Abstract
HER2 positive Breast Cancers (BC) have aggressive behavior and poor prognosis. Previously, we have identified miR-342-5p as an upstream regulator of HER2 signaling, as well as inhibitor of HER2 positive BC cell line growth. Here, we aimed to further investigate the molecular mechanisms behind miR-342-5pinduced HER2 pathway deregulation. Two HER2 amplified breast cancer cell lines were transiently transfected with miR-342-5p mimic or negative control, and gene expression was analyzed by Agilent microarrays. Three clinical datasets with BC patients were used to identify correlations between candidate genes and miR-342- 5p, and associations with survival. Pathway analyses of all deregulated genes revealed a significant suppression of the HER2 downstream pathways ERK/MAPK and SAPK/JNK, whereas the miR-342-5p predicted target genes were enriched for pathways associated with cell motility.Biological functions linked to mitochondrial stability were ranked among the top toxicological functions in both gene lists. Among the most deregulated genes, Cytochrome B5 Reductase 3 (CYB5R3) and Rap Guanine Nucleotide Exchange Factor 6 (RAPGEF6) significantly anticorrelated and correlated, respectively, with miR-342-5p in all three clinical BC datasets. Low CYB5R3 levels and high RAPGEF6 levels were significantly associated with survival, although this was not directly associated with HER2 expression. Our data suggest that miR-342-5p overexpression in HER2 positive BC cell lines elicits broad effects on HER2 downstream signaling, cell motility and mitochondrial stability. Together these effects may render cells less proliferative and more sensitive to cellular stress.
Highlights
In recent years, microRNAs have revealed a wealth of new insights into the intrinsic regulation of transcription and translation of protein-coding RNAs
Identification of Differentially Regulated Genes after miR-342-5p Overexpression To investigate the mechanisms behind the inhibitory effects of miR-342-5p on human epidermal growth factor receptor 2 (HER2) signaling and cell growth, gene expression microarrays were performed after miR-3425p overexpression in two HER2 amplified breast cancer cell lines, JIMT-1 and KPL-4
In line with this observation, four of the ten most upregulated genes from the total gene list of 581 genes were associated with cellular contact (Table 3): The guanine exchange factor Rap Guanine Nucleotide Exchange Factor 6 (RAPGEF6), Keratin 80 (KRT80), Claudin 19 (CLDN19) and Delta Like Canonical Notch Ligand 1 (DLL1). 4.3. miR-342-5p can Suppress mRNAs Involved in Mitochondrial Dysfunction and Protection of Oxidative Stress Deregulation of mitochondrial function was enriched among the miR-342-5p regulated genes, within the top toxicological functions (Table 4)
Summary
MicroRNAs (miRNAs) have revealed a wealth of new insights into the intrinsic regulation of transcription and translation of protein-coding RNAs. We identified several miRNAs regulating cell growth and apoptosis, HER2 protein levels and inhibition of AKT and ERK1/2 downstream signaling mediators in HER2 positive cell lines. By combining the gene expression data with miRNA target predictions, we identified Cytochrome B5 Reductase 3 (CYB5R3) and Rap Guanine Nucleotide Exchange Factor 6 (RAPGEF6) as potential contributors to the tumor-inhibiting effects of miR-342-5p. Conclusion: Our data suggest that miR-342-5p overexpression in HER2 positive BC cell lines elicits broad effects on HER2 downstream signaling, cell motility and mitochondrial stability. Together these effects may render cells less proliferative and more sensitive to cellular stress.
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