Identification of differentially expressed microRNAs in human male breast cancer

Abstract

Background: The discovery of small non-coding RNAs and the subsequent analysis of microRNA expression patterns in human cancer specimens have provided completely new insights into cancer biology. Genetic and epigenetic data indicate oncogenic or tumor suppressor function of these pleiotropic regulators. Therefore, many studies analyzed the expression and function of microRNA in human breast cancer, the most frequent malignancy in females. However, nothing is known so far about microRNA expression in male breast cancer, accounting for approximately 1% of all breast cancer cases.

Methods: The expression of 319 microRNAs was analyzed in 9 primary human male breast tumors and in epithelial cells from 15 male gynecomastia specimens using fluorescence-labeled bead technology. For identification of differentially expressed microRNAs data were analyzed by cluster analysis and selected statistical methods.Expression levels were validated for the most up- or down-regulated microRNAs in this training cohort using real-time PCR methodology as well as in an independent test cohort comprising 12 cases of human male breast cancer.

Results: Unsupervised cluster analysis separated very well male breast cancer samples and control specimens according to their microRNA expression pattern indicating cancer-specific alterations of microRNA expression in human male breast cancer. miR-21, miR519d, miR-183, miR-197, and miR-493-5p were identified as most prominently up-regulated, miR-145 and miR-497 as most prominently down-regulated in male breast cancer.

Conclusions: Male breast cancer displays several differentially expressed microRNAs. Not all of them are shared with breast cancer biopsies from female patients indicating male breast cancer specific alterations of microRNA expression.

Figure 1

Unsupervised cluster analysis of the expression level of 319 microRNAs in 9 male breast cancer and 4 pools of gynecomastia specimens (pools of 3 or 4 individual samples each). For this analysis the normalization was performed as described by Blenkiron et al. [26] with inter-pool normalization.

Figure 2

t-test (p = 0.01) for the identification of differentially expressed microRNAs in human male breast cancer.

Figure 3

Expression of the two most strongly up-regulated and the two most strongly down-regulated microRNAs in male breast cancer and control samples as determined by real-time PCR. Relative expression levels for every sample were calculated by normalizing to the mean expression level of the three small RNA species RNU24, U54, and Z-30. All measurements were performed independently two times. Statistical significance of differences between cancer (MBC) and control samples (C) were calculated using Mann-Whitney-U-test.

Figure 4

Expression of miR-21- target genes in male breast cancer in comparison to the control samples (A), correlation of miR-21 expression with the mRNA level of MASPIN (B) and PDCD4 (C) in male breast cancer specimens. Relative expression levels for every sample were calculated by normalizing to the mean expression level of two reference genes (βGUS and TBP). For every target gene the mean of the expression level in the control group was set equal to 1. Every individual measurement in the tumor samples was then normalized to this expression level. All measurements were performed independently two times. Error bars indicate standard deviation of the mean.