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Potential clinical significance of ERβ ON promoter methylation in sporadic breast cancer

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Abstract

The aim of the study was to assess how hypermethylation of the ON promoter of the estrogen receptor beta (ERβ) gene affects its expression (at the mRNA and protein level) and to correlate these with some clinical and histopathological parameters. A total of 131 samples of frozen breast cancer tissue was analyzed. A custom-designed, two-step PCR method was used to measure the methylation index of the ERβ gene ON promoter region. Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) was performed to quantify mRNA of the ERβ1 isoform, while ERβ1 protein was determined using the Western blot method. There was a significant difference in the methylation index of the ERβ gene ON promoter between the groups of patients with negative and positive axillary lymph node status (P = 0.03). In addition, the methylation index of the ON promoter was positively correlated with estrogen receptor alfa (ERα) protein levels (ρ = 0.31, P = 0.02). There was a significant difference in the methylation index of the ON promoter between the progesterone receptor (PR)-negative and PR-positive groups of patients (P = 0.01). ERβ1 protein levels were negatively correlated with ERα protein (ρ = −0.27, P < 0.01). The methylation index of the ON promoter could be a more reliable additional parameter for prediction and/or prognosis in breast cancer than ERβ1-mRNA and/or protein levels.

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Acknowledgments

This work was supported by the Ministry of Education and Science, Republic of Serbia, grants ON173049 (Mandusić, V., Božović, A., Dimitrijević, B., Jovanović-Ćupić, S., Krajnović, M.) and ON175068 (Markicevic, M. and Lukić, S.).

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Authors and Affiliations

  1. Vinča Institute of Nuclear Sciences, University of Belgrade, Mike Alasa 12-14, 11001, Belgrade, Serbia

    Ana Božović, Bogomir Dimitrijević, Snežana Jovanović Ćupić, Milena Krajnović & Vesna Mandušić

  2. Institute for Oncology and Radiology of Serbia, Pasterova 14, 11000, Belgrade, Serbia

    Milan Markićević & Silvana Lukić

  3. Department for Radiobiology and Molecular Genetics, Vinča Institute of Nuclear Sciences, University of Belgrade, Mike Petrovića Alasa 12-14, 11000, Belgrade, Serbia

    Ana Božović

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  1. Ana Božović
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Correspondence to Ana Božović.

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12032_2013_642_MOESM1_ESM.jpg

Online Resource 1. Melting curve profile analysis of PCR product amplified from samples of tumor DNA for: a) total reference, amplified using ERβ-EXT-F2 and ERβ-EXT-R primers; b) methylated target DNA, amplified using MSP primers: ERβ-M-F and ERβ-M-R (JPEG 241 kb)

12032_2013_642_MOESM2_ESM.jpg

Online Resource 2. Panels a), b) and c): The ERβ immunoblots of the same tumor sample probed with three different antibodies, after immunoprecipitation: a) Estrogen receptor beta antibody [9.88] (ab16813, Abcam, Cambridge, MA USA) showing two protein bands at MW~52 KDa and one at MW~76 KDa; b) Mouse antihuman estrogen receptor beta 1 (MCA1974S, AbD Serotec, Oxford, UK), an antibody that recognizes the C terminus of the ERβ1 isoform, showing one protein band at MW ~ 52 kDa; c) Novocastra lyophilized mouse monoclonal antibody estrogen receptor (beta) (NCL-ER-beta, Leica Biosystems Newcastle Ltd, United Kingdom), specific for ERβ1, showing one band at MW ~ 52 kDa.; d) Representative ERβ immunoblot of the tumor samples (lanes T1-T8), NC-negative control, MCF7-positive control, K calibrator (lane K), showing one protein band at MW~ 52 kDa corresponding to wt ERβ1 and some lower MW bands which represent different ERβ isoforms, as well as higher MW bands that are probably complexes of ERβ with other proteins; e) panel showing the corresponding GAPDH immunoblot of the samples listed above. Lane M has the Amersham High Range Rainbow Molecular Weight Marker (RPN756E, GE Healthcare, UK) (JPEG 147 kb)

12032_2013_642_MOESM3_ESM.jpg

Online Resource 3. Histogram showing the distribution pattern of the ERβ ON promoter methylation index in the analyzed samples (JPEG 441 kb)

12032_2013_642_MOESM4_ESM.jpg

Online Resource 4. Histogram showing the distribution pattern of ERβ1 protein levels in the analyzed samples (JPEG 195 kb)

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Božović, A., Markićević, M., Dimitrijević, B. et al. Potential clinical significance of ERβ ON promoter methylation in sporadic breast cancer. Med Oncol 30, 642 (2013). https://doi.org/10.1007/s12032-013-0642-4

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