By Maurice Walker,2014-06-06 12:07
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    Tumor suppressor BRCA1 epigenetically controls oncogenic microRNA-155RESULTS

    R1699Q variant affects ES cell survival and differentiation

    we observed tenfold lower survival of R1699Q BRCA1–expressing Brca1ko/ko ES cells compared with wild-type BRCA1

    To determine the effect of R1699Q mutation

    differentiated the ES cells into embryoid carried out comparative gene expression


    Histological analysisseveral altered signaling pathways

    The cells of the outer layer of the R1699Q R1699Q ES cells are prone to spontaneous

    embryoid bodies were TUNEL+differentiation

    miR-155 is upregulated in R1699Q mutant cells

    Dicer1-null ES cells have a very similar phenotype to that of the R1699Q ES cells, this phenotypic similarity led us to investigate whether miRNA expression is misregulated in wild-type and We confirmed miR-155 upregulation in In situ hybridization revealed ~40-fold

    R1699Q cells by real-time PCRupregulation of miR-155

    R1699Q embryoid bodies

    tested the relation of the upregulation and the

    differentiation defects observed in the R1699Q ES cells

    Using a tet-inducible system we overexpressed miR-155 in wild-type ES cells

    H&E-stained control embryoid bodies are not exactly like wild-type embryoid

    bodies (Fig. 2e), probably as a result of leaky expression of miR-155.

    miR-155, at least in part, contributed to the

    differentiation defects observed in R1699Q ES cells

    miR-155 is upregulated

    in BRCA1-deficient cells

    test this possibility that BRCA1 controls miR-155 expression we measured miR-155 expression in tumors from Brca1ko/+;Trp53ko/+;TgR1699Q mice, which have Brca1 and Trp53 knockout alleles on the same

    chromosomeloss of wild-type Brca1 is required for miR-155 upregulationtested a panel of human breast cancer cell lines with different BRCA1 genotypes to examine whether the deregulation of miR-155 is specific to R1699Q or whether the complete loss of BRCA1 has a similar effect

miR-155 expression analysis in the primary tumors from the Brca1 conditional knockout mouse

    model35 showed that miR-155 was consistently upregulated in BRCA1-deficient tumorsonly one of the four tumors from Her2/Neu transgenic mice overexpressed miR-155

    BRCA1 negatively controls miR-155 and that loss of BRCA1 function leads to the upregulation of miR-155.

    BRCA1 epigenetically

    represses miR-155 promoterexamined at which step(s) BRCA1 is involved

    BRCA1 controls miR-155 expression by regulating the transcription of pri-miR-155.;一?

    hypothesized that BRCA1 associates with the miR-155

    promoter and suppresses its transcription

    Sequence analysis of the mouse miR-155 promoter showed two regions with

    putative BRCA1-binding sites38 (BRCA1-1 and BRCA1-2

    ChIP analysis of the promoter using primers showed that BRCA1 binds to the BRCA1-2 region

    carried out mutation analysis

    Each mutant led to miR-155 double mutant did not show any additive

    promoter activationeffect

    the binding of R1699Q BRCA1 to the miR-155 promoter was comparable to wild-type

    reasoned that BRCA1 may recruit other transcriptional repressors and that the interaction

    is disrupted without functional BRCA1

    None of the known co-repressors of BRCA1 affected BRCA1-dependent repression of miR-155 promoter AND R1699Q BRCA1 had no effect on the expression of other known transcriptional targets

    BRCA1 may regulate miR-155 by a different mechanism


    BRCA1 is associated with the histone deacetylase (HDAC) complex, we

    examined the possibility that BRCA1 epigenetically controls miR-155.Treatment with HDAC inhibitors in in the two BRCA1-deficient cell lines,miR-

    MDA-MB-468 (BRCA1+) led to a two- 155 expression did not increase in response

    to fourfold increase of miR-155to the HDAC inhibitors

    miR-155 promoter is controlled epigenetically and that the regulation is abrogated

    without BRCA1 supported by luciferase reporter analysis and mutation reaction


    tested whether BRCA1 affects the acetylation of one or more histones on the miR-155 promoter, which in turn could activate or silence the gene.

    ChIP analysis using a series of antibodies recognizing acetylated histonesH2A and H3 were more acetylated in BRCA1-deficient MDA-MB-436 cells than in BRCA1+ MCF7 cells indicating that BRCA1 is responsible for these acetylation changes.the acetylation of H2A and H3 was lower when wild-type BRCA1 was expressed in MDA-MB-436 cells, whereas R1699Q overexpression did not have this effectdemonstrate that BRCA1 negatively controls the miR-155 promoter by decreasing the acetylation of H2A and H3.

    identify the HDAC involved in the deacetylation of histones H2A and H3 at the miR-155 promoter by BRCA1


    ChIP analysis using antibodies to HDAC1–HDAC5 and

    HDAC7a specific association between HDAC2 and the miR-155 promoter in cells expressing wild-type BRCA1

    in R1699Q cells, HDAC2 association with miR-155 promoter was similar to the IgG controltested the interaction between HDAC2 and BRCA1 by

    coimmunoprecipitation in mouse mammary epithelial cells

    found interaction between HDAC2 and wild-type or M1652I BRCA1

    but not between HDAC2 and R1699Q BRCA1

    Because R1699Q BRCA1 associated with the miR-155 promoter but did not interact with HDAC2 , we questioned whether R1699Q BRCA1 has a dominant-negative effect on miR-155 regulation

    coexpressed R1699Q mutant with wild-type BRCA1

    R1699Q BRCA1 does not have a dominant-negative effect

    Effect of

    miR-155 the epigenetic regulation of miR-155 promoter by BRCA1 is specific.on



    examine the effect of overexpression of miR-155 observed in BRCA1 mutant tumors or tumor cell lines

    stably expressed miR-155 in BRCA1+, low miR-155–expressing MDA-MB-468 cellsmiR-155 is oncogenic in this breast cancer cell line, consistent with a recent report showing that miR-155 supports the growth of MDA-MB-231 cells

    tested the effect of miR-155 inhibition in BRCA1-deficient cells on in vivo tumor cell growth;二?

    the knockdown of miR-155 inhibits in vivo tumor growth of BRCA1-deficient cells.miR-155 upregulation in BRCA1-mutant human breast tumors

    examine the correlation between BRCA1 status and miR-155 expression in human breast cancer

    screened miR-155+ tumors by in situ assessed BRCA1 status by immunohistochemistry

    hybridizationclassified the tumors into groups of 50 BRCA1+

    20 cases showing marked upregulationtumors and 16 BRCA1-weak or BRCA1? tumors

    These results show a significant correlation between loss of BRCA1 and upregulation of miR-155examined human tumors with mutation in BRCA1

    show that miR-155 upregulation is correlated with BRCA1 mutation in human breast tumorsDISCUSSION

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