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. 2012;7(5):e37361.
doi: 10.1371/journal.pone.0037361. Epub 2012 May 21.

Identification of microRNAs inhibiting TGF-β-induced IL-11 production in bone metastatic breast cancer cells

Sirkku Pollari  1 , Suvi-Katri Leivonen, Merja Perälä, Vidal Fey, Sanna-Maria Käkönen, Olli Kallioniemi
Affiliations

Identification of microRNAs inhibiting TGF-β-induced IL-11 production in bone metastatic breast cancer cells

Sirkku Pollari et al. PLoS One. 2012.

Abstract

Development of bone metastases is dependent on the cancer cell-bone cell interactions in the bone microenvironment. Transforming growth factor β (TGF-β) is released from bone during osteoclastic bone resorption and induces production of osteolytic factors, such as interleukin 11 (IL-11), in breast cancer cells. IL-11 in turn increases osteolysis by stimulating osteoclast function, launching a vicious cycle of cancer growth and bone destruction. We aimed to identify and functionally characterize microRNAs (miRNAs) that mediate the bone metastatic process, focusing on miRNAs that regulate the TGF-β induction of IL-11. First, we profiled the expression of 455 miRNAs in a highly bone metastatic MDA-MB-231(SA) variant as compared to the parental MDA-MB-231 breast cancer cell line and found 16 miRNAs (3.5%) having a >3-fold expression difference between the two cell types. We then applied a cell-based overexpression screen with Pre-miRNA constructs to functionally identify miRNAs regulating TGF-β-induced IL-11 production. This analysis pinpointed miR-204, miR-211, and miR-379 as such key regulators. These miRNAs were shown to directly target IL11 by binding to its 3' UTR. MiR-379 also inhibited Smad2/3/4-mediated transcriptional activity. Gene expression analysis of miR-204 and miR-379-transfected cells indicated that these miRNAs downregulated the expression of several genes involved in TGF-β signaling, including prostaglandin-endoperoxide synthase 2 (PTGS2). In addition, there was a significant correlation between the genes downregulated by miR-379 and a set of genes upregulated in basal subtype of breast cancer. Taken together, the functional evidence and clinical correlations imply novel mechanistic links between miRNAs and the key steps in the bone metastatic process in breast cancer, with potential clinical relevance.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Effects of 55 miRNA precursors and a negative control on IL-11 secretion in MDA-MB-231(SA) cells.
Cells were transfected with miRNA precursors in 96-well plates. Medium was changed 24 hours after transfection (+/− TGF-β), and IL-11 concentration in the conditioned medium was measured 24 hours later. IL-11 concentration was normalized to the number of viable cells in the well. The scale of reduction (blue) or increase (red) in IL-11 concentration in the conditioned medium is represented in the color graph in the upper right hand corner of the figure.
Figure 2
Figure 2. The effects of miR-204, -211 and -379 on IL-11 secretion and IL11 mRNA expression.
A. IL-11 concentration in the conditioned medium of MDA-MB-231(SA) cells overexpressing miR-204, -211, or -379 (n = 3). B. IL11 mRNA expression changes in MDA-MB-231(SA) cells overexpressing miR-204, -211, or -379 (n = 3). C. IL-11 concentration in the conditioned medium of parental MDA-MB-231 cells whose miR-204, -211, or -379 expression was inhibited by specific Anti-miR inhibitors (n = 3). * p<0.05, ** p<0.01, *** p<0.001, as compared to the negative control Pre-miR or Anti-miR.
Figure 3
Figure 3. IL11 3′ UTR luciferase assay.
Luciferase reporter constructs, each containing one fragment of the IL11 3′ UTR, were co-transfected with a Renilla luciferase construct and the miRNA precursors into MDA-MB-231(SA) cells (n = 5), and luciferase activity was measured 28 hours later. * p<0.05, ** p<0.01, *** p<0.001, as compared to the negative control Pre-miR.
Figure 4
Figure 4. Genes over 1.5-fold downregulated in response to miR-204 and/or -379 versus negative control Pre-miR.
RNA samples for genome-wide gene expression analysis were collected 24 hours after MDA-MB-231(SA) cells had been transfected with miRNA precursors. The predicted targets of miR-204 are typed in red and the predicted targets of miR-379 in blue. Expression fold changes, gene names, and more detailed information about the target predictions are listed in the Tables S3 and S4.
Figure 5
Figure 5. Effects of miR-204, -211, and -379 on Smad signaling.
Smad signaling was quantified using a luciferase construct which encodes the firefly luciferase reporter gene under the control of a minimal (m)CMV promoter and tandem repeats of the Smad transcriptional response element. The luciferase reporter construct and miRNA precursors were co-transfected into MDA-MB-231(SA) cells (n = 3), and the medium was replaced with serum-free medium 16 hours after transfection. TGF-β was added 8 hours later. Activity of the firefly luciferase reporter and Renilla luciferase was measured after 16 hour TGF-β induction. * p<0.05, ** p<0.01, as compared to the Pre-miR negative control and Smad reporter-transfected cells.
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References

    1. Nevins JR, Potti A. Mining gene expression profiles: expression signatures as cancer phenotypes. Nat Rev Genet. 2007;8:601–609. DOI: 10.1038/nrg2137. - DOI - PubMed
    1. Gregory PA, Bert AG, Paterson EL, Barry SC, Tsykin A, et al. The miR-200 family and miR-205 regulate epithelial to mesenchymal transition by targeting ZEB1 and SIP1. Nat Cell Biol. 2008;10:593–601. DOI: 10.1038/ncb1722. - DOI - PubMed
    1. Wang L, Wang J. MicroRNA-mediated breast cancer metastasis: from primary site to distant organs. Oncogene. DOI: 10.1038/ 2011;onc.2011.444 10.1038/onc.2011.444. - PubMed
    1. Tavazoie SF, Alarcon C, Oskarsson T, Padua D, Wang Q, et al. Endogenous human microRNAs that suppress breast cancer metastasis. Nature. 2008;451:147–152. DOI: 10.1038/nature06487. - DOI - PMC - PubMed
    1. Dangi-Garimella S, Yun J, Eves EM, Newman M, Erkeland SJ, et al. Raf kinase inhibitory protein suppresses a metastasis signalling cascade involving LIN28 and let-7. EMBO J. 2009;28:347–358. DOI: 10.1038/emboj.2008.294. - DOI - PMC - PubMed

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