Integrated miRNA and mRNA expression profiling of mouse mammary tumor models identifies miRNA signatures associated with mammary tumor lineage

Abstract

Background: MicroRNAs (miRNAs) are small, non-coding, endogenous RNAs involved in regulating gene expression and protein translation. miRNA expression profiling of human breast cancers has identified miRNAs related to the clinical diversity of the disease and potentially provides novel diagnostic and prognostic tools for breast cancer therapy. In order to further understand the associations between oncogenic drivers and miRNA expression in sub-types of breast cancer, we performed miRNA expression profiling on mammary tumors from eight well-characterized genetically engineered mouse (GEM) models of human breast cancer, including MMTV-H-Ras, -Her2/neu, -c-Myc, -PymT, -Wnt1 and C3(1)/SV40 T/t-antigen transgenic mice, BRCA1(fl/fl);p53(+/-);MMTV-cre knock-out mice and the p53(fl/fl);MMTV-cre transplant model.

Results: miRNA expression patterns classified mouse mammary tumors according to luminal or basal tumor subtypes. Many miRNAs found in luminal tumors are expressed during normal mammary development. miR-135b, miR-505 and miR-155 are expressed in both basal human and mouse mammary tumors and many basal-associated miRNAs have not been previously characterized. miRNAs associated with the initiating oncogenic event driving tumorigenesis were also identified. miR-10b, -148a, -150, -199a and -486 were only expressed in normal mammary epithelium and not tumors, suggesting that they may have tumor suppressor activities. Integrated miRNA and mRNA gene expression analyses greatly improved the identification of miRNA targets from potential targets identified in silico.

Conclusions: This is the first large-scale miRNA gene expression study across a variety of relevant GEM models of human breast cancer demonstrating that miRNA expression is highly associated with mammary tumor lineage, differentiation and oncogenic pathways.

Figure 1

Unsupervised hierarchical clustering analysis of miRNA gene expression of 41 mammary tumors derived from 8 genetically engineered mouse models and samples of 5 normal mammary glands from 17.5-day-pregnant FVB/N mice. The heatmap shows the expression of 1,336 mouse miRNAs at the probe level. Heatmap colors represent relative miRNA expression as indicated in the color key.

Figure 2

Hierarchical clustering analysis of basal- and luminal-specific miRNA gene expression among mouse mammary tumor subtypes. miRNAs that distinguished basal from luminal tumor subtypes were identified and used in this hierarchical clustering of all tumor samples. A color-coded matrix below the dendrogram identifies each sample: red, basal like; green, luminal. The normal mammary samples were then integrated into the heatmap for comparison.

Figure 3

Heatmap of GEM-specific miRNA expression signatures associated with eight GEM models and normal mammary glands. In-house z-score-based methods are used with P-value < 0.001, FDR by permutation less than or close to 1%, and FDR-BH (false discovery rate-Benjamini and Hochberg) < 5% as described in Materials and methods.

Figure 4

Over-expression of (a) miR-494 and (b) miR-412 inhibits expression of Birc4 and Bmpr1a, respectively. M6 cells and DB-7 cells were transduced with lentivirus expressing miR-494 and miR-412, respectively. Control cells were transduced with lentivirus expressing scrambled miRNA. Following infection, cells were FACS sorted for RFP and RNA was extracted. RT-PCR was then performed to examine the expression of Birc4 in M6 cells and Bmpr1a in DB-7 cells. The error bar represents the standard deviation.