**3. MicroRNA in mammary gland development and lactation biology**

#### **3.1. Occurrence of microRNA in ruminant mammary gland and in milk**

The regulatory roles of miRNAs in livestock species have emerged and are growing quickly [46, 47]. The most recent release of miRBase (release 21, http://www.mirbase.org/, [38]) contains 793 mature miRNAs for cattle, 436 for goat and 153 for sheep [38] (**Table 1**). However, with the increase in the application of RNA sequencing in expression profiling of miRNAs in different livestock species, the number of novel livestock miRNAs is expected to increase.

#### *3.1.1. Cattle*

and (3) preferential incorporation of one strand (the 'guide' miRNA strand) onto the RNAinduced silencing complex (RISC) [25]. Most miRNA genes located in introns of protein-coding genes share the promoter of the host gene [29]. MiRNAs often have multiple transcription start sites and regulate gene expression through inhibition of translation initiation or elongation, co-

Since the discovery of the first miRNA, lin-4, in 1993 [31] and aided by deep sequencing technologies and developments in bioinformatics processing of deep sequence data, thousands of miRNAs have been detected in humans, mouse, farm animal species and plants and deposited in the miRNA data base (**Table 1**). Due to the crucial regulatory roles of miRNAs in many biological processes across species, they are being considered as candidate biomarkers of various human diseases, such as autoimmune [32], metabolic [33] and cardiovascular

**Precursor Mature Transcripts Genes**

.

Long non-coding RNAs are a diverse collection of non-coding RNAs with emerging regulatory roles in many biological processes in every branch of life [26, 40–42]. LncRNA transcripts are >200 nucleotides long and constitute the largest portion of the mammalian non-coding RNA transcriptome [40]. LncRNA closely resembles mRNA than other classes of ncRNA in terms of their biogenesis pathways and form. Most lncRNAs are transcribed by the activities of RNA polymerase II, have a 5′ terminal methylguanosine cap and are often spliced and polyadenylated [41]. Some non-polyadenylated lncRNAs arise through alternative pathways probably expressed from RNA polymerase III promoters [43, 44] or arise during splicing and small nucleolar RNA production [45]. Furthermore, some lncRNAs are regulated in different ways at different stages of their biogenesis, maturation and decay [26]. Thousands of genes encoding lncRNAs have been identified in mammalian genomes (including livestock species),

\*Data source: MiRBase release 21 (http://www.mirbase.org/[38], and NONCODE database (www.noncode.org, Noncode

translational protein degradation and premature termination of translation [25, 30].

Cattle 808 793 22,386 23,696

Chicken 740 994 13,085 9681 Human 1881 2588 141,353 90,062 Mouse 1193 1915 117,405 79,940

**Table 1.** Number of detected miRNAs and lncRNAs in farm animal species, mouse and human\*

diseases [34], and various types of cancers [35–37].

**Species MiRNA lncRNA**

Sheep 106 153 – Goat 267 436 – Pig 382 411 –

**2.2. Long non-coding RNAs**

2016 [39]).

58 Current Topics in Lactation

The profiles of miRNAs in bovine MG tissue or milk have been investigated using different approaches, such as microarray [48, 49], genome sequencing [4] and RNA sequencing [50– 57]. A total of 496 miRNA genes were identified following sequencing of the cattle genome of which 135 were novel [4].The expression profiles of miRNAs in MG tissues and cells facilitate discovery of novel miRNAs and also identification of candidate miRNAs for different cell types, lactation stages, periods, disease response and so on. Before the release of the bovine genome sequence, Gu et al. [49] pioneered miRNA discovery in the bovine MG by cloning and sequencing small RNAs from MG tissue followed by identification of 59 distinct bovine miRNAs. Using next-generation sequencing techniques, Chen et al. [58] identified 230 and 213 known miRNAs in cow colostrum and mature milk, respectively. The authors also observed that 108 and 8 miRNAs were upregulated and downregulated, respectively, in colostrum compared to mature milk [58]. Using microarray, Izumi et al. [59] identified 100 and 53 known miRNAs in colostrum and mature milk, respectively. Using Solexa sequencing method, Li et al. [60] reported 884 unique miRNAs sequences in the bovine MG (283 known, 505 novel and 96 conserved miRNAs). Le Guillou et al. [61] identify 167 novel miR-NAs in the bovine MG, many of which were also detected in mouse MG. Analysing three milk fractions (fat, whey and cells) and mammary gland tissues, we reported 210, 200 and 249 known and 33, 31 and 36 novel miRNAs in milk fat, whey and cells, respectively, and 321 known and 176 novel miRNAs in mammary gland tissues [62]. Deep sequencing the milk fat across the lactation curve, we also identified a total of 475 known and 238 novel miRNAs [63].

#### *3.1.2. Goat*

A total of 487 miRNAs were identified when the goat genome was sequenced and the largest miRNA clusters were found on chromosome 21 [6]. Using the Illumina-Solexa high-throughput sequencing technology to analyse goat MG tissues during early lactation, Ji et al. [64] reported 131 novel and 300 conserved miRNAs. Using the same method (Illumina-Solexa sequencing), Li et al. [65] reported 346 conserved and 95 novel miRNAs in goat MG tissues from dry off and peak lactation does.

#### *3.1.3. Sheep*

Most miRNAs identified in sheep come from tissues other than the MG. For example, Caiment et al. [66] identified 747 miRNAs from the skeletal muscle through deep sequencing, whereas McBride et al. [67] reported 212 miRNAs from sheep ovarian follicles and corpus lutea at various reproductive stages. In the MG, Galio et al. [68] showed the presence of three known miRNAs including miR-21, miR-205 and miR-200 family in pregnant and lactating sheep.

#### **3.2. MicroRNA function in ruminant mammary gland and milk synthesis**

#### *3.2.1. Expression patterns of microRNAs in lactation stages*

#### *3.2.1.1. Temporal and spatial expression of microRNAs*

Indication of involvement of miRNAs in MG functions was gained through observation of differences in type and expression levels of miRNAs between lactation stages, under different nutritional regimes and presence of disease pathogens. Li et al. [50] identified 56 miRNAs that were significantly differentially expressed between lactation and non-lactation periods. Similarly, Wang et al. [48] detected 12 downregulated miRNAs (miR-10a, miR-15b, miR-16, miR-21, miR-33b, miR-145, miR-146b, miR-155, miR-181a, miR-205, miR-221 and miR-223) in the dry period (30 days prepartum) compared to early lactation period (7 days postpartum) and one upregulated miRNA (miR-31) in early lactation compared to the dry period. Previously, we examined miRNA expression pattern during a lactation cycle to explore it regulatory mechanisms during lactation using milk fat as input tissue for sampling [63]. In a previous investigation, we have shown that milk fat miRNA transcriptome closely resemble the miRNome of MG tissue [62]. We collected samples at the lactogenesis (LAC) (day 1 and 7), galactopoiesis (GAL) (day 30, 70, 130, 170 and 230) and involution (INV) (day 290 and when milk production dropped to 5 kg/day) stages from nine cows for deep sequencing [63]. We observed that 15 miRNAs (miR-30a-5p, miR-30d, miR-21-5p, miR-26a, miR-148a, let-7a-5p, let-7b, let-7f, let-7g, miR-99a-5p, miR-191, miR-200a, miR-200c, miR-186, miR-92a) were highly expressed across lactation stages [63]. MiR-148a and miR-26a were the most abundantly expressed accounting for more than 10% of the read counts in each stage of lactation. We also performed a differential expression (DE) analysis and detected miR-29b/ miR-363 and miR-874/miR-6254 as important mediators of transition signals from LAC to GAL and from GAL to INV stages, respectively [63]. Furthermore, DE analysis indicated various patterns of miRNA expression across the lactation curve. For instance, some miR-NAs were highly expressed during early lactation (lactogenesis) followed by decreased expression at later stages, whereas others were slightly expressed during early lactation but showed increased expression during mid-lactation and decreased expression during late lactation and vice versa [63] (**Figure 2**).

Non-Coding RNA Roles in Ruminant Mammary Gland Development and Lactation http://dx.doi.org/10.5772/67194 61


reported 131 novel and 300 conserved miRNAs. Using the same method (Illumina-Solexa sequencing), Li et al. [65] reported 346 conserved and 95 novel miRNAs in goat MG tissues

Most miRNAs identified in sheep come from tissues other than the MG. For example, Caiment et al. [66] identified 747 miRNAs from the skeletal muscle through deep sequencing, whereas McBride et al. [67] reported 212 miRNAs from sheep ovarian follicles and corpus lutea at various reproductive stages. In the MG, Galio et al. [68] showed the presence of three known miRNAs including miR-21, miR-205 and miR-200 family in pregnant and

Indication of involvement of miRNAs in MG functions was gained through observation of differences in type and expression levels of miRNAs between lactation stages, under different nutritional regimes and presence of disease pathogens. Li et al. [50] identified 56 miRNAs that were significantly differentially expressed between lactation and non-lactation periods. Similarly, Wang et al. [48] detected 12 downregulated miRNAs (miR-10a, miR-15b, miR-16, miR-21, miR-33b, miR-145, miR-146b, miR-155, miR-181a, miR-205, miR-221 and miR-223) in the dry period (30 days prepartum) compared to early lactation period (7 days postpartum) and one upregulated miRNA (miR-31) in early lactation compared to the dry period. Previously, we examined miRNA expression pattern during a lactation cycle to explore it regulatory mechanisms during lactation using milk fat as input tissue for sampling [63]. In a previous investigation, we have shown that milk fat miRNA transcriptome closely resemble the miRNome of MG tissue [62]. We collected samples at the lactogenesis (LAC) (day 1 and 7), galactopoiesis (GAL) (day 30, 70, 130, 170 and 230) and involution (INV) (day 290 and when milk production dropped to 5 kg/day) stages from nine cows for deep sequencing [63]. We observed that 15 miRNAs (miR-30a-5p, miR-30d, miR-21-5p, miR-26a, miR-148a, let-7a-5p, let-7b, let-7f, let-7g, miR-99a-5p, miR-191, miR-200a, miR-200c, miR-186, miR-92a) were highly expressed across lactation stages [63]. MiR-148a and miR-26a were the most abundantly expressed accounting for more than 10% of the read counts in each stage of lactation. We also performed a differential expression (DE) analysis and detected miR-29b/ miR-363 and miR-874/miR-6254 as important mediators of transition signals from LAC to GAL and from GAL to INV stages, respectively [63]. Furthermore, DE analysis indicated various patterns of miRNA expression across the lactation curve. For instance, some miR-NAs were highly expressed during early lactation (lactogenesis) followed by decreased expression at later stages, whereas others were slightly expressed during early lactation but showed increased expression during mid-lactation and decreased expression during late

**3.2. MicroRNA function in ruminant mammary gland and milk synthesis**

*3.2.1. Expression patterns of microRNAs in lactation stages*

*3.2.1.1. Temporal and spatial expression of microRNAs*

lactation and vice versa [63] (**Figure 2**).

from dry off and peak lactation does.

*3.1.3. Sheep*

60 Current Topics in Lactation

lactating sheep.

**Figure 2.** Differential miRNA expression patterns during a bovine lactation curve. (a) Fold change values of six miRNAs whose expression patterns changed significantly during each lactation switch and (b) box plots of their normalized read count values by lactation day. <sup>1</sup> LAC: lactogenesis; GAL: galactopoiesis; INV: involution; <sup>2</sup> D: downregulated and U: upregulated.

The temporal expression pattern of miRNAs has been reported in other ruminant species. For example, Galio et al. [68] reported a change in the expression pattern of miR-21, miR-205 and miR-200 family in MG tissues from pregnant and lactating sheep. From the early, middle and late stages of pregnancy and during lactation, the expression of miR-21 and miR-25 decreased, whereas miR-200 family (miR-200a, miR-200b, miR-200c, miR-141 and miR-429) showed increased expression [68]. Similarly, investigating the expression pattern of miR-NAs during early and peak lactation and dry period, Li et al. [65] identified 15 differentially expressed miRNAs when comparing peak lactation and dry period including three significantly highly expressed miRNAs (miR-2887, miR-451 and miR-2478) during peak lactation and 12 significantly highly expressed miRNAs (miR-199b, miR-128, miR-25, miR-145, miR-98, miR-222, miR-181b, miR-199a-3p, miR-93, miR-221, let-7b and let-7c) during the dry period.

#### *3.2.1.2. MicroRNAs synergistically regulate lactation control mechanisms*

A wealth of evidence indicates that several miRNAs can work together to regulate target genes in the same or different biological pathways [69, 70]. We have successfully characterized a group of highly interacting miRNAs (modules) using a weighted co-expression network analysis [71] and correlated important miRNA modules to milk yield and milk components [72]. We identified three consensus (BLUE [62 miRNAs], TURQUOISE [133 miRNAs] and BROWN [59 miRNAs]) modules and the GREY module reserved for unclassified genes, throughout lactation stages (**Figure 3**). Based on module trait relationship, we were able to determine important modules (with absolute correlation >0.6) for milk components at each lactation stage. The BROWN and BLUE modules were highly related to protein and somatic cell count, respectively, in early lactation, the BLUE module to somatic cells in middle lactation and the BLUE module to urea and lactose in late lactation stage. We also found the most important component or hub miRNAs, which potentially coordinated miRNA synergetic mechanisms in their respective modules. MiR-149-5b and miR-874 were hub miRNAs in the BLUE module for milk somatic cells at early and middle lactation, respectively, whereas miR-330 was the hub miRNA in the BLUE module for milk urea and lactose at late lactation (**Figure 3**). Three miRNAs (mir-149-5b, miR-874 and miR-30) in the BLUE module play important roles in cell cycle [73–77], so it could be expected that these miRNAs regulate secretion of somatic cells in milk from MG.

**Figure 3.** Important consensus modules and their hub miRNAs for milk component traits in different lactation periods. (a) Dynamic cut tree (dendrogram) based on topological overlap distance in gene expression profile; (b) module trait relationship in early, middle and late lactation and (c) hub miRNAs in the modules. GREY colour is for genes that do not belong to a specific module.

#### *3.2.2. Networks and pathways regulated by microRNAs during a lactation cycle*

Through their target genes, miRNAs have been shown to control signal transduction in different species [78]. MiRNA roles in important pathways such as transforming growth factor beta (TGF-β), prolactin and protein kinase signalling in MG development and lactation have been reviewed by several authors [79–83]. MiRNA regulation of three important signalling pathways (NOTCH, PTEN and HIPPO) in MG and breast cancer cells was recently reviewed [15]. Important miRNAs regulating these pathways include mir-34, mir-29, mir-146, mir-199 and mir-200 families for NOTCH signalling pathway, miR-21 and miR-155 for PTEN signalling pathway and miR-934 for HIPPO pathway. In Canadian Holstein cows, we performed the enrichment of differentially expressed miRNA target genes to signalling pathways and noted that relevant signalling pathways for transition between lactation stages are involved in apoptosis (PTEN and SAPK/JNK), intracellular signalling (protein kinase A, TGF-β and ERK5), cell cycle regulation (STAT3), cytokines (prolactin), hormone and growth factors (growth hormone and glucocorticoid receptor). *PTEN* is an important target gene for miR-29b in the regulation of mammary gland development [84]. PTEN signalling is crucial for the activities of prolactin autocrine [85]. The initiation of lactation is known to require induction of autocrine prolactin, and the level of this autocrine is known to be endogenously regulated by the signal of PTEN-PI3K-AKT pathway [85]. **Figure 4** is an illustration of some miRNAs that target genes in relevant signalling pathways during lactation [63]. Pathways, such as PTEN and growth hormone signalling, have been identified as important for regulatory mechanisms during lactation [85, 86].

We identified three consensus (BLUE [62 miRNAs], TURQUOISE [133 miRNAs] and BROWN [59 miRNAs]) modules and the GREY module reserved for unclassified genes, throughout lactation stages (**Figure 3**). Based on module trait relationship, we were able to determine important modules (with absolute correlation >0.6) for milk components at each lactation stage. The BROWN and BLUE modules were highly related to protein and somatic cell count, respectively, in early lactation, the BLUE module to somatic cells in middle lactation and the BLUE module to urea and lactose in late lactation stage. We also found the most important component or hub miRNAs, which potentially coordinated miRNA synergetic mechanisms in their respective modules. MiR-149-5b and miR-874 were hub miRNAs in the BLUE module for milk somatic cells at early and middle lactation, respectively, whereas miR-330 was the hub miRNA in the BLUE module for milk urea and lactose at late lactation (**Figure 3**). Three miRNAs (mir-149-5b, miR-874 and miR-30) in the BLUE module play important roles in cell cycle [73–77], so it could be expected that these miRNAs regulate secretion of somatic cells in milk from MG.

*3.2.2. Networks and pathways regulated by microRNAs during a lactation cycle*

not belong to a specific module.

62 Current Topics in Lactation

Through their target genes, miRNAs have been shown to control signal transduction in different species [78]. MiRNA roles in important pathways such as transforming growth factor beta

**Figure 3.** Important consensus modules and their hub miRNAs for milk component traits in different lactation periods. (a) Dynamic cut tree (dendrogram) based on topological overlap distance in gene expression profile; (b) module trait relationship in early, middle and late lactation and (c) hub miRNAs in the modules. GREY colour is for genes that do

**Figure 4.** Illustration of miRNA-gene-pathway networks obtained from dynamic differentially expressed miRNAs during a bovine lactation curve. The outer layer shows miRNAs (blue arrow heads), which targets at least two genes (white dots) in significantly enriched pathways (red dots).

#### *3.2.3. Functional validation of microRNA target genes*

Since *in vivo* experiments for functional validation of MG miRNAs are not feasible, such studies have mostly relied on the use of knock-out/mimics and MG-specific cell types. Using bovine mammary epithelial cells (BMEC), miR-15a was shown to regulate growth hormone receptor, viability of BMEC and the expression of casein genes [86]. MiR-486 regulation of lactation by targeting the PTEN gene in cow MGs has been demonstrated [87]. Bian et al. [88] recently reported that epigenetic regulation of miR-29s affects the lactation activity of BMEC. MiR-181a was shown to regulate the biosynthesis of bovine milk fat through targeting acyl-CoA synthetase long-chain family member 1 (*ACSL1*) [89]. MiR-103 was reported to control milk fat accumulation in goat MG during lactation [90]. Moreover, miR-27a was shown to suppress triglyceride accumulation as well as altered gene expression associated with fat metabolism in dairy goat mammary epithelial cells (GMEC) [91]. In another study, miR-135a was reported to target and regulate prolactin receptor (*PRLR*) gene in GMEC [92]. Inhibition of the expression of miR-145 in GMEC was shown to increase methylation levels of fatty acid synthase (*FASN*), stearoyl-CoA desaturase 1 (*SCD1*), peroxisome proliferator-activated receptor gamma (*PPARG*) and sterol regulatory element binding transcription factor 1 (*SREBF1*) [93]. MiR-24 control of triacylglycerol synthesis in goat mammary epithelial cells by targeting *FASN* gene has been demonstrated [94]. The ability of miR-145 to regulate lipogenesis in GMEC through targeting insulin-induced gene 1 (*INSIG1*) and epigenetic regulation of lipidrelated genes has been demonstrated [93]. MiR-143 was shown to inhibit proliferation as well as induce apoptosis of GMEC [95]. MiR130b regulation of PPARγ coactivator-1α suppressed fat metabolism in GMEC [96]. In non-ruminant species, many miRNAs, including let-7 family members, mir-17/92, miR-30b, miR-93, miR-99a and miR-b, miR-101a, miR-126-3p, miR-138, miR-146b, miR-200 family members, mir-203, miR-205, miR-206, miR-210, miR-212/132, miR-221 and miR-424/50, have been reported to play roles in mammary gland development and disease [15]. Some miRNAs with functionally validated targets are summarized in **Table 2**.

#### **3.3. Nutritional modulation of microRNA expression and function**

The miRNA expression profile in response to dietary treatments has been studied in adipose tissues of lambs and cattle and bovine mammary gland tissues [56, 100–102]. A change in diet that interferes with energy balance has been shown to change miRNA expression pattern in cow liver [103]. Wang et al. [104] fed cows with high- and low-quality forage diets (corn stover and rice straw) and showed that miR-125b, miR-141, miR-181a, miR-221 and miR-15b changed their expression patterns across different tissues including MG. We have examined the expression pattern of miRNAs following MG adaptation to dietary supplementation with 5% linseed oil or 5% safflower oil using miRNA sequencing and identified seven differentially regulated miRNAs, including six upregulated (miR-199c, miR-199a-3p, miR-98, miR-378, miR-148b and miR-21-5p) and one downregulated (miR-200a) by both linseed and safflower oil. The target genes of these seven miRNAs have functions related to gene expression and general cellular metabolism and are enriched in four pathways of lipid metabolism (3-phosphoinositide biosynthesis, 3-phosphoinositide degradation, D-myo-inisitol-5-phosphate metabolism and the superpathway of inositol phosphate compounds) [51]. The largest number of target genes (39) were associated with two functions (synthesis of lipid and concentration of lipid) related with lipogenesis. In goat, Mobuchon et al. [105] detected 30 miRNAs with expression patterns potentially modulated by food deprivation (14 and 16 were upregulated and downregulated, respectively). Among them, miR-204-5p and miR-223-3p were most remarkably affected by food deprivation and potentially played roles in the nutritional regulation of gene expression in the MG.


**Table 2.** MicroRNAs with functionally validated target genes using ruminant mammary gland cells.

### **3.4. MicroRNA functions in mammary gland health**

*3.2.3. Functional validation of microRNA target genes*

64 Current Topics in Lactation

**3.3. Nutritional modulation of microRNA expression and function**

The miRNA expression profile in response to dietary treatments has been studied in adipose tissues of lambs and cattle and bovine mammary gland tissues [56, 100–102]. A change in diet that interferes with energy balance has been shown to change miRNA expression pattern in cow liver [103]. Wang et al. [104] fed cows with high- and low-quality forage diets (corn stover and rice straw) and showed that miR-125b, miR-141, miR-181a, miR-221 and miR-15b changed their expression patterns across different tissues including MG. We have examined the expression pattern of miRNAs following MG adaptation to dietary supplementation with 5% linseed oil or 5% safflower oil using miRNA sequencing and identified seven differentially regulated miRNAs, including six upregulated (miR-199c, miR-199a-3p, miR-98, miR-378, miR-148b and miR-21-5p) and one downregulated (miR-200a) by both linseed and safflower oil. The target genes of these seven miRNAs have functions related to gene expression and general cellular metabolism and are enriched in four pathways of lipid metabolism (3-phosphoinositide biosynthesis, 3-phosphoinositide degradation, D-myo-inisitol-5-phosphate metabolism and the superpathway of inositol phosphate compounds) [51]. The largest number of target genes

Since *in vivo* experiments for functional validation of MG miRNAs are not feasible, such studies have mostly relied on the use of knock-out/mimics and MG-specific cell types. Using bovine mammary epithelial cells (BMEC), miR-15a was shown to regulate growth hormone receptor, viability of BMEC and the expression of casein genes [86]. MiR-486 regulation of lactation by targeting the PTEN gene in cow MGs has been demonstrated [87]. Bian et al. [88] recently reported that epigenetic regulation of miR-29s affects the lactation activity of BMEC. MiR-181a was shown to regulate the biosynthesis of bovine milk fat through targeting acyl-CoA synthetase long-chain family member 1 (*ACSL1*) [89]. MiR-103 was reported to control milk fat accumulation in goat MG during lactation [90]. Moreover, miR-27a was shown to suppress triglyceride accumulation as well as altered gene expression associated with fat metabolism in dairy goat mammary epithelial cells (GMEC) [91]. In another study, miR-135a was reported to target and regulate prolactin receptor (*PRLR*) gene in GMEC [92]. Inhibition of the expression of miR-145 in GMEC was shown to increase methylation levels of fatty acid synthase (*FASN*), stearoyl-CoA desaturase 1 (*SCD1*), peroxisome proliferator-activated receptor gamma (*PPARG*) and sterol regulatory element binding transcription factor 1 (*SREBF1*) [93]. MiR-24 control of triacylglycerol synthesis in goat mammary epithelial cells by targeting *FASN* gene has been demonstrated [94]. The ability of miR-145 to regulate lipogenesis in GMEC through targeting insulin-induced gene 1 (*INSIG1*) and epigenetic regulation of lipidrelated genes has been demonstrated [93]. MiR-143 was shown to inhibit proliferation as well as induce apoptosis of GMEC [95]. MiR130b regulation of PPARγ coactivator-1α suppressed fat metabolism in GMEC [96]. In non-ruminant species, many miRNAs, including let-7 family members, mir-17/92, miR-30b, miR-93, miR-99a and miR-b, miR-101a, miR-126-3p, miR-138, miR-146b, miR-200 family members, mir-203, miR-205, miR-206, miR-210, miR-212/132, miR-221 and miR-424/50, have been reported to play roles in mammary gland development and disease [15]. Some miRNAs with functionally validated targets are summarized in **Table 2**.

> MiRNAs have been shown to play roles in bovine infection and immunity in a wide range of tissues [54, 106–113]. For mammary gland, Naeem et al. [114] studied the expression of 14 miRNAs (miR-10a, miR-15b, miR-16a, miR-17, miR-21, miR-31, miR-145, miR-146a, miR-146b, miR-155, miR-181a, miR-205, miR-221 and miR-223) in MG tissue challenged with *Streptococcus uberis* and identified three downregulated miRNAs (miR-181a, miR-16 and miR-31) and one upregulated miRNA (miR-223) in infected versus healthy tissue. Lawless et al. [107] showed that 21 miRNAs were differentially expressed upon *Streptococcus uberis* infection of bovine primary epithelial cells. Using BMEC, Jin et al. [108] reported a differential expression of nine miRNAs (miR-184, miR-24-3p, miR-148, miR-486, let-7a-5p, miR-2339, miR-499, miR-23a and miR-99b) upon challenge with heat inactivated *Escherichia coli* and *Staphylococcus aureus* bacteria. Hou et al. [115] identified three upregulated miRNAs (miR-296, miR-2430 and miR-671) and one downregulated miRNA (miR-2318) in mastitis affected compared with healthy mammary gland tissues. Li et al. [111] sequenced RNA isolated

from *S. aureus*-induced mastitis and control cows and identified 77 miRNAs with significant expression differences between the two groups. Li et al. [116] showed that miR-23 might be an important immune miRNA through its target mastitis candidate gene, high mobility group box 1 *(HMGB1)*.

#### **3.5. MicroRNA function in milk recipients**

Recent evidence suggesting that milk-derived miRNAs may have potential regulatory roles in modulating the immune system or metabolic processes of milk recipients still remain controversial [117–124]. Currently, there are two hypotheses about miRNA function in infants/offspring: the first proposes that milk miRNAs exert physiological regulatory functions after transferring to offspring, and the second assumes that miRNAs do not have any function but merely provide nutrition. According to Zhang et al. [117], the rice-derived miRNA, miR-168a, can bind to the mRNA of human/mouse low-density lipoprotein receptor adapter protein 1 (*LDLRAP1*) and inhibit its expression in the liver, and consequently decrease LDL removal from mouse plasma. Baier et al. [118] reported that miR-29b-3p and miR-200c-3p could be absorbed by humans in biologically meaningful amounts, which could affect related gene expression in peripheral blood mononuclear cells while Izumi et al. [125] confirmed that whey exosomes containing miRNAs and mRNA could be absorbed by human macrophages. These results opened a new aspect of the nutritional control of metabolism [119]. However, other studies have not succeeded to validate the hypothesis that milk miRNAs exert physiological regulatory functions after transferring to offspring [126–129]. For instance, Auerbach et al. [129] observed that drinking bovine milk increased circulating levels of miRNAs (miR-29b-3p and miR-200c-3p) but found no evidence that they significantly altered miRNA signals after milk ingestion. These authors concluded that milk miRNAs likely serve as a source of nutrition but not as post-transcriptional regulators in recipients.
