Abstract
Aberrant microRNA expression contributes to breast cancer progression and endocrine resistance. We reported that although tamoxifen stimulated miR-29b-1/a transcription in tamoxifen (TAM)-resistant breast cancer cells, ectopic expression of miR-29b-1/a did not drive TAM-resistance in MCF-7 breast cancer cells. However, miR-29b-1/a overexpression significantly repressed TAM-resistant LCC9 cell proliferation, suggesting that miR-29b-1/a is not mediating TAM resistance but acts as a tumor suppressor in TAM-resistant cells. The target genes mediating this tumor suppressor activity were unknown. Here, we identify miR-29b-1 and miR-29a target transcripts in both MCF-7 and LCC9 cells. We find that miR-29b-1 and miR-29a regulate common and unique transcripts in each cell line. The cell-specific and common downregulated genes were characterized using the MetaCore Gene Ontology (GO) enrichment analysis algorithm. LCC9-sepecific miR-29b-1/a-regulated GO processes include oxidative phosphorylation, ATP metabolism, and apoptosis. Extracellular flux analysis of cells transfected with anti- or pre- miR-29a confirmed that miR-29a inhibits mitochondrial bioenergetics in LCC9 cells. qPCR,luciferase reporter assays, and western blot also verified the ATP synthase subunit genes ATP5G1 and ATPIF1 as bone fide miR29b-1/a targets. Our results suggest that miR-29 repression of TAM-resistant breast cancer cell proliferation is mediated in part through repression of genes important in mitochondrial bioenergetics.
Highlights
MicroRNAs are 22 nt non-coding RNAs that recognize and bind complementary seed sequences in the 3′-UTR region of a target messenger RNA[1, 2]
To identify potential miR-29b-1 and miR-29a targets in breast cancer cells and their possible role in tumor suppression, TAM- sensitive (TAM-S) MCF-7 and TAM-R LCC9 cells were grown in hormonally depleted medium and transfected with anti-miR-29a and pre-miR-29b-1 or pre-miR-29a (Supplementary Fig. 1)
MiR-29b-1 and miR-29a downregulated 447 and 139 genes in MCF-7 cells respectively (Fig. 1A) and 1,751 and 1,959 genes in LCC9 cells (Fig. 1B; p < 0.05), respectively. The identity of these genes with MetaCore pathway analysis is provided in Supplementary Tables 1 and 2. These data suggest that miR-29b-1 and miR-29a regulate more transcripts in TAM-R LCC9 cells compared to TAM-S MCF-7 cells
Summary
MicroRNAs (miRNA, miRs) are 22 nt non-coding RNAs that recognize and bind complementary seed sequences in the 3′-UTR region of a target messenger RNA (mRNA)[1, 2]. This results in translational repression and/or transcriptional degradation of the target gene. We recently reported that ERα mediates 4-hydroxyTAM (4-OHT) repression of miR-29b-1/a in MCF-7 and upregulation in TAM-R LCC9 and LY2 BC cells. MetaCoreTM functional analysis of the RNA-seq data identified several metabolic processes including oxidative phosphorylation (OXPHOS) uniquely regulated by both miR-29b-1 and miR-29a in LCC9 cells. We used qPCR, a luciferase reporter assay, and western blot to validate that two subunits of ATP synthase (Complex V in the oxidative phosphorylation respiratory chain), ATP5G1 and ATPIF1, are bona fide miR-29b-1/a targets
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