J. Anim. Breed. Genet. ISSN 0931-2668
ORIGINAL ARTICLE
Characterization of microRNA profile in mammary tissue of dairy and beef breed heifers ska1 & T. Motyl1 Z. Wicik1,2, M. Gajewska1, A. Majewska1, D. Walkiewicz3, E. Osin 1 Department of Physiological Sciences, Faculty of Veterinary Medicine, Warsaw University of Life Sciences, Warsaw, Poland 2 Department of Human Epigenetics, Mossakowski Medical Research Centre Polish Academy of Sciences, Warsaw, Poland 3 Department of Geriatrics and Gerontology, Medical Center of Postgraduate Education, Warsaw, Poland
Summary
Keywords Dairy and beef cattle breeds; mammary gland; mammary stem cells; microRNA. Correspondence T. Motyl, Department of Physiological Sciences, Faculty of Veterinary Medicine, Warsaw University of Life Sciences, Nowoursynowska 159, 02-776 Warsaw, Poland. Tel: +48 22 847 24 52; Fax: +48 22 847 24 52; E-mail: [email protected] Received: 18 September 2014; accepted: 11 February 2015
MicroRNAs (miRNAs) are small non-coding RNAs that participate in the regulation of gene expression. Their role during mammary gland development is still largely unknown. In this study, we performed a microarray analysis to identify miRNAs associated with high mammogenic potential of the bovine mammary gland. We identified 54 significantly differentially expressed miRNAs between the mammary tissue of dairy (Holstein-Friesian, HF) and beef (Limousin, LM) postpubertal heifers. Fifty-two miRNAs had higher expression in the mammary tissue of LM heifers. The expression of the top candidate miRNAs (bta-miR-10b, bta-miR-29b, bta-miR101, bta-miR-375, bta-miR-2285t, bta-miR-146b, bta-let7b, bta-miR-107, bta-miR-1434-3p) identified in the microarray experiment was additionally evaluated by qPCR. Enrichment analyses for targeted genes revealed that the major differences between miRNA expression in the mammary gland of HF versus LM were associated with the regulation of signalling pathways that are crucial for mammary gland development, such as TGFbeta, insulin, WNT and inflammatory pathways. Moreover, a number of genes potentially targeted by significantly differentially expressed miRNAs were associated with the activity of mammary stem cells. These data indicate that the high developmental potential of the mammary gland in dairy cattle, leading to high milk productivity, depends also on a specific miRNA expression pattern.
Introduction MicroRNAs (miRNAs) are short, ~22 nucleotide long, non-coding RNAs that are involved in the regulation of gene expression by affecting mRNA stability and translation. After maturation, miRNAs are incorporated into an RNA-induced silencing complex and recognize the target genes through imperfect base pairing. As partial complementarity is sufficient for an miRNA to target an mRNA, each miRNA has the ability to regulate a large number of genes (Filipowicz et al. 2008). miRNAs have been identified in many types of cells as well as in biological fluids such as milk. According to the miRBase, 808 precursors and © 2015 Blackwell Verlag GmbH
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783 mature miRNAs have been identified so far for the Bos taurus species (Kozomara & Griffiths-Jones 2014). The total number of miRNAs in bovine milk is about twice as high as it is in serum, which indicates that mammary alveolar cells express their own miRNAs (Chen et al. 2010). In the mammary gland, most of the miRNAs studied have functions related to immunity and show alterations in their expression pattern during lactation (Zhou et al. 2012). Previous studies have indicated that miRNAs could play an important role during the development of the mammary gland tissue, especially the epithelium, through the regulation of gene expression (Avril-Sassen et al. 2009; Tanaka et al. 2009). miRNAs also have an doi:10.1111/jbg.12172
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miRNA expression profiles in cattle mammary gland
important role in regulating self-renewal and differentiation of stem cells (Gangaraju & Lin 2009). The majority of studies on miRNA function have been focused on human-related diseases and rodent animal models. On the other hand, studies performed on farm mammals have mainly focused on miRNAs that potentially regulate gene expression in the lactating, not virgin mammary gland. Knowledge about the development of mammary glands is essential in terms of further improvement of dairy cattle production capacity in the future. The potential of mammary gland development differs between beef and dairy cattle, and the intensity of development, as well as the productivity of the gland in beef breeds, is incomparably smaller (Keys et al. 1989; Akers 2006). Our recent study revealed that these differences may not only result from various levels of genetic and central endocrine regulation, but may also depend on intramammary factors, including the number of mammary stem cells and expression of genes involved in the development of the mammary stem cell niche (Osi nska et al. 2014). This study provides the first global comparison of the mammary miRNA profiles between postpubertal dairy (Holstein-Friesian, HF) and beef heifers (Limousin, LM). These results may be helpful for further elucidation of complex regulatory networks between miRNAs and mRNAs, and for future studies on mammary gland physiology and dairy potential. Special attention was paid to miRNAs associated with the activity of mammary gland stem cells.
labelled and hybridized in accordance with Agilent’s instructions. Microarrays were scanned using the G2505C Microarray Scanner (Agilent Technologies, Santa Clara, CA, USA), then gridded, and the quality of hybridization was initially determined by the AGILENT FEATURE EXTRACTION software. Raw data were then analysed using GENESPRING 12.0 (Agilent Technologies, Sanat Clara, CA, USA). To minimize the biological variability across arrays, raw data were 1.0-thresholded, log2transformed and 75-percentile-shifted with baseline transformation (mean of all samples). Then, all miRNAs were filtered by flags (detected, non-detected) and statistically analysed. Statistical analysis was performed using unpaired t-test with Benjamini–Hochberg FDR multiple testing corrections (p ≤ 0.05). Further ontological analyses were conducted on genes showing significant changes after FDR correction. The area of the analyses covered in this publication has been deposited in NCBI’s Gene Expression Omnibus and is accessible via GEO Series accession number GSE61227. Clustering analysis
Hierarchical cluster analysis was performed in Genespring 12.6 (Agilent Technologies, Santa Clara, USA). The expression data were log-transformed and grouped using a hierarchical clustering algorithm by normalized intensity values on miRNAs and conditions (HF and LM). A similarity measure was performed using squared Euclidean measure with average linkage rule. The analysis was performed on non-averaged lists of genes with corrected p-value ≤0.05.
Material and methods Tissue sampling
Mammary tissue was obtained at the slaughterhouse from 2-year-old non-pregnant heifers (four HFs and four LMs) free of clinical signs of mastitis. Udders were removed, and mammary tissue was collected, fixed in RNAlater solution (Qiagen, Hilden, Germany) and stored at 80°C until further analysis. miRNA microarray analysis
Total RNA containing miRNA was isolated from samples using the miRNeasy Mini Kit (Qiagen, Hilden, Germany) in accordance with the manufacturer’s protocol. Total RNA quantity and purity were assessed using NanoDrop, and RNA integrity was verified using an Agilent 2100 Bioanalyzer. The miRNA profiling was performed using an Agilent custom microarray (8 9 60 K format) containing probes of 763 B. taurus miRNAs. Total RNA from each sample was amplified, 32
Target gene prediction of differential expression microRNAs
miRNA target prediction was performed using the TargetScan database integrated with GeneSpring 12.0 (Agilent Technologies, Santa Clara, CA, USA). Analysis was performed for the list of all significantly differentially expressed miRNAs. It revealed which genes (targets) could be most significantly regulated by analysed miRNA. Scanning was performed for context score percentile (50) and conserved/non-conserved miRNA families and target sites. Ontological analyses of miRNA target genes
Ontological analyses revealing biological processes and molecular functions of miRNA targets were carried out using the binomial overrepresentation test with Bonferroni correction in PANTHER (Protein ANalysis THrough Evolutionary Relationships) © 2015 Blackwell Verlag GmbH
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Table 2 (continued) (b)
Target scan p-value
Gene symbol
Gene description
Fold change obtained from our previous ska study (Osin et al. 2014)
1.16E-04
IL6
Interleukin 6 (interferon, beta 2)
276.9
1.92E-02
IL1B
Interleukin 1, beta
4.13E-02
CSF2
2.98E-02
CEBPD
5.84E-03
NGF
6.06E-04
EDN1
Colony-stimulating factor 2 (granulocyte–macrophage) CCAAT/enhancer binding protein (C/EBP), delta Nerve growth factor (beta polypeptide) Endothelin 1
1.9
3.37E-05
EZH2
Enhancer of zeste homolog 2
2.2
11.4 2.9 2.6 2.1
Figure 4 Comparison of expression of selected miRNAs in the mammary gland samples from LM (n = 4) and HF (n = 4) heifers. The expression was analysed using microarray and qPCR methods. In the case of qPCR, the levels of miRNA expression were normalized according to the relative expression of housekeeping miRNAs – bta-miR-10b and bta-miR181b. Fold changes of gene expression determined by qPCR were calculated using delta delta Ct method and are shown as mean SD.
gestation, lactation, as well as early and late involution). Some of the miRNAs identified, which were upregulated in virgin animals, were also present in our © 2015 Blackwell Verlag GmbH
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Annotated miRNAs systematic names bta-miR-26b,bta-let-7f,bta-miR-376b,bta-miR-26a,bta-let-7c,bta-let-7e, bta-let-7b,bta-let-7d,bta-let-7i,bta-let-7a-5p,bta-miR-1434-3p bta-miR-204,bta-miR-222,bta-miR-27a-3p,bta-miR-183,bta-miR-221, bta-miR-27b,bta-miR-24-3p bta-let-7c,bta-let-7e,bta-let-7b,bta-let-7a-5p,bta-miR-199a-5p, bta-miR-199b bta-miR-101,bta-miR-27a-3p,bta-miR-199c,bta-miR-27b bta-let-7f,bta-let-7c,bta-let-7e,bta-let-7b,bta-let-7d,bta-let-7i, bta-let-7a-5p bta-miR-199c,bta-miR-185,bta-let-7a-5p,bta-miR-199a-3p,bta-let-7f, bta-let-7c,bta-let-7e,bta-let-7d,bta-let-7b,bta-let-7i,bta-miR-1434-3p, bta-miR-101,bta-miR-194,bta-miR-411a bta-miR-26b,bta-miR-199c,bta-let-7f,bta-miR-26a,bta-let-7c,bta-let-7e, bta-let-7b,bta-let-7d,bta-let-7i, bta-miR-25,bta-let-7a-5p,bta-miR-101
data set, that is let-7b, let-7c, let-7d, let-7e, let-7f, let-7i, miR-146b, miR-199b, miR-222, miR-24, miR25, miR-26a, miR-26b, miR-27b, miR-29a, miR-29b, miR-29c and miR-338. The identified members of the let-7 family were previously shown to be depleted in mouse mammary epithelial progenitors, whereas enforced let-7 expression led to inhibition of selfrenewal capacities of cells (Ibarra et al. 2007; Yu et al. 2007). It is worth noting that these miRNAs had lower expression in HF heifers’ mammary tissue compared to the LM mammary gland (Figure 1). The lower expression of let-7 miRNAs corresponds to higher productivity of the HF breed and suggests that this property may also be connected with enhanced selfrenewal capacity of HF’s mammary tissue. Furthermore, ontological analysis of miRNA targets revealed that the major interbreed differences were associated with metabolic processes and signalling pathways that are closely related to stem cell activity and mammary gland development (Figures 2 and 3). This was observed on many levels, including highly regulated genes associated with stem cell maintenance and renewal, epithelium development or signalling pathways known to play a crucial role during mammogenesis. Among the most strongly regulated pathways were insulin, Wnt, TGF-beta, MAPK, EGFR and interleukin signalling pathways (Figure 3). Wnt, EGFR, TGFB and insulin signalling pathways are known to play an important role in normal development of the mammary gland (Musters et al. 2004; 39
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Figure 1 A heat map based on the mean expression levels of miRNAs. Each column corresponds to the expression profile of the heifer breed, Limousin (LM) and Holstein-Friesian (HF) in quadruplicate, and each row corresponds to a miRNA accession number. Red indicates lower expression and green indicates higher expression of analysed miRNAs, whereas black indicates mean level of expression. Fold changes for each gene are shown on the color scale on the right side of the graph. Blue color indicates downregulation; gradation from yellow to red indicates upregulation of gene expression.
Table 1 List of miRNAs, differing between Limousin and Holstein-Friesian heifers above two fold change (FC) with FDR-corrected p-value cut-off ≤0.05 Systematic_name
mirbase accession No
p-Value
FC
bta-miR-375 bta-miR-218 bta-miR-29b_v12.0 bta-miR-24 bta-miR-183 bta-miR-147 bta-miR-204 bta-miR-421 bta-miR-155 bta-miR-146b bta-miR-218_v13.0 bta-miR-29b bta-miR-154c bta-miR-101 bta-miR-194 bta-miR-1434-3p bta-miR-2285t
MIMAT0009303 MIMAT0003798 MIMAT0003828 MIMAT0009250 MIMAT0009245 MIMAT0009237 MIMAT0004338 MIMAT0009314 MIMAT0009241 MIMAT0009235 MIMAT0003798 MIMAT0003828 MIMAT0025542 MIMAT0003520 MIMAT0009254 MIMAT0012040 MIMAT0025577
0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.002 0.005 0.002 0.029 0.042 0.014 0.001 0.015 0.020
157.70 106.44 83.58 72.89 47.06 29.73 6.53 2.39 2.28 2.24 2.23 2.17 2.07 2.03 2.02 2.29 4.34
heifers could influence target genes involved in stem cell activity, differentiation and immune response (Figure 3). Furthermore, pathways involved in the regulation of senescence and autophagy, Id signalling, adipogenesis and androgen receptor signalling were also shown to be potentially significantly regulated. Analysis of the potential of miRNAs to influence mammary stem cells and their niche
Special attention was given to the role of identified miRNAs in post-transcriptional regulation of genes, whose products might affect mammary stem/progenitor cells. We identified 20 targeted genes involved in cell maintenance, stem cell renewal and stem cell development (HAS2, EZH2, MED6, HOXA9, VEGFA, COL1A1, CNOT2, KDR, BCL9, EIF2AK3, MED21, IRS1, HNF1B, RIF1, WNT3A, LRP6, SALL4, SLC12A2, TFAP2C and IGF1R) and 7 other genes associated with stem cell niche activity (IL6, IL1B, CSF2, CEBPD, NGF, EDN1 and EZH2), as described in our previous publication (Osi nska et al. 2014). The characteristics of these target genes are presented in Table 2a, b. © 2015 Blackwell Verlag GmbH
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Validation of the results using qPCR
Verification of the miRnomic results was carried out using the qPCR method. The miRNAs for validation were selected upon analysing their involvement in the major processes associated with mammary gland development. Analysis confirmed the changes in expression of all 8 genes tested (Figure 4); however, in the case of bta-mir-375 and bta-miR-29b, we observed a significant difference in the values of expression obtained in qPCR compared to the microarray results (but the direction of changes remained the same). This was due to the fact that the expression of bta-mir-375 and bta-miR-29b was not detected on microarrays for the HF samples (lack of hybridization). Therefore, the obtained values of fold change were very high compared to the expression in LM heifers. We also found four other miRNAs (bta-miR147, bta-miR-183, bta-miR-218 and bta-miR-24), which yielded similar results in the microarray analysis. In our opinion, these genes should not be excluded from the analysis, but it is necessary to consider that the changes in their expression may in fact not be as large. Discussion MicroRNAs and their role in mammary gland development are currently intensively studied, especially in humans, but there is still a limited understanding of their function in bovine mammary glands, influence on organ development and dairy potential. In this study, an extensive miRNA expression profile in the mammary glands of dairy (HF) and beef (LM) postpubertal heifers was investigated. Microarray analysis revealed 54 miRNAs that significantly differed between the two investigated breeds of cattle (Figure 1). Fifty-two of the miRNAs identified showed higher expression in the mammary tissue of LM heifers, which is an important finding given the inhibitory role of miRNAs in the regulation of genes that are involved in mammary gland development. To our knowledge, this is the first study comparing the expression of miRNAs involved in the regulation of the mammary gland between two cattle breeds with a different productivity type. 35
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(a)
(b)
Figure 2 Major categories of (a) biological processes and (b) molecular functions regulated by targets of miRNAs with significantly differing expression between dairy heifers (Holstein-Friesian) and beef heifers (Limousin). Analysis was performed using Panther overrepresentation test with Bonferroni correction (p ≤ 0.05).
Figure 3 Top 10 signaling pathways (p ≤ 0.05) regulated by targets of miRNAs with significantly differing expression between beef heifers (Limousin) and dairy heifers (HolsteinFriesian). Analysis was performed using Single Experiment Analysis tool in GENESPRING software.
We compared the data obtained in our microarray analysis with the data published in the available literature and found that nearly 40 of the 54 miRNAs showing significant breed-dependent differences in expression were also detected in the studies of other authors on bovine (Li et al. 2012), goat (Dong et al. 2013), mouse (Avril-Sassen et al. 2009) and human (Silveri et al. 2006) mammary glands. Most of the studies compared the expression of miRNA in the mammary tissue at different stages of development during the postnatal life (Avril-Sassen et al. 2009; Li et al. 2012) or focused specifically on the lactation period (Dong et al. 2013; Le Guillou et al. 2014). Li et al. (2012) compared miRNA profiles from lactating and non-lactating bovine mammary glands and showed that the expression of 56 miRNAs was significantly different between lactation and non-lactation periods. Among these miRNAs, only 6 were highly 36
expressed in the non-lactation period: miR-107-3p, miR-23b-3p, PC-320c-2-3p, PC-574-3p, CN-100-1-5p and CN-190-5p (Li et al. 2012). Two of these miRNAs, that is miR-107 and miR-23b, were also identified in our microarray study and showed lower expression in HF than in LM heifers. Interestingly, it has been suggested that miR-107 is an important regulator of proliferative properties of cells, decreasing the rates of cell division with cell cycle arrest (Finnerty et al. 2010), whereas miR-23b may repress the growth of the ductal system (Rogler et al. 2009; Li et al. 2012). This suggests that the higher milk production potential of HF cattle also results from a more favourable pattern of miRNA expression, which stimulates the development of the mammary gland. Avril-Sassen et al. (2009) compared the expression of miRNA in the murine mammary gland at different stages of development (juvenile, puberty, mature virgin, © 2015 Blackwell Verlag GmbH
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Table 2 (a) List of target genes regulated by miRNAs, differing between Limousin (LM) and Holstein-Friesian (HF) heifers, associated with mammary gland development and stem cell activity; miRNAs in bold showed expression fold change over 2. (b) List of significantly targeted genes associated ska et al. 2014). These factors were significantly regulated on mRNA level in the with stem cell niche, as described in our previous publication (Osin mammary tissue of HF, in comparison with LM heifers. Analysis was performed for genes significantly expressed over a 1.3 absolute fold change (FC) with FDR-corrected p-value cut-off ≤0.05 (a) Target scan p-value
Gene symbol
2.40E-05
Gene description
Major gene functions (GO)
Annotated miRNAs systematic names
HAS2
Hyaluronan synthase 2
Stem cell development Positive regulation of cell proliferation Positive regulation of cell migration
3.37E-05
EZH2
Enhancer of zeste homolog 2
5.27E-05
MED6
Mediator complex subunit 6
9.54E-04
HOXA9
Homeobox A9
Stem cell development Positive regulation of epithelial-to-mesenchymal transition Regulation of cell proliferation Stem cell maintenance Positive regulation of transcription from RNA polymerase II promoter Mammary gland development Endothelial cell activation
bta-mir-27a-3p, bta-mir-16a, bta-miR-375, bta-let-7a-5p, bta-miR-27b, bta-miR-497, bta-miR-204, bta-miR-186, bta-miR-376b, bta-miR-26a, bta-let-7c, bta-let-7b, bta-let-7d, bta-miR-15a, bta-miR-25, bta-miR-23b-3p, ↓bta-miR-1434-3p, bta-miR-101, bta-miR-16b bta-miR-26b, bta-miR-199c, bta-let-7f, bta-miR-26a, bta-let-7c, bta-let-7e, bta-let-7b, bta-let-7d, bta-let-7i, bta-miR-25, bta-let-7a-5p, bta-miR-101
6.38E-03
VEGFA
Vascular endothelial growth factor A
8.81E-03
COL1A1
Collagen, type I, alpha 1
1.36E-02
CNOT2
1.36E-02
KDR
1.41E-02
BCL9
CCR4-NOT transcription complex, subunit 2 Kinase insert domain receptor; vascular endothelial growth factor receptor 2 B-cell CLL/lymphoma 9
1.41E-02
EIF2AK3
1.51E-02
MED21
Eukaryotic translation initiation factor 2-alpha kinase 3 Mediator complex subunit 21
Lactation Mammary gland alveolus development Positive regulation of endothelial cell proliferation Positive regulation of endothelial cell migration Positive regulation of epithelial-to-mesenchymal transition Blood vessel development Positive regulation of canonical Wnt signalling pathway Regulation of stem cell maintenance Negative regulation of oestrogen receptor signalling pathway Branching morphogenesis of an epithelial tube Calcium ion homoeostasis Endothelial cell differentiation Somatic stem cell maintenance Canonical Wnt pathway
Lactation Fat cell differentiation Insulin-like growth factor receptor signalling pathway Stem cell maintenance Blastocyst development Positive regulation of transcription from RNA polymerase II promoter
bta-mir-27a-3p, bta-let-7f, bta-miR-186, bta-miR-375, bta-let-7c, bta-let-7e, bta-let-7d, bta-let-7b, bta-let-7i, bta-let-7a-5p, bta-miR-27b bta-miR-26b, bta-miR-199c, bta-miR-375, bta-let-7a-5p, bta-miR-2288, bta-let-7f, bta-miR-186, bta-miR-376b, bta-miR-26a, bta-let-7c, bta-let-7e, bta-let-7d, bta-let-7b, bta-let-7i, bta-miR-421 bta-miR-199c, bta-mir-16a, bta-miR-375, bta-miR-185, bta-miR-199a-5p, bta-miR-497, bta-miR-29a, bta-miR-186, bta-miR-15a, bta-miR-29c, bta-miR-16b, bta-miR-199b, bta-miR-29b
bta-miR-338, bta-let-7a-5p, bta-miR-218, bta-miR-29a, bta-miR-186, bta-let-7f, bta-let-7c, bta-let-7e, bta-let-7d, bta-let-7b, bta-let-7i, bta-miR-29c, bta-miR-29b bta-miR-222, bta-let-7a-5p, bta-miR-218, bta-let-7f, bta-let-7c, bta-let-7e, bta-let-7d, bta-let-7b, bta-let-7i, bta-miR-23b-3p, bta-miR-221 bta-miR-222, bta-miR-26b, bta-miR-183, bta-mir-16a, bta-miR-24-3p, bta-miR-497, bta-miR-186, bta-miR-26a, bta-miR-15a, bta-miR-221, bta-miR-16b bta-miR-199c, bta-miR-338, bta-mir-16a, bta-miR-218, bta-miR-497, bta-miR-204, bta-miR-15a, ↓bta-miR-1434-3p, bta-miR-101, bta-miR-16b bta-miR-222, bta-miR-26b, bta-miR-199c, bta-miR-155, bta-miR-24-3p, bta-miR-26a, bta-miR-101, bta-miR-409a, bta-miR-221, bta-miR-421 bta-miR-199c, bta-miR-155, bta-let-7a-5p, bta-miR-107, bta-let-7f, bta-miR-186, bta-let-7c, bta-let-7e, bta-let-7b, bta-let-7d, bta-let-7i
(continued)
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Table 2 (continued) (a) Target scan p-value
Gene symbol
1.60E-02
Gene description
Major gene functions (GO)
Annotated miRNAs systematic names
IRS1
Insulin receptor substrate 1
Mammary gland development Lipid catabolic proces Negative regulation of insulin secretion
2.67E-02
HNF1B
HNF1 homeobox B
2.67E-02
RIF1
RAP1 interacting factor homolog (yeast)
Epithelial cell proliferation Branching morphogenesis of an epithelial tube Insulin secretion Regulation of Wnt signalling pathway Stem cell maintenance Chromosome telomeric region
bta-miR-183, bta-mir-16a, bta-miR-146b, bta-miR-107, bta-miR-186, bta-miR-29c, bta-miR-29b, bta-mir-27a-3p, bta-miR-199c, bta-miR-27b, bta-miR-497, bta-miR-29a, bta-miR-15a, bta-miR-23b-3p, bta-miR-101, bta-miR-16b bta-miR-26b, bta-miR-199c, bta-miR-375, bta-miR-199a-3p, bta-miR-218, bta-miR-24-3p, bta-miR-26a, bta-miR-25, bta-miR-101, bta-miR-194
3.13E-02
WNT3A
Wingless-type MMTV integration site family, member 3A
3.50E-02
LRP6
Low-density lipoprotein receptor-related protein 6
3.81E-02
SALL4
Sal-like 4
Mammary gland development Wnt signalling pathway Negative regulation of fat cell differentiation Branching involved in mammary gland duct morphogenesis Mammary placode formation Negative regulation of fat cell differentiation Stem cell maintenance
3.81E-02
SLC12A2
Solute carrier family 12 (sodium/ potassium/chloride transporters), member 2
Branching involved in cmammary gland duct morphogenesis Mammary duct terminal end bud growth
3.81E-02
TFAP2C
Transcription factor AP-2 gamma (activating enhancer binding protein 2 gamma)
4.41E-02
IGF1R
Insulin-like growth factor 1 receptor
Dichotomous subdivision of terminal units involved in mammary gland duct morphogenesis Epithelial cell proliferation involved in mammary gland duct elongation Somatic stem cell maintenance Germ-line stem cell maintenance Mammary gland development Insulin-like growth factor receptor signalling pathway Negative regulation of apoptotic process
bta-miR-155, bta-mir-16a, bta-miR-29c, bta-miR-199b, bta-miR-29b, bta-miR-375, bta-miR-218, bta-miR-199a-5p, bta-miR-497, bta-miR-15a, bta-miR-23b-3p, bta-miR-411a, bta-miR-16b bta-mir-27a-3p, bta-mir-16a, bta-miR-185, bta-miR-27b, bta-miR-497, bta-miR-107, bta-miR-15a, bta-miR-101, bta-miR-16b bta-miR-183, bta-mir-16a, bta-miR-2288, bta-miR-186, ↓bta-miR-1434-3p, bta-miR-29c, bta-miR-29b, bta-miR-26b, bta-miR-497, bta-miR-26a, bta-miR-15a, bta-miR-101, bta-miR-411a, bta-miR-16b bta-mir-16a, bta-miR-15a, bta-miR-16b, bta-miR-497, bta-miR-107 bta-mir-16a, bta-miR-107, bta-miR-186, bta-miR-26b, bta-mir-27a-3p, bta-miR-199c, bta-miR-218, bta-miR-27b, bta-miR-497, bta-miR-204, bta-miR-26a, bta-miR-25, bta-miR-15a, bta-miR-101, bta-miR-16b bta-mir-27a-3p, bta-miR-27b, bta-miR-542-5p, bta-miR-29a, bta-miR-186, bta-miR-24, bta-miR-29c, bta-miR-29b, bta-miR-421
bta-miR-185, bta-let-7a-5p, bta-let-7f, bta-miR-186, bta-miR-376b, bta-let-7c, bta-let-7e, bta-let-7b, bta-let-7d, bta-let-7i, bta-miR-25, bta-miR-194
(continued)
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Table 2 (continued) (b)
Target scan p-value
Gene symbol
Gene description
Fold change obtained from our previous ska study (Osin et al. 2014)
1.16E-04
IL6
Interleukin 6 (interferon, beta 2)
276.9
1.92E-02
IL1B
Interleukin 1, beta
4.13E-02
CSF2
2.98E-02
CEBPD
5.84E-03
NGF
6.06E-04
EDN1
Colony-stimulating factor 2 (granulocyte–macrophage) CCAAT/enhancer binding protein (C/EBP), delta Nerve growth factor (beta polypeptide) Endothelin 1
1.9
3.37E-05
EZH2
Enhancer of zeste homolog 2
2.2
11.4 2.9 2.6 2.1
Figure 4 Comparison of expression of selected miRNAs in the mammary gland samples from LM (n = 4) and HF (n = 4) heifers. The expression was analysed using microarray and qPCR methods. In the case of qPCR, the levels of miRNA expression were normalized according to the relative expression of housekeeping miRNAs – bta-miR-10b and bta-miR181b. Fold changes of gene expression determined by qPCR were calculated using delta delta Ct method and are shown as mean SD.
gestation, lactation, as well as early and late involution). Some of the miRNAs identified, which were upregulated in virgin animals, were also present in our © 2015 Blackwell Verlag GmbH
• J. Anim. Breed. Genet. 133 (2016) 31–42
Annotated miRNAs systematic names bta-miR-26b,bta-let-7f,bta-miR-376b,bta-miR-26a,bta-let-7c,bta-let-7e, bta-let-7b,bta-let-7d,bta-let-7i,bta-let-7a-5p,bta-miR-1434-3p bta-miR-204,bta-miR-222,bta-miR-27a-3p,bta-miR-183,bta-miR-221, bta-miR-27b,bta-miR-24-3p bta-let-7c,bta-let-7e,bta-let-7b,bta-let-7a-5p,bta-miR-199a-5p, bta-miR-199b bta-miR-101,bta-miR-27a-3p,bta-miR-199c,bta-miR-27b bta-let-7f,bta-let-7c,bta-let-7e,bta-let-7b,bta-let-7d,bta-let-7i, bta-let-7a-5p bta-miR-199c,bta-miR-185,bta-let-7a-5p,bta-miR-199a-3p,bta-let-7f, bta-let-7c,bta-let-7e,bta-let-7d,bta-let-7b,bta-let-7i,bta-miR-1434-3p, bta-miR-101,bta-miR-194,bta-miR-411a bta-miR-26b,bta-miR-199c,bta-let-7f,bta-miR-26a,bta-let-7c,bta-let-7e, bta-let-7b,bta-let-7d,bta-let-7i, bta-miR-25,bta-let-7a-5p,bta-miR-101
data set, that is let-7b, let-7c, let-7d, let-7e, let-7f, let-7i, miR-146b, miR-199b, miR-222, miR-24, miR25, miR-26a, miR-26b, miR-27b, miR-29a, miR-29b, miR-29c and miR-338. The identified members of the let-7 family were previously shown to be depleted in mouse mammary epithelial progenitors, whereas enforced let-7 expression led to inhibition of selfrenewal capacities of cells (Ibarra et al. 2007; Yu et al. 2007). It is worth noting that these miRNAs had lower expression in HF heifers’ mammary tissue compared to the LM mammary gland (Figure 1). The lower expression of let-7 miRNAs corresponds to higher productivity of the HF breed and suggests that this property may also be connected with enhanced selfrenewal capacity of HF’s mammary tissue. Furthermore, ontological analysis of miRNA targets revealed that the major interbreed differences were associated with metabolic processes and signalling pathways that are closely related to stem cell activity and mammary gland development (Figures 2 and 3). This was observed on many levels, including highly regulated genes associated with stem cell maintenance and renewal, epithelium development or signalling pathways known to play a crucial role during mammogenesis. Among the most strongly regulated pathways were insulin, Wnt, TGF-beta, MAPK, EGFR and interleukin signalling pathways (Figure 3). Wnt, EGFR, TGFB and insulin signalling pathways are known to play an important role in normal development of the mammary gland (Musters et al. 2004; 39
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Figure 5 Summarizing scheme showing significantly differentially expressed miRNAs, between dairy (Holstein-Friesian) and beef (Limousin) heifers, in context of their influences on specified processes and signalling pathways associated with mammary gland development. miRNAs marked with a red are known for their involvement in the lactation network (Li et al. 2012).
Akers et al. 2005; Roarty & Serra 2007; Mukhopadhyay et al. 2013). The MAPK pathway is one of the key regulators of mammary epithelial cell differentiation and function (Whyte et al. 2009). Moreover, seven of the miRNAs identified in our study (miR-199a-5p, miR-25, miR-27b, miR-29a and miR-29b, miR-375 m and miR-107) which are present in the interaction network performed by us (Figure 5.) were also present in the lactation interaction network created by Li et al. (2012). Nearly half of the gene targets involved in our interaction network had significance of targeting lower than 0.01. Genes with the highest degree of connections with selected processes were also shown to be the most significantly regulated by the significantly differentially expressed miRNA. These included NLK (27 connections), TP53 (26), FZD4 (25), GAB1 (24), MITF (23), CDC42, WNT3A (21), TAB 2 (20), MAP4K3, PIK3R1 and FERMT2 (19). Comparing results from network and gene target scan analysis revealed 8 genes which are significantly specific (p ≤ 0.0001) targets of the identified miRNAs and have the highest degree of connection with processes specifically involved in mammary gland functions: CAPN3, COL1A2, FZD4, 40
HAS2, MED6, NLK, TAB 2, WASL. Some of these miRNA target genes identified in this analysis were shown to be associated with stem cell activity (HAS2, MED6), as well as cytoskeleton and extracellular matrix (FERMT2, COL1A2, WASL and CAPN3). Moreover, we also identified some mutual interactions between the gene targets, for example CAPN3 gene expression may be regulated by MITF, whereas CDC42 activity might be regulated by the WASL gene product. Additionally, we observed a strong breed-dependent influence of miRNAs on signalling pathways connected with inflammatory processes. These data and our previous findings (Osi nska et al. 2014) suggest that the activity of inflammatory signalling pathways may be associated with regulation of stem cell activity and adaptation of dairy cattle resulting in protection against mastitis. For example, the levels of miRNA regulating the expression of interleukin 6 (IL6) (Table 2b) were lower in HF heifers, which corresponds with our previous results showing that IL6 expression was over 200-fold higher in this cattle breed (Osi nska et al. 2014). Furthermore, miR-146b has been associated with regulation of innate immune © 2015 Blackwell Verlag GmbH
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response, interleukin signalling and inflammation (Avril-Sassen et al. 2009). In our further analyses, we specifically focused on the potential role of miRNAs in the regulation of the mammary gland stem cell niche, which is required for intensive regenerative processes during puberty and pregnancy, preparing the gland for milk production and secretion during lactation (Table 2a). We previously showed that the high mammogenic potential of HF heifers is manifested by a higher number of stem/ progenitor cells and upregulation of genes involved in the formation of a favourable niche for the maintenance, renewal, proliferation and differentiation of stem/progenitor cells (Osi nska et al. 2014). Consistently, the result of the present study revealed downregulation of miRNAs involved in inhibition of the translation of genes responsible for the maintenance and activity of the mammary stem cells in HF heifers. Our analysis showed that genes potentially regulated by the identified miRNAs are involved in a network of signalling pathways, and several miRNAs involved in regulation of these pathways may be important for the maintenance of the self-renewal properties of the mammary gland and its development (Figure 5). miR-146b appears to be one of the key regulators, and its function has already been documented in previous studies. It has been suggested that high expression of miR-146b is connected with differentiation of mammary epithelial cells, as its expression levels increase during pregnancy, especially in the luminal progenitors compared to the basal/stem cells (Elsarraj et al. 2013). Furthermore, miR-146b showed significantly higher expression in the luminal progenitors in pregnant mice than in the mammary glands of virgin animals, indicating that a low level of miR-146b is required for the maintenance of the undifferentiated state of the mammary stem cells (Elsarraj et al. 2013). In our study, the expression of this miRNA was lower in the mammary glands of HF heifers that show a high mammogenic potential, compared to LM heifers with a lower mammogenic potential. Conclusions In conclusion, the present study has shown that the high potential of growth and development of the mammary gland in dairy cattle, as well as the high lactogenic potential of dairy breeds, depends not only on genetic but also on epigenetic control by miRNA. It is particularly interesting that miRNAs-dependent inhibition of the translation of genes connected with mammary stem cell functions is less pronounced in dairy heifers than it is in beef heifers, promoting a © 2015 Blackwell Verlag GmbH
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gene expression pattern responsible for more intensive development of the mammary gland in dairy cattle. Acknowledgement This study was supported by grant No. NN308594138 from the National Research Centre. References Akers R.M. (2006) Major advances associated with hormone and growth factor regulation of mammary growth and lactation in dairy cows. J. Dairy Sci., 89, 1222–1234. Akers R.M., Ellis S.E., Berry S.D. (2005) Ovarian and IGF-I axis control of mammary development in prepubertal heifers. Domest. Anim. Endocrinol., 29, 259–267. Avril-Sassen S., Goldstein L.D., Stingl J., Blenkiron C., Le Quesne J., Spiteri I., Karagavriilidou K., Watson C.J., Tavare S., Miska E.A., Caldasm C. (2009) Characterisation of microRNA expression in post-natal mouse mammary gland development. BMC Genom., 10, 548. Bauer-Mehren A. (2013) Integration of genomic information with biological networks using Cytoscape. Methods Mol. Biol., 1021, 37–61. Busk P.K. (2014) A tool for design of primers for microRNAspecific quantitative RT-qPCR. BMC Bioinformatics, 15, 29. Chen X., Gao C., Li H., Huang L., Sun Q., Dong Y., Tian C., Gao S., Dong H., Guan D., Hu X., Zhao S., Li L., Zhu L., Yan Q., Zhan J., Zen K., Zhang C.Y. (2010) Identification and characterization, of microRNAs in raw milk during different periods, of lactation, commercial fluid, and powdered milk products. Cell Res., 20, 1128–1137. Davoren P.A., McNeill R.E., Lowery A.J., Kerin M.J., Miller N. (2008) Identification of suitable endogenous control genes for microRNA gene expression analysis in human breast cancer. BMC Mol. Biol., 9, 76. Dong F., Ji Z.B., Chen C.X., Wang G.Z., Wang J.M. (2013) Target gene and function prediction of differentially expressed micrornas in lactating mammary glands of dairy goats. Int. J. Genomics, 2013, 917342. Elsarraj H.S., Hong Y., Valdez K., Carletti M., Salah S.M., Raimo M., Taverna D., Prochasson P., Bharadwaj U., Tweardy D.J., Christenson L.K., Behbod F. (2013) A novel role of microRNA146b in promoting mammary alveolar progenitor cell maintenance. J. Cell Sci., 126(Pt 11), 2446–2458. Filipowicz W., Bhattacharyya S.N., Sonenberg N. (2008) Mechanisms of post-transcriptional regulation by microRNAs: are the answers in sight? Nat. Rev. Genet., 9, 102–114. Finnerty J.R., Wang W.X., Hebert S.S., Wilfred B.R., Mao G., Nelson P.T. (2010) The miR-15/107 group of microRNA genes: evolutionary biology, cellular
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Ratert N., Meyer H.A., Jung M., Mollenkopf H.J., Wagner I., Miller K., Kilic E., Erbersdobler A., Weikert S., Jung K. (2012) Reference miRNAs for miRNAome analysis of urothelial carcinomas. PLoS One, 7, e39309. Roarty K., Serra R. (2007) Wnt5a is required for proper mammary gland development and TGF-beta-mediated inhibition of ductal growth. Development, 134, 3929–3939. Rogler C.E., Levoci L., Ader T., Massimi A., Tchaikovskaya T., Norel R., Rogler L.E. (2009) MicroRNA-23b cluster microRNAs regulate transforming growth factor-beta/ bone morphogenetic protein signaling and liver stem cell differentiation by targeting Smads. Hepatology, 50, 575–584. Silveri L., Tilly G., Vilotte J.L., Le Provost F. (2006) MicroRNA involvement in mammary gland development and breast cancer. Reprod. Nutr. Dev., 46, 549–556. Tanaka T., Haneda S., Imakawa K., Sakai S., Naqaoka K. (2009) A microRNA, miR-101a, controls mammary gland development by regulating cyclooxygenase- 2 expression. Differentiation, 77, 181–187. Whyte J., Bergin O., Bianchi A., McNally S., Martin F. (2009) Key signaling nodes in mammary gland development and cancer. Mitogen-activated protein kinase signaling in experimental models of breast cancer progression and in mammary gland development. Breast Cancer Res., 11, 209. Yu F., Yao H., Zhu P., Zhang X., Pan Q., Gong C., Huang Y., Hu X., Su F., Lieberman J., Song E. (2007) let-7 regulates self renewal and tumorigenicity of breast cancer cells. Cell, 131, 1109–1123. Zhou Q., Li M., Wang X., Li Q., Wang T., Zhu Q., Zhou X., Wang X., Gao X., Li X. (2012) Immune-related microRNAs are, abundant in breast milk exosomes. Int. J. Biol. Sci., 8, 118–123.
Supporting Information Additional Supporting Information may be found in the online version of this article: Table S1 Primers used in qPCR analysis.
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ORIGINAL ARTICLE
Characterization of microRNA profile in mammary tissue of dairy and beef breed heifers ska1 & T. Motyl1 Z. Wicik1,2, M. Gajewska1, A. Majewska1, D. Walkiewicz3, E. Osin 1 Department of Physiological Sciences, Faculty of Veterinary Medicine, Warsaw University of Life Sciences, Warsaw, Poland 2 Department of Human Epigenetics, Mossakowski Medical Research Centre Polish Academy of Sciences, Warsaw, Poland 3 Department of Geriatrics and Gerontology, Medical Center of Postgraduate Education, Warsaw, Poland
Summary
Keywords Dairy and beef cattle breeds; mammary gland; mammary stem cells; microRNA. Correspondence T. Motyl, Department of Physiological Sciences, Faculty of Veterinary Medicine, Warsaw University of Life Sciences, Nowoursynowska 159, 02-776 Warsaw, Poland. Tel: +48 22 847 24 52; Fax: +48 22 847 24 52; E-mail: [email protected] Received: 18 September 2014; accepted: 11 February 2015
MicroRNAs (miRNAs) are small non-coding RNAs that participate in the regulation of gene expression. Their role during mammary gland development is still largely unknown. In this study, we performed a microarray analysis to identify miRNAs associated with high mammogenic potential of the bovine mammary gland. We identified 54 significantly differentially expressed miRNAs between the mammary tissue of dairy (Holstein-Friesian, HF) and beef (Limousin, LM) postpubertal heifers. Fifty-two miRNAs had higher expression in the mammary tissue of LM heifers. The expression of the top candidate miRNAs (bta-miR-10b, bta-miR-29b, bta-miR101, bta-miR-375, bta-miR-2285t, bta-miR-146b, bta-let7b, bta-miR-107, bta-miR-1434-3p) identified in the microarray experiment was additionally evaluated by qPCR. Enrichment analyses for targeted genes revealed that the major differences between miRNA expression in the mammary gland of HF versus LM were associated with the regulation of signalling pathways that are crucial for mammary gland development, such as TGFbeta, insulin, WNT and inflammatory pathways. Moreover, a number of genes potentially targeted by significantly differentially expressed miRNAs were associated with the activity of mammary stem cells. These data indicate that the high developmental potential of the mammary gland in dairy cattle, leading to high milk productivity, depends also on a specific miRNA expression pattern.
Introduction MicroRNAs (miRNAs) are short, ~22 nucleotide long, non-coding RNAs that are involved in the regulation of gene expression by affecting mRNA stability and translation. After maturation, miRNAs are incorporated into an RNA-induced silencing complex and recognize the target genes through imperfect base pairing. As partial complementarity is sufficient for an miRNA to target an mRNA, each miRNA has the ability to regulate a large number of genes (Filipowicz et al. 2008). miRNAs have been identified in many types of cells as well as in biological fluids such as milk. According to the miRBase, 808 precursors and © 2015 Blackwell Verlag GmbH
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783 mature miRNAs have been identified so far for the Bos taurus species (Kozomara & Griffiths-Jones 2014). The total number of miRNAs in bovine milk is about twice as high as it is in serum, which indicates that mammary alveolar cells express their own miRNAs (Chen et al. 2010). In the mammary gland, most of the miRNAs studied have functions related to immunity and show alterations in their expression pattern during lactation (Zhou et al. 2012). Previous studies have indicated that miRNAs could play an important role during the development of the mammary gland tissue, especially the epithelium, through the regulation of gene expression (Avril-Sassen et al. 2009; Tanaka et al. 2009). miRNAs also have an doi:10.1111/jbg.12172
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important role in regulating self-renewal and differentiation of stem cells (Gangaraju & Lin 2009). The majority of studies on miRNA function have been focused on human-related diseases and rodent animal models. On the other hand, studies performed on farm mammals have mainly focused on miRNAs that potentially regulate gene expression in the lactating, not virgin mammary gland. Knowledge about the development of mammary glands is essential in terms of further improvement of dairy cattle production capacity in the future. The potential of mammary gland development differs between beef and dairy cattle, and the intensity of development, as well as the productivity of the gland in beef breeds, is incomparably smaller (Keys et al. 1989; Akers 2006). Our recent study revealed that these differences may not only result from various levels of genetic and central endocrine regulation, but may also depend on intramammary factors, including the number of mammary stem cells and expression of genes involved in the development of the mammary stem cell niche (Osi nska et al. 2014). This study provides the first global comparison of the mammary miRNA profiles between postpubertal dairy (Holstein-Friesian, HF) and beef heifers (Limousin, LM). These results may be helpful for further elucidation of complex regulatory networks between miRNAs and mRNAs, and for future studies on mammary gland physiology and dairy potential. Special attention was paid to miRNAs associated with the activity of mammary gland stem cells.
labelled and hybridized in accordance with Agilent’s instructions. Microarrays were scanned using the G2505C Microarray Scanner (Agilent Technologies, Santa Clara, CA, USA), then gridded, and the quality of hybridization was initially determined by the AGILENT FEATURE EXTRACTION software. Raw data were then analysed using GENESPRING 12.0 (Agilent Technologies, Sanat Clara, CA, USA). To minimize the biological variability across arrays, raw data were 1.0-thresholded, log2transformed and 75-percentile-shifted with baseline transformation (mean of all samples). Then, all miRNAs were filtered by flags (detected, non-detected) and statistically analysed. Statistical analysis was performed using unpaired t-test with Benjamini–Hochberg FDR multiple testing corrections (p ≤ 0.05). Further ontological analyses were conducted on genes showing significant changes after FDR correction. The area of the analyses covered in this publication has been deposited in NCBI’s Gene Expression Omnibus and is accessible via GEO Series accession number GSE61227. Clustering analysis
Hierarchical cluster analysis was performed in Genespring 12.6 (Agilent Technologies, Santa Clara, USA). The expression data were log-transformed and grouped using a hierarchical clustering algorithm by normalized intensity values on miRNAs and conditions (HF and LM). A similarity measure was performed using squared Euclidean measure with average linkage rule. The analysis was performed on non-averaged lists of genes with corrected p-value ≤0.05.
Material and methods Tissue sampling
Mammary tissue was obtained at the slaughterhouse from 2-year-old non-pregnant heifers (four HFs and four LMs) free of clinical signs of mastitis. Udders were removed, and mammary tissue was collected, fixed in RNAlater solution (Qiagen, Hilden, Germany) and stored at 80°C until further analysis. miRNA microarray analysis
Total RNA containing miRNA was isolated from samples using the miRNeasy Mini Kit (Qiagen, Hilden, Germany) in accordance with the manufacturer’s protocol. Total RNA quantity and purity were assessed using NanoDrop, and RNA integrity was verified using an Agilent 2100 Bioanalyzer. The miRNA profiling was performed using an Agilent custom microarray (8 9 60 K format) containing probes of 763 B. taurus miRNAs. Total RNA from each sample was amplified, 32
Target gene prediction of differential expression microRNAs
miRNA target prediction was performed using the TargetScan database integrated with GeneSpring 12.0 (Agilent Technologies, Santa Clara, CA, USA). Analysis was performed for the list of all significantly differentially expressed miRNAs. It revealed which genes (targets) could be most significantly regulated by analysed miRNA. Scanning was performed for context score percentile (50) and conserved/non-conserved miRNA families and target sites. Ontological analyses of miRNA target genes
Ontological analyses revealing biological processes and molecular functions of miRNA targets were carried out using the binomial overrepresentation test with Bonferroni correction in PANTHER (Protein ANalysis THrough Evolutionary Relationships) © 2015 Blackwell Verlag GmbH
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© 2015 Blackwell Verlag GmbH
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Table 2 (continued) (b)
Target scan p-value
Gene symbol
Gene description
Fold change obtained from our previous ska study (Osin et al. 2014)
1.16E-04
IL6
Interleukin 6 (interferon, beta 2)
276.9
1.92E-02
IL1B
Interleukin 1, beta
4.13E-02
CSF2
2.98E-02
CEBPD
5.84E-03
NGF
6.06E-04
EDN1
Colony-stimulating factor 2 (granulocyte–macrophage) CCAAT/enhancer binding protein (C/EBP), delta Nerve growth factor (beta polypeptide) Endothelin 1
1.9
3.37E-05
EZH2
Enhancer of zeste homolog 2
2.2
11.4 2.9 2.6 2.1
Figure 4 Comparison of expression of selected miRNAs in the mammary gland samples from LM (n = 4) and HF (n = 4) heifers. The expression was analysed using microarray and qPCR methods. In the case of qPCR, the levels of miRNA expression were normalized according to the relative expression of housekeeping miRNAs – bta-miR-10b and bta-miR181b. Fold changes of gene expression determined by qPCR were calculated using delta delta Ct method and are shown as mean SD.
gestation, lactation, as well as early and late involution). Some of the miRNAs identified, which were upregulated in virgin animals, were also present in our © 2015 Blackwell Verlag GmbH
• J. Anim. Breed. Genet. 133 (2016) 31–42
Annotated miRNAs systematic names bta-miR-26b,bta-let-7f,bta-miR-376b,bta-miR-26a,bta-let-7c,bta-let-7e, bta-let-7b,bta-let-7d,bta-let-7i,bta-let-7a-5p,bta-miR-1434-3p bta-miR-204,bta-miR-222,bta-miR-27a-3p,bta-miR-183,bta-miR-221, bta-miR-27b,bta-miR-24-3p bta-let-7c,bta-let-7e,bta-let-7b,bta-let-7a-5p,bta-miR-199a-5p, bta-miR-199b bta-miR-101,bta-miR-27a-3p,bta-miR-199c,bta-miR-27b bta-let-7f,bta-let-7c,bta-let-7e,bta-let-7b,bta-let-7d,bta-let-7i, bta-let-7a-5p bta-miR-199c,bta-miR-185,bta-let-7a-5p,bta-miR-199a-3p,bta-let-7f, bta-let-7c,bta-let-7e,bta-let-7d,bta-let-7b,bta-let-7i,bta-miR-1434-3p, bta-miR-101,bta-miR-194,bta-miR-411a bta-miR-26b,bta-miR-199c,bta-let-7f,bta-miR-26a,bta-let-7c,bta-let-7e, bta-let-7b,bta-let-7d,bta-let-7i, bta-miR-25,bta-let-7a-5p,bta-miR-101
data set, that is let-7b, let-7c, let-7d, let-7e, let-7f, let-7i, miR-146b, miR-199b, miR-222, miR-24, miR25, miR-26a, miR-26b, miR-27b, miR-29a, miR-29b, miR-29c and miR-338. The identified members of the let-7 family were previously shown to be depleted in mouse mammary epithelial progenitors, whereas enforced let-7 expression led to inhibition of selfrenewal capacities of cells (Ibarra et al. 2007; Yu et al. 2007). It is worth noting that these miRNAs had lower expression in HF heifers’ mammary tissue compared to the LM mammary gland (Figure 1). The lower expression of let-7 miRNAs corresponds to higher productivity of the HF breed and suggests that this property may also be connected with enhanced selfrenewal capacity of HF’s mammary tissue. Furthermore, ontological analysis of miRNA targets revealed that the major interbreed differences were associated with metabolic processes and signalling pathways that are closely related to stem cell activity and mammary gland development (Figures 2 and 3). This was observed on many levels, including highly regulated genes associated with stem cell maintenance and renewal, epithelium development or signalling pathways known to play a crucial role during mammogenesis. Among the most strongly regulated pathways were insulin, Wnt, TGF-beta, MAPK, EGFR and interleukin signalling pathways (Figure 3). Wnt, EGFR, TGFB and insulin signalling pathways are known to play an important role in normal development of the mammary gland (Musters et al. 2004; 39
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Figure 1 A heat map based on the mean expression levels of miRNAs. Each column corresponds to the expression profile of the heifer breed, Limousin (LM) and Holstein-Friesian (HF) in quadruplicate, and each row corresponds to a miRNA accession number. Red indicates lower expression and green indicates higher expression of analysed miRNAs, whereas black indicates mean level of expression. Fold changes for each gene are shown on the color scale on the right side of the graph. Blue color indicates downregulation; gradation from yellow to red indicates upregulation of gene expression.
Table 1 List of miRNAs, differing between Limousin and Holstein-Friesian heifers above two fold change (FC) with FDR-corrected p-value cut-off ≤0.05 Systematic_name
mirbase accession No
p-Value
FC
bta-miR-375 bta-miR-218 bta-miR-29b_v12.0 bta-miR-24 bta-miR-183 bta-miR-147 bta-miR-204 bta-miR-421 bta-miR-155 bta-miR-146b bta-miR-218_v13.0 bta-miR-29b bta-miR-154c bta-miR-101 bta-miR-194 bta-miR-1434-3p bta-miR-2285t
MIMAT0009303 MIMAT0003798 MIMAT0003828 MIMAT0009250 MIMAT0009245 MIMAT0009237 MIMAT0004338 MIMAT0009314 MIMAT0009241 MIMAT0009235 MIMAT0003798 MIMAT0003828 MIMAT0025542 MIMAT0003520 MIMAT0009254 MIMAT0012040 MIMAT0025577
0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.002 0.005 0.002 0.029 0.042 0.014 0.001 0.015 0.020
157.70 106.44 83.58 72.89 47.06 29.73 6.53 2.39 2.28 2.24 2.23 2.17 2.07 2.03 2.02 2.29 4.34
heifers could influence target genes involved in stem cell activity, differentiation and immune response (Figure 3). Furthermore, pathways involved in the regulation of senescence and autophagy, Id signalling, adipogenesis and androgen receptor signalling were also shown to be potentially significantly regulated. Analysis of the potential of miRNAs to influence mammary stem cells and their niche
Special attention was given to the role of identified miRNAs in post-transcriptional regulation of genes, whose products might affect mammary stem/progenitor cells. We identified 20 targeted genes involved in cell maintenance, stem cell renewal and stem cell development (HAS2, EZH2, MED6, HOXA9, VEGFA, COL1A1, CNOT2, KDR, BCL9, EIF2AK3, MED21, IRS1, HNF1B, RIF1, WNT3A, LRP6, SALL4, SLC12A2, TFAP2C and IGF1R) and 7 other genes associated with stem cell niche activity (IL6, IL1B, CSF2, CEBPD, NGF, EDN1 and EZH2), as described in our previous publication (Osi nska et al. 2014). The characteristics of these target genes are presented in Table 2a, b. © 2015 Blackwell Verlag GmbH
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Validation of the results using qPCR
Verification of the miRnomic results was carried out using the qPCR method. The miRNAs for validation were selected upon analysing their involvement in the major processes associated with mammary gland development. Analysis confirmed the changes in expression of all 8 genes tested (Figure 4); however, in the case of bta-mir-375 and bta-miR-29b, we observed a significant difference in the values of expression obtained in qPCR compared to the microarray results (but the direction of changes remained the same). This was due to the fact that the expression of bta-mir-375 and bta-miR-29b was not detected on microarrays for the HF samples (lack of hybridization). Therefore, the obtained values of fold change were very high compared to the expression in LM heifers. We also found four other miRNAs (bta-miR147, bta-miR-183, bta-miR-218 and bta-miR-24), which yielded similar results in the microarray analysis. In our opinion, these genes should not be excluded from the analysis, but it is necessary to consider that the changes in their expression may in fact not be as large. Discussion MicroRNAs and their role in mammary gland development are currently intensively studied, especially in humans, but there is still a limited understanding of their function in bovine mammary glands, influence on organ development and dairy potential. In this study, an extensive miRNA expression profile in the mammary glands of dairy (HF) and beef (LM) postpubertal heifers was investigated. Microarray analysis revealed 54 miRNAs that significantly differed between the two investigated breeds of cattle (Figure 1). Fifty-two of the miRNAs identified showed higher expression in the mammary tissue of LM heifers, which is an important finding given the inhibitory role of miRNAs in the regulation of genes that are involved in mammary gland development. To our knowledge, this is the first study comparing the expression of miRNAs involved in the regulation of the mammary gland between two cattle breeds with a different productivity type. 35
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(a)
(b)
Figure 2 Major categories of (a) biological processes and (b) molecular functions regulated by targets of miRNAs with significantly differing expression between dairy heifers (Holstein-Friesian) and beef heifers (Limousin). Analysis was performed using Panther overrepresentation test with Bonferroni correction (p ≤ 0.05).
Figure 3 Top 10 signaling pathways (p ≤ 0.05) regulated by targets of miRNAs with significantly differing expression between beef heifers (Limousin) and dairy heifers (HolsteinFriesian). Analysis was performed using Single Experiment Analysis tool in GENESPRING software.
We compared the data obtained in our microarray analysis with the data published in the available literature and found that nearly 40 of the 54 miRNAs showing significant breed-dependent differences in expression were also detected in the studies of other authors on bovine (Li et al. 2012), goat (Dong et al. 2013), mouse (Avril-Sassen et al. 2009) and human (Silveri et al. 2006) mammary glands. Most of the studies compared the expression of miRNA in the mammary tissue at different stages of development during the postnatal life (Avril-Sassen et al. 2009; Li et al. 2012) or focused specifically on the lactation period (Dong et al. 2013; Le Guillou et al. 2014). Li et al. (2012) compared miRNA profiles from lactating and non-lactating bovine mammary glands and showed that the expression of 56 miRNAs was significantly different between lactation and non-lactation periods. Among these miRNAs, only 6 were highly 36
expressed in the non-lactation period: miR-107-3p, miR-23b-3p, PC-320c-2-3p, PC-574-3p, CN-100-1-5p and CN-190-5p (Li et al. 2012). Two of these miRNAs, that is miR-107 and miR-23b, were also identified in our microarray study and showed lower expression in HF than in LM heifers. Interestingly, it has been suggested that miR-107 is an important regulator of proliferative properties of cells, decreasing the rates of cell division with cell cycle arrest (Finnerty et al. 2010), whereas miR-23b may repress the growth of the ductal system (Rogler et al. 2009; Li et al. 2012). This suggests that the higher milk production potential of HF cattle also results from a more favourable pattern of miRNA expression, which stimulates the development of the mammary gland. Avril-Sassen et al. (2009) compared the expression of miRNA in the murine mammary gland at different stages of development (juvenile, puberty, mature virgin, © 2015 Blackwell Verlag GmbH
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Table 2 (a) List of target genes regulated by miRNAs, differing between Limousin (LM) and Holstein-Friesian (HF) heifers, associated with mammary gland development and stem cell activity; miRNAs in bold showed expression fold change over 2. (b) List of significantly targeted genes associated ska et al. 2014). These factors were significantly regulated on mRNA level in the with stem cell niche, as described in our previous publication (Osin mammary tissue of HF, in comparison with LM heifers. Analysis was performed for genes significantly expressed over a 1.3 absolute fold change (FC) with FDR-corrected p-value cut-off ≤0.05 (a) Target scan p-value
Gene symbol
2.40E-05
Gene description
Major gene functions (GO)
Annotated miRNAs systematic names
HAS2
Hyaluronan synthase 2
Stem cell development Positive regulation of cell proliferation Positive regulation of cell migration
3.37E-05
EZH2
Enhancer of zeste homolog 2
5.27E-05
MED6
Mediator complex subunit 6
9.54E-04
HOXA9
Homeobox A9
Stem cell development Positive regulation of epithelial-to-mesenchymal transition Regulation of cell proliferation Stem cell maintenance Positive regulation of transcription from RNA polymerase II promoter Mammary gland development Endothelial cell activation
bta-mir-27a-3p, bta-mir-16a, bta-miR-375, bta-let-7a-5p, bta-miR-27b, bta-miR-497, bta-miR-204, bta-miR-186, bta-miR-376b, bta-miR-26a, bta-let-7c, bta-let-7b, bta-let-7d, bta-miR-15a, bta-miR-25, bta-miR-23b-3p, ↓bta-miR-1434-3p, bta-miR-101, bta-miR-16b bta-miR-26b, bta-miR-199c, bta-let-7f, bta-miR-26a, bta-let-7c, bta-let-7e, bta-let-7b, bta-let-7d, bta-let-7i, bta-miR-25, bta-let-7a-5p, bta-miR-101
6.38E-03
VEGFA
Vascular endothelial growth factor A
8.81E-03
COL1A1
Collagen, type I, alpha 1
1.36E-02
CNOT2
1.36E-02
KDR
1.41E-02
BCL9
CCR4-NOT transcription complex, subunit 2 Kinase insert domain receptor; vascular endothelial growth factor receptor 2 B-cell CLL/lymphoma 9
1.41E-02
EIF2AK3
1.51E-02
MED21
Eukaryotic translation initiation factor 2-alpha kinase 3 Mediator complex subunit 21
Lactation Mammary gland alveolus development Positive regulation of endothelial cell proliferation Positive regulation of endothelial cell migration Positive regulation of epithelial-to-mesenchymal transition Blood vessel development Positive regulation of canonical Wnt signalling pathway Regulation of stem cell maintenance Negative regulation of oestrogen receptor signalling pathway Branching morphogenesis of an epithelial tube Calcium ion homoeostasis Endothelial cell differentiation Somatic stem cell maintenance Canonical Wnt pathway
Lactation Fat cell differentiation Insulin-like growth factor receptor signalling pathway Stem cell maintenance Blastocyst development Positive regulation of transcription from RNA polymerase II promoter
bta-mir-27a-3p, bta-let-7f, bta-miR-186, bta-miR-375, bta-let-7c, bta-let-7e, bta-let-7d, bta-let-7b, bta-let-7i, bta-let-7a-5p, bta-miR-27b bta-miR-26b, bta-miR-199c, bta-miR-375, bta-let-7a-5p, bta-miR-2288, bta-let-7f, bta-miR-186, bta-miR-376b, bta-miR-26a, bta-let-7c, bta-let-7e, bta-let-7d, bta-let-7b, bta-let-7i, bta-miR-421 bta-miR-199c, bta-mir-16a, bta-miR-375, bta-miR-185, bta-miR-199a-5p, bta-miR-497, bta-miR-29a, bta-miR-186, bta-miR-15a, bta-miR-29c, bta-miR-16b, bta-miR-199b, bta-miR-29b
bta-miR-338, bta-let-7a-5p, bta-miR-218, bta-miR-29a, bta-miR-186, bta-let-7f, bta-let-7c, bta-let-7e, bta-let-7d, bta-let-7b, bta-let-7i, bta-miR-29c, bta-miR-29b bta-miR-222, bta-let-7a-5p, bta-miR-218, bta-let-7f, bta-let-7c, bta-let-7e, bta-let-7d, bta-let-7b, bta-let-7i, bta-miR-23b-3p, bta-miR-221 bta-miR-222, bta-miR-26b, bta-miR-183, bta-mir-16a, bta-miR-24-3p, bta-miR-497, bta-miR-186, bta-miR-26a, bta-miR-15a, bta-miR-221, bta-miR-16b bta-miR-199c, bta-miR-338, bta-mir-16a, bta-miR-218, bta-miR-497, bta-miR-204, bta-miR-15a, ↓bta-miR-1434-3p, bta-miR-101, bta-miR-16b bta-miR-222, bta-miR-26b, bta-miR-199c, bta-miR-155, bta-miR-24-3p, bta-miR-26a, bta-miR-101, bta-miR-409a, bta-miR-221, bta-miR-421 bta-miR-199c, bta-miR-155, bta-let-7a-5p, bta-miR-107, bta-let-7f, bta-miR-186, bta-let-7c, bta-let-7e, bta-let-7b, bta-let-7d, bta-let-7i
(continued)
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Table 2 (continued) (a) Target scan p-value
Gene symbol
1.60E-02
Gene description
Major gene functions (GO)
Annotated miRNAs systematic names
IRS1
Insulin receptor substrate 1
Mammary gland development Lipid catabolic proces Negative regulation of insulin secretion
2.67E-02
HNF1B
HNF1 homeobox B
2.67E-02
RIF1
RAP1 interacting factor homolog (yeast)
Epithelial cell proliferation Branching morphogenesis of an epithelial tube Insulin secretion Regulation of Wnt signalling pathway Stem cell maintenance Chromosome telomeric region
bta-miR-183, bta-mir-16a, bta-miR-146b, bta-miR-107, bta-miR-186, bta-miR-29c, bta-miR-29b, bta-mir-27a-3p, bta-miR-199c, bta-miR-27b, bta-miR-497, bta-miR-29a, bta-miR-15a, bta-miR-23b-3p, bta-miR-101, bta-miR-16b bta-miR-26b, bta-miR-199c, bta-miR-375, bta-miR-199a-3p, bta-miR-218, bta-miR-24-3p, bta-miR-26a, bta-miR-25, bta-miR-101, bta-miR-194
3.13E-02
WNT3A
Wingless-type MMTV integration site family, member 3A
3.50E-02
LRP6
Low-density lipoprotein receptor-related protein 6
3.81E-02
SALL4
Sal-like 4
Mammary gland development Wnt signalling pathway Negative regulation of fat cell differentiation Branching involved in mammary gland duct morphogenesis Mammary placode formation Negative regulation of fat cell differentiation Stem cell maintenance
3.81E-02
SLC12A2
Solute carrier family 12 (sodium/ potassium/chloride transporters), member 2
Branching involved in cmammary gland duct morphogenesis Mammary duct terminal end bud growth
3.81E-02
TFAP2C
Transcription factor AP-2 gamma (activating enhancer binding protein 2 gamma)
4.41E-02
IGF1R
Insulin-like growth factor 1 receptor
Dichotomous subdivision of terminal units involved in mammary gland duct morphogenesis Epithelial cell proliferation involved in mammary gland duct elongation Somatic stem cell maintenance Germ-line stem cell maintenance Mammary gland development Insulin-like growth factor receptor signalling pathway Negative regulation of apoptotic process
bta-miR-155, bta-mir-16a, bta-miR-29c, bta-miR-199b, bta-miR-29b, bta-miR-375, bta-miR-218, bta-miR-199a-5p, bta-miR-497, bta-miR-15a, bta-miR-23b-3p, bta-miR-411a, bta-miR-16b bta-mir-27a-3p, bta-mir-16a, bta-miR-185, bta-miR-27b, bta-miR-497, bta-miR-107, bta-miR-15a, bta-miR-101, bta-miR-16b bta-miR-183, bta-mir-16a, bta-miR-2288, bta-miR-186, ↓bta-miR-1434-3p, bta-miR-29c, bta-miR-29b, bta-miR-26b, bta-miR-497, bta-miR-26a, bta-miR-15a, bta-miR-101, bta-miR-411a, bta-miR-16b bta-mir-16a, bta-miR-15a, bta-miR-16b, bta-miR-497, bta-miR-107 bta-mir-16a, bta-miR-107, bta-miR-186, bta-miR-26b, bta-mir-27a-3p, bta-miR-199c, bta-miR-218, bta-miR-27b, bta-miR-497, bta-miR-204, bta-miR-26a, bta-miR-25, bta-miR-15a, bta-miR-101, bta-miR-16b bta-mir-27a-3p, bta-miR-27b, bta-miR-542-5p, bta-miR-29a, bta-miR-186, bta-miR-24, bta-miR-29c, bta-miR-29b, bta-miR-421
bta-miR-185, bta-let-7a-5p, bta-let-7f, bta-miR-186, bta-miR-376b, bta-let-7c, bta-let-7e, bta-let-7b, bta-let-7d, bta-let-7i, bta-miR-25, bta-miR-194
(continued)
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Table 2 (continued) (b)
Target scan p-value
Gene symbol
Gene description
Fold change obtained from our previous ska study (Osin et al. 2014)
1.16E-04
IL6
Interleukin 6 (interferon, beta 2)
276.9
1.92E-02
IL1B
Interleukin 1, beta
4.13E-02
CSF2
2.98E-02
CEBPD
5.84E-03
NGF
6.06E-04
EDN1
Colony-stimulating factor 2 (granulocyte–macrophage) CCAAT/enhancer binding protein (C/EBP), delta Nerve growth factor (beta polypeptide) Endothelin 1
1.9
3.37E-05
EZH2
Enhancer of zeste homolog 2
2.2
11.4 2.9 2.6 2.1
Figure 4 Comparison of expression of selected miRNAs in the mammary gland samples from LM (n = 4) and HF (n = 4) heifers. The expression was analysed using microarray and qPCR methods. In the case of qPCR, the levels of miRNA expression were normalized according to the relative expression of housekeeping miRNAs – bta-miR-10b and bta-miR181b. Fold changes of gene expression determined by qPCR were calculated using delta delta Ct method and are shown as mean SD.
gestation, lactation, as well as early and late involution). Some of the miRNAs identified, which were upregulated in virgin animals, were also present in our © 2015 Blackwell Verlag GmbH
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Annotated miRNAs systematic names bta-miR-26b,bta-let-7f,bta-miR-376b,bta-miR-26a,bta-let-7c,bta-let-7e, bta-let-7b,bta-let-7d,bta-let-7i,bta-let-7a-5p,bta-miR-1434-3p bta-miR-204,bta-miR-222,bta-miR-27a-3p,bta-miR-183,bta-miR-221, bta-miR-27b,bta-miR-24-3p bta-let-7c,bta-let-7e,bta-let-7b,bta-let-7a-5p,bta-miR-199a-5p, bta-miR-199b bta-miR-101,bta-miR-27a-3p,bta-miR-199c,bta-miR-27b bta-let-7f,bta-let-7c,bta-let-7e,bta-let-7b,bta-let-7d,bta-let-7i, bta-let-7a-5p bta-miR-199c,bta-miR-185,bta-let-7a-5p,bta-miR-199a-3p,bta-let-7f, bta-let-7c,bta-let-7e,bta-let-7d,bta-let-7b,bta-let-7i,bta-miR-1434-3p, bta-miR-101,bta-miR-194,bta-miR-411a bta-miR-26b,bta-miR-199c,bta-let-7f,bta-miR-26a,bta-let-7c,bta-let-7e, bta-let-7b,bta-let-7d,bta-let-7i, bta-miR-25,bta-let-7a-5p,bta-miR-101
data set, that is let-7b, let-7c, let-7d, let-7e, let-7f, let-7i, miR-146b, miR-199b, miR-222, miR-24, miR25, miR-26a, miR-26b, miR-27b, miR-29a, miR-29b, miR-29c and miR-338. The identified members of the let-7 family were previously shown to be depleted in mouse mammary epithelial progenitors, whereas enforced let-7 expression led to inhibition of selfrenewal capacities of cells (Ibarra et al. 2007; Yu et al. 2007). It is worth noting that these miRNAs had lower expression in HF heifers’ mammary tissue compared to the LM mammary gland (Figure 1). The lower expression of let-7 miRNAs corresponds to higher productivity of the HF breed and suggests that this property may also be connected with enhanced selfrenewal capacity of HF’s mammary tissue. Furthermore, ontological analysis of miRNA targets revealed that the major interbreed differences were associated with metabolic processes and signalling pathways that are closely related to stem cell activity and mammary gland development (Figures 2 and 3). This was observed on many levels, including highly regulated genes associated with stem cell maintenance and renewal, epithelium development or signalling pathways known to play a crucial role during mammogenesis. Among the most strongly regulated pathways were insulin, Wnt, TGF-beta, MAPK, EGFR and interleukin signalling pathways (Figure 3). Wnt, EGFR, TGFB and insulin signalling pathways are known to play an important role in normal development of the mammary gland (Musters et al. 2004; 39
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Figure 5 Summarizing scheme showing significantly differentially expressed miRNAs, between dairy (Holstein-Friesian) and beef (Limousin) heifers, in context of their influences on specified processes and signalling pathways associated with mammary gland development. miRNAs marked with a red are known for their involvement in the lactation network (Li et al. 2012).
Akers et al. 2005; Roarty & Serra 2007; Mukhopadhyay et al. 2013). The MAPK pathway is one of the key regulators of mammary epithelial cell differentiation and function (Whyte et al. 2009). Moreover, seven of the miRNAs identified in our study (miR-199a-5p, miR-25, miR-27b, miR-29a and miR-29b, miR-375 m and miR-107) which are present in the interaction network performed by us (Figure 5.) were also present in the lactation interaction network created by Li et al. (2012). Nearly half of the gene targets involved in our interaction network had significance of targeting lower than 0.01. Genes with the highest degree of connections with selected processes were also shown to be the most significantly regulated by the significantly differentially expressed miRNA. These included NLK (27 connections), TP53 (26), FZD4 (25), GAB1 (24), MITF (23), CDC42, WNT3A (21), TAB 2 (20), MAP4K3, PIK3R1 and FERMT2 (19). Comparing results from network and gene target scan analysis revealed 8 genes which are significantly specific (p ≤ 0.0001) targets of the identified miRNAs and have the highest degree of connection with processes specifically involved in mammary gland functions: CAPN3, COL1A2, FZD4, 40
HAS2, MED6, NLK, TAB 2, WASL. Some of these miRNA target genes identified in this analysis were shown to be associated with stem cell activity (HAS2, MED6), as well as cytoskeleton and extracellular matrix (FERMT2, COL1A2, WASL and CAPN3). Moreover, we also identified some mutual interactions between the gene targets, for example CAPN3 gene expression may be regulated by MITF, whereas CDC42 activity might be regulated by the WASL gene product. Additionally, we observed a strong breed-dependent influence of miRNAs on signalling pathways connected with inflammatory processes. These data and our previous findings (Osi nska et al. 2014) suggest that the activity of inflammatory signalling pathways may be associated with regulation of stem cell activity and adaptation of dairy cattle resulting in protection against mastitis. For example, the levels of miRNA regulating the expression of interleukin 6 (IL6) (Table 2b) were lower in HF heifers, which corresponds with our previous results showing that IL6 expression was over 200-fold higher in this cattle breed (Osi nska et al. 2014). Furthermore, miR-146b has been associated with regulation of innate immune © 2015 Blackwell Verlag GmbH
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response, interleukin signalling and inflammation (Avril-Sassen et al. 2009). In our further analyses, we specifically focused on the potential role of miRNAs in the regulation of the mammary gland stem cell niche, which is required for intensive regenerative processes during puberty and pregnancy, preparing the gland for milk production and secretion during lactation (Table 2a). We previously showed that the high mammogenic potential of HF heifers is manifested by a higher number of stem/ progenitor cells and upregulation of genes involved in the formation of a favourable niche for the maintenance, renewal, proliferation and differentiation of stem/progenitor cells (Osi nska et al. 2014). Consistently, the result of the present study revealed downregulation of miRNAs involved in inhibition of the translation of genes responsible for the maintenance and activity of the mammary stem cells in HF heifers. Our analysis showed that genes potentially regulated by the identified miRNAs are involved in a network of signalling pathways, and several miRNAs involved in regulation of these pathways may be important for the maintenance of the self-renewal properties of the mammary gland and its development (Figure 5). miR-146b appears to be one of the key regulators, and its function has already been documented in previous studies. It has been suggested that high expression of miR-146b is connected with differentiation of mammary epithelial cells, as its expression levels increase during pregnancy, especially in the luminal progenitors compared to the basal/stem cells (Elsarraj et al. 2013). Furthermore, miR-146b showed significantly higher expression in the luminal progenitors in pregnant mice than in the mammary glands of virgin animals, indicating that a low level of miR-146b is required for the maintenance of the undifferentiated state of the mammary stem cells (Elsarraj et al. 2013). In our study, the expression of this miRNA was lower in the mammary glands of HF heifers that show a high mammogenic potential, compared to LM heifers with a lower mammogenic potential. Conclusions In conclusion, the present study has shown that the high potential of growth and development of the mammary gland in dairy cattle, as well as the high lactogenic potential of dairy breeds, depends not only on genetic but also on epigenetic control by miRNA. It is particularly interesting that miRNAs-dependent inhibition of the translation of genes connected with mammary stem cell functions is less pronounced in dairy heifers than it is in beef heifers, promoting a © 2015 Blackwell Verlag GmbH
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gene expression pattern responsible for more intensive development of the mammary gland in dairy cattle. Acknowledgement This study was supported by grant No. NN308594138 from the National Research Centre. References Akers R.M. (2006) Major advances associated with hormone and growth factor regulation of mammary growth and lactation in dairy cows. J. Dairy Sci., 89, 1222–1234. Akers R.M., Ellis S.E., Berry S.D. (2005) Ovarian and IGF-I axis control of mammary development in prepubertal heifers. Domest. Anim. Endocrinol., 29, 259–267. Avril-Sassen S., Goldstein L.D., Stingl J., Blenkiron C., Le Quesne J., Spiteri I., Karagavriilidou K., Watson C.J., Tavare S., Miska E.A., Caldasm C. (2009) Characterisation of microRNA expression in post-natal mouse mammary gland development. BMC Genom., 10, 548. Bauer-Mehren A. (2013) Integration of genomic information with biological networks using Cytoscape. Methods Mol. Biol., 1021, 37–61. Busk P.K. (2014) A tool for design of primers for microRNAspecific quantitative RT-qPCR. BMC Bioinformatics, 15, 29. Chen X., Gao C., Li H., Huang L., Sun Q., Dong Y., Tian C., Gao S., Dong H., Guan D., Hu X., Zhao S., Li L., Zhu L., Yan Q., Zhan J., Zen K., Zhang C.Y. (2010) Identification and characterization, of microRNAs in raw milk during different periods, of lactation, commercial fluid, and powdered milk products. Cell Res., 20, 1128–1137. Davoren P.A., McNeill R.E., Lowery A.J., Kerin M.J., Miller N. (2008) Identification of suitable endogenous control genes for microRNA gene expression analysis in human breast cancer. BMC Mol. Biol., 9, 76. Dong F., Ji Z.B., Chen C.X., Wang G.Z., Wang J.M. (2013) Target gene and function prediction of differentially expressed micrornas in lactating mammary glands of dairy goats. Int. J. Genomics, 2013, 917342. Elsarraj H.S., Hong Y., Valdez K., Carletti M., Salah S.M., Raimo M., Taverna D., Prochasson P., Bharadwaj U., Tweardy D.J., Christenson L.K., Behbod F. (2013) A novel role of microRNA146b in promoting mammary alveolar progenitor cell maintenance. J. Cell Sci., 126(Pt 11), 2446–2458. Filipowicz W., Bhattacharyya S.N., Sonenberg N. (2008) Mechanisms of post-transcriptional regulation by microRNAs: are the answers in sight? Nat. Rev. Genet., 9, 102–114. Finnerty J.R., Wang W.X., Hebert S.S., Wilfred B.R., Mao G., Nelson P.T. (2010) The miR-15/107 group of microRNA genes: evolutionary biology, cellular
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Supporting Information Additional Supporting Information may be found in the online version of this article: Table S1 Primers used in qPCR analysis.
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