Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2011 May;3(5):279-90.
doi: 10.1002/emmm.201100136. Epub 2011 Apr 5.

Down-regulation of BRCA1 expression by miR-146a and miR-146b-5p in triple negative sporadic breast cancers

Amandine I Garcia  1 , Monique Buisson, Pascale Bertrand, Ruth Rimokh, Etienne Rouleau, Bernard S Lopez, Rosette Lidereau, Ivan Mikaélian, Sylvie Mazoyer
Affiliations

Down-regulation of BRCA1 expression by miR-146a and miR-146b-5p in triple negative sporadic breast cancers

Amandine I Garcia et al. EMBO Mol Med. 2011 May.

Abstract

Germ-line mutations in the BRCA1 gene strongly predispose women to breast cancer (lifetime risk up to 80%). Furthermore, the BRCA1 protein is absent or present at very low levels in about one third of sporadic breast cancers. However, the mechanisms underlying BRCA1 somatic inactivation appear multiple and are still not fully understood. We report here the involvement of miR-146a and miR-146b-5p that bind to the same site in the 3'UTR of BRCA1 and down-regulate its expression as demonstrated using reporter assays. 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. This work provides further evidence for the involvement of miRNAs in sporadic breast cancer through down-regulation of BRCA1.

PubMed Disclaimer

Figures

Figure 1
Figure 1. Binding of miR-146a and miR-146b-5p to BRCA1 3′UTR

  1. Relative luciferase activity after cotransfection into HeLa cells of the Luc-BRCA1 3′UTR reporter vector and of an empty miR-Vec construct (control vector), or of miR-Vec constructs expressing different miRNAs, as indicated. Error bars represent standard error of the mean (SEM) of four independent experiments. *p < 0.05; ***p < 0.001 (Student's t-test).

  2. Sequence alignment of miR-146a and miR-146b-5p and their complementary site in the schematically represented BRCA1 3′UTR. The seed sequence is bolded.

  3. Repression of luciferase activity after cotransfection into HeLa cells of the wild-type (wt) or mutated (mut146) Luc-BRCA1 3′UTR reporter vector and of control or miR-146 synthetic precursors, as indicated. Error bars represent SEM of four independent experiments.

  4. Western blot analysis with an antibody against IRAK1 or BRCA1 of proteins extracted from HeLa cells transfected with a control, miR-146a, miR-146b-5p or both miR-146a and miR-146b-5p precursors. The bands corresponding to BRCA1 were quantified relative to the α-tubulin loading control (BRCA1 normalized level) using the GelDoc™XR+ Imager (Bio-Rad) and the Image Lab™ software. The results shown are representative of at least three independent experiments.

Figure 2
Figure 2. miR-146 a/b-5p and BRCA1 expression levels in mammary cell lines

  1. Expression level of miR-146a and miR-146b-5p determined by quantitative RT-PCR in three mammary normal cell lines, eight tumour cell lines with a luminal transcriptional profile and seven tumour cell lines with a basal-like transcriptional profile. miR-146a and miR-146b-5p expression were normalized using RNU44 RNA expression. Error bars represent standard deviations (SD) for triplicates of one representative experiment.

  2. Western blot analysis with an antibody against BRCA1 of proteins extracted from a mammary normal and of seven tumour cell lines with a basal-like transcriptional profile. The bands were quantified relative to the α-tubulin loading control using the UVP BioImaging system (EC3) and the Quality One Software.

Figure 3
Figure 3. Modulation of miR-146a/b-5p and BRCA1 expression in three tumour mammary cell lines
Western blot analysis with an antibody against BRCA1 of proteins extracted from MDA-MB-468 (A), MDA-MB-436 (B) or MDA-MB-157 (C) cells.

  1. A. MDA-MB-468 cells were transfected with a control, miR-146a, miR-146b-5p or both miR-146a and miR-146b-5p precursors, a scrambled siRNA or a siRNA targeting the BRCA1 gene.

  2. B, C. MDA-MB-436 or MDA-MB-157 cellswere transfected with a control, anti-miR-146a, anti-miR-146b-5p or both anti-miR-146a and anti-miR-146b-5p LNA. The bands were quantified relative to the α-tubulin loading control as in Fig 1D. The results shown are representative of at least three independent experiments.

Figure 4
Figure 4. Proliferation and homologous recombination rate of cells transfected with miR-146a/b-5p precursors

  1. Proliferation rate of HeLa cells transfected with a control or miR-146a and miR-146b-5p precursors. Proliferation rate was also measured after cotransfection with a BRCA1 expressing vector lacking the BRCA1 3′UTR [pBRCA1 (1–24)]. Error bars represent SEM for four independent experiments.

  2. Proliferation rate of MDA-MB-468 cells transfected with a control or miR-146a and miR-146b-5p precursors. Error bars represent SEM for four independent experiments.

  3. Rate of induced recombinant GFP positive cells (GFP+) either mock transfected or cotransfected with a control ormiR-146a and miR-146b-5p precursors and an I-SceI expressing plasmid. Error bars represent SEM for three independent experiments.

Figure 5
Figure 5. Relationship between miR-146a/b-5p expression and hormonal status in 167 mammary tumours
Expression level of miR-146a or miR-146b-5b in 167 mammary tumours and in different subgroups classified according to hormonal status (triple negative, i.e. ER negative/PR negative/no ERBB2 expression), or to SBR grades (I–III). Each point represents the expression level in one tumour while the line represents the median expression. p < 0.01; p < 0.001 (one-way analysis of variance with Tukey's multiple comparison test (ANOVA)).

  1. Expression level of miR-146a.

  2. Expression level of miR-146b-5p.

See this image and copyright information in PMC

References

    1. Abbott DW, Thompson ME, Robinson-Benion C, Tomlinson G, Jensen RA, Holt JT. BRCA1 expression restores radiation resistance in BRCA1-defective cancer cells through enhancement of transcription-coupled DNA repair. J Biol Chem. 1999;274:18808–18812. - PubMed
    1. Anczukow O, Ware MD, Buisson M, Zetoune AB, Stoppa-Lyonnet D, Sinilnikova OM, Mazoyer S. Does the nonsense-mediated mRNA decay mechanism prevent the synthesis of truncated BRCA1, CHK2, and p53 proteins. Hum Mutat. 2008;29:65–73. - PubMed
    1. Anglian Breast Cancer Study Group. Prevalence and penetrance of BRCA1 and BRCA2 mutations in a population-based series of breast cancer cases. Br J Cancer. 2000;83:1301–1308. - PMC - PubMed
    1. Aprelikova ON, Fang BS, Meissner EG, Cotter S, Campbell M, Kuthiala A, Bessho M, Jensen RA, Liu ET. BRCA1-associated growth arrest is RB-dependent. Proc Natl Acad Sci USA. 1999;96:11866–11871. - PMC - PubMed
    1. Baldassarre G, Battista S, Belletti B, Thakur S, Pentimalli F, Trapasso F, Fedele M, Pierantoni G, Croce CM, Fusco A. Negative regulation of BRCA1 gene expression by HMGA1 proteins accounts for the reduced BRCA1 protein levels in sporadic breast carcinoma. Mol Cell Biol. 2003;23:2225–2238. - PMC - PubMed

Publication types