Down-regulation of BRCA1 expression by miR-146a and miR-146b-5p in triple negative sporadic breast cancers.
Bottom Line: This was further confirmed with the endogenous BRCA1 gene by transfecting microRNA (miRNA) precursors or inhibitors in mammary cell lines.This down-regulation was accompanied by an increased proliferation and a reduced homologous recombination rate, two processes controlled by BRCA1.Furthermore, we showed that the highest levels of miR-146a and/or miR-146b-5p are found in basal-like mammary tumour epithelial cell lines and in triple negative breast tumours, which are the closest to tumours arising in carriers of BRCA1 mutations.
Affiliation: CNRS UMR5286 Inserm U1052, "Equipe Labellisée LIGUE 2008", University Lyon, Cancer Research Center of Lyon, Centre Léon Bérard, France.Show MeSH
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Mentions: We first tested the influence of miR-9, miR-17-5p, miR-146a, and miR-146b-5p on their predicted messenger target by using a reporter vector into which we inserted the entire 3′UTR of BRCA1 downstream of the firefly luciferase open reading frame (ORF). This reporter vector, that we named Luc-BRCA1 3′UTR, was transfected into HeLa cells with a control vector encoding no miRNA, with a miR-Vec construct encoding let-7i for which no binding site in BRCA1 3′UTR is predicted by any of the four algorithms used, or with miR-Vec constructs encoding miR-9, miR-17-5p or miR-146a (Voorhoeve et al, 2006). These latter are expressed at low levels or are not expressed in HeLa cells according to Cheng and colleagues (Cheng et al, 2005) and/or Nelson and colleagues (Nelson et al, 2004). Whereas miR-146a expression reduced luciferase activity by ∼20% compared to control vector transfection, weak or no statistical effect was observed with miR-9, miR-17-5p or with let-7i (Fig 1A). Although miR-146a and miR-146b-5p are encoded by two different genes (located on different chromosomes), their seed region is identical and their mature sequences differ by only 2 nt (Fig 1B). The unique target site on the 3′UTR of BRCA1 (nt 489–507) predicted by three algorithms (MicroInspector, TargetScan 3.1 and RNA22) is common to both miRNAs (Fig 1B). To confirm the effect of miR-146a and to explore that of miR-146b-5p, we then transfected Luc-BRCA1 3′UTR into HeLa cells with miR-146a or miR-146b-5p synthetic precursors or with a negative control precursor that does not target any known mRNA within the human transcriptome. With both miRNAs, the degree of luciferase inhibition reached 50–60% compared to the control precursor (Fig 1C). This higher level of inhibition was expected as synthetic precursors have been shown to be more effectively delivered and more active than plasmids expressing miRNAs. As expected, considering the fact that miR-146a and miR-146b-5p share the same binding site on the 3′ UTR of BRCA1, cotransfection of both synthetic precursors did not increase the extent of inhibition (Fig 1C). When the Luc-BRCA1 3′UTR vector was mutated within this target site, miR-146a- or miR-146b-5p-mediated repression was no longer observed in cotransfection experiments (Fig 1C), suggesting specificity of the repression effect.
Affiliation: CNRS UMR5286 Inserm U1052, "Equipe Labellisée LIGUE 2008", University Lyon, Cancer Research Center of Lyon, Centre Léon Bérard, France.