MicroRNA expression profiling of human breast cancer identifies new markers of tumor subtype

Abstract

Background: MicroRNAs (miRNAs), a class of short non-coding RNAs found in many plants and animals, often act post-transcriptionally to inhibit gene expression.

Results: Here we report the analysis of miRNA expression in 93 primary human breast tumors, using a bead-based flow cytometric miRNA expression profiling method. Of 309 human miRNAs assayed, we identify 133 miRNAs expressed in human breast and breast tumors. We used mRNA expression profiling to classify the breast tumors as luminal A, luminal B, basal-like, HER2+ and normal-like. A number of miRNAs are differentially expressed between these molecular tumor subtypes and individual miRNAs are associated with clinicopathological factors. Furthermore, we find that miRNAs could classify basal versus luminal tumor subtypes in an independent data set. In some cases, changes in miRNA expression correlate with genomic loss or gain; in others, changes in miRNA expression are likely due to changes in primary transcription and or miRNA biogenesis. Finally, the expression of DICER1 and AGO2 is correlated with tumor subtype and may explain some of the changes in miRNA expression observed.

Conclusion: This study represents the first integrated analysis of miRNA expression, mRNA expression and genomic changes in human breast cancer and may serve as a basis for functional studies of the role of miRNAs in the etiology of breast cancer. Furthermore, we demonstrate that bead-based flow cytometric miRNA expression profiling might be a suitable platform to classify breast cancer into prognostic molecular subtypes.

Figure 1

Unsupervised hierarchical clustering (Pearson correlation, average linkage) over 137 detected miRNAs. Heatmap colors represent relative miRNA expression as indicated in the color key. (a) Clustering of 21 cell lines (orange), 5 normal breast samples (green) and 93 primary tumors (blue). (b) Clustering of 93 primary tumors with ER status as shown.

Figure 2

Supervised hierarchical clustering over selected miRNAs (Pearson correlation, average linkage). Heatmap colors represent relative miRNA expression as indicated in the color key for each panel. Brackets in the right margin indicate members of the same miRNA family. (a) Clustering of 51 tumor samples that could be classified as basal-like (red), HER2+ (pink), luminal A (dark blue), luminal B (light blue) or normal-like (green) over 38 miRNAs with Benjamini-Hochberg adjusted Kruskal-Wallis p < 0.05. (b) Clustering of 24 tumor samples classified as luminal A (dark blue) or luminal B (light blue) over 9 miRNAs with Benjamini-Hochberg adjusted Wilcoxon p < 0.05.

Figure 3

Association of individual miRNAs and tumor subtype or clinicopathological factors. Shown are 31 miRNAs and three factors with at least one association at adjusted p < 0.01. Differential expression was assessed by a non-parametric Wilcoxon rank sum test for comparison between two groups or a non-parametric Kruskal-Wallis test for comparison between multiple groups. To address the issue of multiple testing for the same factor, p values were adjusted by Benjamini and Hochberg's method [102]. Heatmap colors represent relative miRNA expression as shown in the color key. The expression values for a given sample group of interest were summarized by their mean. Brackets in the left margin indicate members of the same miRNA family. Significance levels are shown in the right margins: * adjusted p < 0.05; ** adjusted p < 0.01; *** adjusted p < 0.001. Abbreviations for subtype: B, basal-like; H, HER2+; LA, luminal A; LB, luminal B; N, normal-like.

Figure 4

Association of miRNA expression and DNA copy number. miRNAs mapping to regions of genomic aberration were plotted according to chromosome and genomic location. Heatmap colors represent relative miRNA expression as shown in the color key. Expression values for samples with genomic loss (L), unaltered samples (N), samples with genomic gain (G) and amplification (A) were summarized by their mean, respectively, with numbers of samples as indicated. miRNAs transcribed from multiple loci are indicated in blue. Adjacent miRNAs not separated by a black line are less than 50 kb apart. Significance levels correspond to unadjusted p values obtained by a non-parametric Wilcoxon rank sum test (* p < 0.05, ** p < 0.01, *** p < 0.001). Given the high dependence of the performed tests, p values were not adjusted for multiple testing.

Figure 5

Expression of clustered miRNAs is coordinated. Shown are pairwise scatter plots of expression values for mature miRNAs transcribed from genomic regions within 50 kb of each other. (a) miR-15a, miR-15b and miR-16 transcribed from two intronic clusters at C3q26.1 (SMC4L1) and C13q14.3 (DLEU2). (b) miR-25, miR-93 and miR-106b transcribed from an intronic cluster at C7q22.1 (MCM7). (c) miR-199a, miR-199a*, miR-199b and miR-214 transcribed from one intergenic cluster at C1q24.3 and two intergenic stem-loops at C9q34.11 and C19p13.2. Pearson correlation coefficients (r) and data points shown are based on samples with available array CGH data and no identified genomic loss or gain at the relevant locus (Additional data file 1). Genome plots are drawn to scale as shown in the legend (bottom right), except where missing regions are indicated by vertical bars. Positive and negative strands are depicted by the top and bottom plots, respectively. Gene loci and miRNA stem-loop regions are colored in blue and red, respectively. The location of exons is marked by greater line width.

Figure 6

Genes required for miRNA biogenesis are differentially expressed according to molecular subtype and ER status. Shown are boxplots of Illumina log₂ intensities for (a) AGO2 (EIF2C2), (b) DICER1, (c) DROSHA (RNASEN). Data are based on 58 samples that could be classified according to molecular subtype (17 basal-like (red), 5 HER2+ (pink), 18 luminal A (dark blue), 8 luminal B (light blue), 10 normal-like (green)) and 99 samples with known ER status (31 ER- (blue), 68 ER+ (yellow)). (d) Boxplots of mean miRNA expression after control-based normalization. Data are based on 51 samples that could be classified according to molecular subtype (16 basal-like (red), 5 HER2+ (pink), 15 luminal A (dark blue), 9 luminal B (light blue), 6 normal-like (green)) and 93 samples with known ER status (33 ER- (blue), 60 ER+ (yellow)). Black bars indicate the median; boxes interquartile range; whiskers most extreme data points not exceeding 1.5 times the interquartile range; points outliers. P values are based on non-parametric Kruskal-Wallis tests for subtype and Wilcoxon rank sum tests for ER status.