miRNA expression patterns in normal breast tissue and invasive breast cancers of BRCA1 and BRCA2 germ-line mutation carriers

Abstract

miRNA deregulation has been found to promote carcinogenesis. Little is known about miRNA deregulation in hereditary breast tumors as no miRNA expression profiling studies have been performed in normal breast tissue of BRCA1 and BRCA2 mutation carriers. miRNA profiles of 17 BRCA1- and 9 BRCA2-associated breast carcinomas were analyzed using microarrays. Normal breast tissues from BRCA1 and BRCA2 mutation carriers (both n = 5) and non-mutation carriers (n = 10) were also included. Candidate miRNAs were validated by qRT-PCR. Breast carcinomas showed extensive miRNA alteration compared to normal breast tissues in BRCA1 and BRCA2 mutation carriers. Moreover, normal breast tissue from BRCA1 mutation carriers already showed miRNA alterations compared to non-mutation carriers. Chromosomal distribution analysis showed several hotspots containing down- or up-regulated miRNAs. Pathway analysis yielded many similarities between the BRCA1 and BRCA2 axes with miRNAs involved in cell cycle regulation, proliferation and apoptosis. Lesser known pathways were also affected, including cellular movement and protein trafficking. This study provides a comprehensive insight into the potential role of miRNA deregulation in BRCA1/2-associated breast carcinogenesis. The observed extensive miRNA deregulation is likely the result of genome-wide effects of chromosomal instability caused by impaired BRCA1 or BRCA2 function. This study's results also suggest the existence of common pathways driving breast carcinogenesis in both BRCA1 and BRCA2 germ-line mutation carriers.

Keywords: BRCA1; BRCA2; breast cancer; hereditary; miRNA.

Figure 1. Number of differentially expressed miRNAs per chromosome from the comparison between cancers and normal tissue from BRCA1 and BRCA2 carriers, respectively

Explanation: Fisher's exact test: 10.200, p-value 0.989. BRCA1-C = BRCA1-associated breast carcinomas; BRCA2-C = BRCA2-associated breast carcinomas; BRCA1-N = normal breast tissue from BRCA1 germ-line mutation carriers; BRCA2-N = normal breast tissue from BRCA2 germ-line mutation carriers.

Figure 2. Chromosomal location of differentially expressed miRNAs between the normal tissue and cancers of BRCA1 and BRCA2 carriers, respectively

I. Chromosomal distribution of miRNAs differentially expressed between normal tissue and cancers of BRCA1 carriers. II. Chromosomal distribution of miRNAs differentially expressed between normal tissue and cancers of BRCA2 carriers. All differentially expressed miRNAs from both comparisons with fold change ≥ │1.5│and false discovery rate < 0.05 and known exact chromosomal location are presented. Within chromosomes a mixture of up- and down-regulated miRNAs can be seen. Several hotspots (≥ 4 miRNAs at the same locus) can be seen, in which the miRNAs show a similar direction of deregulation. The miRNA locations partly overlap with known hotspots of chromosomal instability in BRCA1- and BRCA2-associated carcinomas and fragile sites in the genome, in which miRNAs are often located.

Figure 2. Chromosomal location of differentially expressed miRNAs between the normal tissue and cancers of BRCA1 and BRCA2 carriers, respectively

I. Chromosomal distribution of miRNAs differentially expressed between normal tissue and cancers of BRCA1 carriers. II. Chromosomal distribution of miRNAs differentially expressed between normal tissue and cancers of BRCA2 carriers. All differentially expressed miRNAs from both comparisons with fold change ≥ │1.5│and false discovery rate < 0.05 and known exact chromosomal location are presented. Within chromosomes a mixture of up- and down-regulated miRNAs can be seen. Several hotspots (≥ 4 miRNAs at the same locus) can be seen, in which the miRNAs show a similar direction of deregulation. The miRNA locations partly overlap with known hotspots of chromosomal instability in BRCA1- and BRCA2-associated carcinomas and fragile sites in the genome, in which miRNAs are often located.

Figure 3. Unsupervised clustering results

Clustering was performed on both all samples and all miRNAs using a Self-Organizing Map algorithm. From the total heatmap, the most distinguishing parts between the classes are shown in this figure. For further information on the figure, see the legend in the top right corner. The clustering indicates that miRNAs can separate carcinomas (BRCA2 > BRCA1) from the normal breast tissue of both BRCA1/2 and non-mutation carriers. In general, normal breast tissue of BRCA1/2-mutation carriers clusters more with normal breast tissue from non-carriers. However, some of them cluster better with BRCA1/2-associated breast carcinomas.

Figure 3. Unsupervised clustering results

Clustering was performed on both all samples and all miRNAs using a Self-Organizing Map algorithm. From the total heatmap, the most distinguishing parts between the classes are shown in this figure. For further information on the figure, see the legend in the top right corner. The clustering indicates that miRNAs can separate carcinomas (BRCA2 > BRCA1) from the normal breast tissue of both BRCA1/2 and non-mutation carriers. In general, normal breast tissue of BRCA1/2-mutation carriers clusters more with normal breast tissue from non-carriers. However, some of them cluster better with BRCA1/2-associated breast carcinomas.

Figure 4. Flowchart showing the article selection strategy to attain breast miRNA expression profiling studies