Balb/c mice injected with 4T1-379 and three injected with 4T1-Luc cells with 2 technical replicates of each sample were collected and proteomes analyzed by LC-MS/MS. The MS data were then processed with MaxQuant and Perseus platform. The Perseus-processed LFQ data was then transferred to R programmer for further analysis. UMAP analysis was performed using the ‘umap’ package. The DEPs were identified using R package ‘limma’ and screened using P-adj < 0.05 as the thresholds. The interaction network of the DEPs was constructed with the STRING database. The genes of the DEPs were functionally annotated using R package ‘org.Mm.eg.db’, ‘msigdbr’ and ‘clusterProfiler’ for GO, KEGG pathways GSEA functional enrichment analysis.

[Results]: For this dataset, UMAP projections yield highly clusterable representations of the data points. A total of 173 differentially expressed proteins (DEPs) were identified, of which 64 were significantly down-regulated and 109 were significantly up-regulated in miR-379 enrichedsamples (4T1-379) relative to negative control (4T1-Luc). Among the 173 DEPs, 4 down-regulated and 33 up-regulated proteins had a fold change > 2 (|log2(FC)| > 1). The DEPs highly interact with each other enriched in GO terms including regulation of angiogenesis, epithelial cell migration, extracellular matrix organization, regulation of bone remodelling, activation of immune response. Expression datasets were subjected to GSEA and enriched in Hallmark and immunogenic gene sets from Human Molecular Signatures Database (MSigDB). The results showed 6 Hallmark gene sets were enriched including DNA repair and Epithelial Mesenchymal Transition (EMT). In addition, the enriched immunogenic gene sets revealed regulation of immune cells including Neutrophils, Macrophages, NKT cells, CD4+ T cells, CD8+T cells, and B cells.

[Conclusion]: In conclusion, the proteomic mapping of miR379-enriched TNBC tumour samples revealed a potential role for this miRNA in regulation of key processes associated with cancer progression. This strongly supports the hypothesis that miR-379 may be a valuable therapeutic target in breast cancer.