Menin Modulates Mammary Epithelial Cell Numbers in Bovine Mammary Glands Through Cyclin D1
Journal of Mammary Gland Biology and Neoplasia
Menin Modulates Mammary Epithelial Cell Numbers in Bovine Mammary Glands Through Cyclin D1
Kerong Shi 0
Xue Liu 0
Honghui Li 0
Xueyan Lin 0
Zhengui Yan 0
Qiaoqiao Cao 0
Meng Zhao 0
Zhongjin Xu 0
Zhonghua Wang 0
0 Shandong Key Laboratory of Animal Bioengineering and Disease Prevention , Taian 271018 , China
1 Zhonghua Wang
Menin, the protein encoded by the MEN1 gene, is abundantly expressed in the epithelial cells of mammary glands. Here, we found MEN1/menin expression slowly decreased with advancing lactation but increased by the end of lactation. It happened that the number of bovine mammary epithelial cells decreases since lactation, suggesting a role of menin in the control of mammary epithelial cell growth. Indeed, reduction of menin expression through MEN1-specific siRNA transfection in the bovine mammary epithelial cells caused cell growth arrest in G1/S phase. Decreased mRNA and protein expression of Cyclin D1 was observed upon MEN1 knockdown. Furthermore, menin was confirmed to physically bind to the promoter region of Cyclin D1 through a ChIP assay, indicating that menin plays a regulatory role in mammary epithelial cell cycle progression. Moreover, lower expression of MEN1/menin induced increased epithelial cell apoptosis and caused extracellular matrix remodeling by down-regulating its associated genes, such as DSG2 and KRT5, suggesting that menin's role may also be involved in the control of cell-cell adhesion in normal mammary glands. Taken together, our data revealed an unknown molecular function of menin in epithelial cell proliferation, which may be important in the regulation of lactation behavior of mammary glands.
MEN1/menin; Mammary epithelial cells; Cell growth; Cyclin D1
Menin, a protein of 610 amino acids encoded by the MEN1
gene, plays an important regulatory role in the metabolism
of organisms. Mutations in MEN1 gene and therefore mutant
protein menin predispose patients to multiple endocrine
neoplasia type 1 (MEN1) syndrome, which is characterized by
Kerong Shi and Xue Liu contributed equally to this article.
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s10911-017-9385-8) contains
supplementary material, which is available to authorized users.
College of Animal Science and Technology, Shandong
Agricultural University, Taian 271018, China
the occurrence of multiple endocrine tumors, mainly
affecting the parathyroid glands, pituitary (anterior lobe) and
pancreatic islets [
]. These endocrine organs secrete
hormones such as prolactin and insulin, which can
physiologically modulate the lactation curve pattern of mammary
glands. Moreover, prolactin and/or insulin could be
downstream factors of menin, which regulates their gene
]. This suggests the existence of a link between
MEN1/menin and hormone-dependent tissues [
Within the mammary gland, menin was recently found
to directly interact with estrogen receptor-α (ERα) in a
hormone-dependent manner in breast cancer cells [
regulating the transcription of estrogen-responsive genes in
these cells [
]. This finding indicated an important
mediator role of menin in mammary gland tissue of dairy cows
]. Moreover, loss of heterozygosity of MEN1 was found
to cause inherited breast-ovarian cancer in humans [
hyperplasia of breast (female) and prostate (male) cells in
]. These findings indicated a possible regulatory
role of MEN1/menin in cell growth and/or cell cycle
progression in mammary gland tissue.
The milk yield and shape of the lactation curve of a dairy
cow are determined by the number of mammary epithelial
cells and their secretory activity. Nearly all of the cells
present in the bovine mammary gland are formed by calving
]. During early lactation, the number of mammary
epithelial cells within the mammary glands is at a peak and
then gradually decreases with advancing lactation [
decline in mammary cell numbers during lactation must at
least partially account for the decline in milk yield after the
peak stage, which occurs approximately 60–90 days after
]. Accompanying the decline in mammary cell
numbers, apoptotic cell death and a degree of cell renewal
and lobular-alveolar remodeling are observed [
change in the rate of the decline in cell numbers and/or
apoptosis could provide a means of modulating milk yield
and mammary gland lactation curve pattern. However, the
molecular mechanisms controlling mammary cell growth/
survival are poorly defined. It was hypothesized that there
were important inherited genetic factors regulating the
epithelial cell growth and/or death, therefore making effects
on the lactation persistency and milk yield of mammary
glands. Hence, the objective of the present investigation
was to assess the molecular mechanisms of MEN1/menin in
regulating epithelial cell growth in bovine mammary glands.
A bovine mammary epithelial cell line, MAC-T, was used
as the experimental model [
], in addition to
mammary gland tissues at different lactation stages.
MEN1/menin Knowdown in Mammary Epithelial
The expression level of MEN1 and it encoded protein menin
were investigated during five different lactation stages
(including the dry period) in mammary glands of dairy
cows, the results indicated both MEN1 mRNA and the menin
protein slowly decreased with advancing lactation, but later
increased after the peak milk stage through the dry period
(or involution stage), with the lowest expression level being
observed around the peak lactation stage (55 ± 4.3 days in
milk, DIM; supplementary Fig. S1).
To assess the possible regulatory function of the MEN1
gene in the mammary glands of dairy cows, MEN1/menin
expression levels were modified in vitro in bovine
mammary epithelial cells (MAC-T cells) to mimic the decrease
in expression after the initiation of lactation in the
mammary gland. In vitro-synthesized siRNAs specific to the
bovine MEN1 gene were transfected either separately
(sibMEN1-1, sibMEN1-2 or sibMEN1-3) or in combination
(sibMEN1-1/2/3, as a siRNA pool) into MAC-T cells (data not
shown). sibMEN1-1/2/3 transfection were shown to result in
the best knockdown efficiency of MEN1 and menin at 24 h
posttransfection via qRT-PCR and western blot detection,
respectively. The siRNA pool (sibMEN1-1/2/3) was
ultimately used for the following experiments because of the
best knockdown level (25.31%±3.86% of the MEN1 mRNA
level, Fig. 1a; and 29.26%±11.10% of the menin protein level
in untransfected MAC-T cells, respectively; Fig. 1b, c).
Knockdown of MEN1/menin Induces Extracellular
Three independent replicates with lower expression
of MEN1/menin were pooled and subjected to
highthroughput sequencing (Fig. 2a). These samples exhibited
21.04%±0.24% (n = 3) of MEN1 mRNA and 22.87%±1.30%
(n = 3) of the menin protein level found in untransfected
MAC-T cells. The results showed 31 genes presented
differential expression upon MEN1 knockdown (Fig. 2b), with
19 genes being down-regulated (Fig. 2c) and 12 being
upregulated (Fig. 2d). GO analysis indicated that most
downregulated genes were mainly enriched into extracellular
matrix remodeling process upon MEN1 knockdown in
mammary epithelial cells (Fig. 2e).
Knockdown of MEN1/menin Arrests Mammary
Epithelial Cells in G1/G0 Phase and Delays the G1/S
Upon MEN1 knockdown in mammary epithelial cells,
propidium iodide (PI) staining was performed in cells for
analysis of the cell phase distribution via flow cytometry, along
with the corresponding control cells. The results showed
that decreased MEN1 expression substantially increased the
percentage of cells by 5.07% in G1/G0 phase (P = 0.026)
and decreased the percentage of cells by 4.88% in S phase
(P = 0.0019; Fig. 3a, b). To further examine the potential
role of menin in controlling epithelial cell growth, we
performed immunofluorescence staining of exponentially
growing menin-knockdown MAC-T cells using annexin V-FITC
and found that the number of annexin V-positive cells was
increased in cells with lower menin expression (Fig. 3c, d),
indicating cell apoptosis (supplementary Fig. S4). These
results suggested that menin suppression causes inhibition
of the G1/S transition of bovine mammary epithelial cells.
Menin Down‑Regulates of Cyclin D1 Expression
Since the suppression of MEN1/menin expression results
in cell growth arrest in the G1/S phase, it was logical to
question whether cell cycle regulatory genes are
modulated by menin. Hence, the expression of candidate genes
that might specifically control the G1/S transition was
detected in MAC-T cells upon MEN1 knockdown (Fig. 3a,
Supplementary Table S2). The time course MEN1
knockdown induced that the mRNA expression of Cyclin D1
significantly decreased (P < 0.05 for all the three time
points), whereas those of CDK4 (P = 0.009 at 48 h),
CDK6 (P = 0.03 at 24 h and P = 0.01 at 48 h) and p18 (a
G1/S phase-specific cell cycle inhibitor; P= 0.04 at 24 h)
increased (Fig. 4a) in various degree upon MEN1/menin
knockdown, compared to their corresponding negative
controls. At 24 h post-transfection, the expression of Cyclin D1
was further confirmed to be significantly down-regulated
in low menin-expressing mammary gland epithelial cells
at both the mRNA (Fig. 4b, P = 0.03) and protein (Fig. 4c,
d, P = 0.04) levels. These findings were validated by
highthroughput sequencing data from epithelial cells, and Cyclin
D1 (also named as CCND1) was one of the down-regulated
genes identified upon MEN1 knockdown (Fig. 2c).
Menin Associates with the Cyclin D1 Promoter
We hypothesized that menin might associate with promoter
regions of Cyclin D1 and repress the transcription of
Cyclin D1, obstructing the recruitment of other transcriptional
regulators. Hence, a ChIP assay was performed using normal
MAC-T cells. We initially designed six amplicons (P1, P2,
P3, P4, P5 and P6) to determine whether menin binds to the
promoter region of Cyclin D1 (Fig. 5a, Supplementary Table
S3). It was found that menin bound to the regions
detectable by P1 (P < 0.001), P2 (P < 0.001), P3 (P = 0.02) and P4
(P < 0.001) (Fig. 5b). Based on the promoter sequence of
Cyclin D1, up to six TATA-alike boxes and a CCAAT box
exist within the P1 region, and a classical GC box with the
conserved sequence GGGCGG exists in the P2 region. Thus,
menin, known as a scaffold protein directly interacting with
a number of different transcription factors [
], may regulate
Cyclin D1 transcription, by associating with the RNA
polymerase II (Pol II) complex, binding to the promoter region
of Cyclin D1 containing TATA boxes, a CCAAT box and
GC box elements near the transcription start site.
Therefore, menin might regulate the G1/S transition of bovine
mammary gland epithelial cells by binding to the Cyclin
Menin is Abundantly Expressed in Mammary
To investigate whether menin is expressesed in epithelial
cells in vivo, peak (+ 55) and late (+ 312) milking-stage
mammary glands from non-pregnant dairy cows were
stained using antibody against menin for
immunohistochemistry analysis. Menin expression was detected in both the
luminal and basal (myoepithelial) cell layers of the
mammary epithelium (Fig. 6a), with much more abundant
staining being observed in tissues from late lactation (+ 312)
The numbers of up- and down-regulated genes are indicated on the
right. c The 19 down-regulated genes (such as DSG2, KRT5, BLVRA
and CCND1) detected upon MEN1/menin knockdown in MAC-T
cells are shown. The MEN1 gene was included in the gene list as a
positive control. d The 12 up-regulated genes detected upon MEN1/
menin knockdown in MAC-T cells are shown. e Gene Ontology
(GO) analysis of the differentially expressed genes (KRT5, LAMB3,
MEST, DSG2, etc.) indicated that extracellular matrix remodeling
was the most enhanced activity upon the reduction of MEN1/menin
in MAC-T cells. The colors present three different ontology domains;
the x-directed bars present the enriched GO terms
Ctrl sibMEN G1 50.37% S 36.07%
cells were also assessed for the distribution of apoptotic status after
annexin V-FITC/propidium iodide (PI) staining. “Norm” represents
the plain cells without any treatments; “Ctrl” and “sibMEN”
represent the cells treated with scramble and MEN1-specific siRNAs,
respectively. **P < 0.01 and *P < 0.05. d Representative images of
apoptotic analyses illustrating the percentage of apoptotic MAC-T
cells upon MEN1/menin treatment. The percentages of cells in
different apoptotic stages are indicated within each panel
Fig. 4 CycinD1 was significantly suppressed upon MEN1/menin
knockdown. a The expression of cell cycle progression associated
genes was assessed in the MAC-T cells after 24, 48 and 72 h of
transfection with MEN1-specific siRNAs, as well as their negative
controls. The dotted line represents the expression levels of each genes
in their negative control cells at the indicated time points. Cyclin D1
expression was significantly inhibited all time points. **P< 0.01,
*P < 0.05. b The suppression of Cyclin D1 mRNA expression was
mammary glands than that in peak milk-stage (+ 55)
mammary glands (Fig. 6b, P < 0.001).
Regulation of the Expression of Cyclin D1 by Menin at G1/S Phase Can Alter the Cell Cycle and Proliferation of Mammary Epithelial Cells
The MEN1 gene encodes menin, a protein that is
primarily nuclearly localized and has been shown to interact with
confirmed by three independent MEN1 knockdown experiments at
24 h posttransfection. The data are shown as relative expression
levels normalized to the internal control β-actin. *P < 0.05. c Cyclin
D1 protein expression was suppressed upon MEN1/menin
knockdown (n = 3). *P < 0.05. d Representative western blot images of the
expression of menin, Cyclin D1 and the loading control β-actin are
a variety of transcriptional factors and other proteins [
], suggesting that menin may act as an adaptor protein
involved in the regulation of gene expression and, in
particular, menin plays a complex role in cell cycle regulation .
It has recently been shown that menin mediates the
repression of Cyclin B2 expression through physical binding to its
promoter and therefore inhibits the G2/M transition and cell
]. Moreover, MEN1/menin was previously
reported to participate in the regulation of cell cycle
progression at G1/S phase [
]. Progression through the
cell cycle depends on the activity of cyclins (such as Cyclin
D1) and/or cyclin-dependent kinase (CDK) inhibitors (such
Fig. 5 Cyclin D1 is a
transcriptional target of menin.
a Six amplicons (P1, P2, P3,
P4, P5 and P6) were used to
detect the indicated regions
of the Cyclin D1 promoter for
menin-ChIP assays (n = 3). The
potential promoter elements to
which RNA polymerase II (Pol
II) usually binds are shown in
grey-filled ovals. There were
up to six TATA-alike boxes and
one CCAAT box (not shown)
in the P1 region, and a classical
GC box with the conserved
sequence GGGCGG was
present in the P2 region. b Results
of quantitative menin-ChIP
PCR assays in MAC-T cells are
shown, as is the negative control
IgG-ChIP PCR. The results
were representative of three
independent ChIP experiments,
showing as the percentage of
input by quantifying the amount
of chromatin obtained from
immunoprecipitation relative to
the amount in the input samples.
**P < 0.01, *P < 0.05
as p18, p21 and p27 proteins), which can cause G1 arrest
and termination of DNA synthesis in S-phase [
quiescent rat pituitary somatolactotrope GH4C1 cells, menin
blocks the progression from G0/G1 phase to S phase by
regulating the expression of the CDK inhibitors p21 and p27
]. Indeed, menin has been shown to directly associate
with the promoters of p27 and p18 and promotes the
methylation of lysine 4 in histone H3 (H3K4) and the expression
of these genes [
]. Additionally, menin has been shown to
repress the activator of S-phase kinase (ASK)-induced cell
proliferation, preventing cells from entering S phase [
]. Thus, menin may play an important regulatory role at
G1/S phase by interacting with promoter regions and certain
proteins that function as transcription factors.
In this study, we identified a cell cycle progression
regulatory role for menin in mammary epithelial cells. Decreased
expression levels of menin caused mammary epithelial cell
growth arrest at G1/S phase as its physical binding to the
promoter region of Cyclin D1, a crucial ligand protein of
CDK4/CDK6 enables cell progression from G1 to S phase
]. In the promoter region of the bovine Cyclin D1
gene where menin potentially binds, there are up to six
TATA-like boxes and CCAAT box in the P1 region and a
classical GC box with the conserved sequence GGGCGG
in the P2 region, which are the preferred promoter elements
where RNA polymerase II (Pol II) usually binds. Thus, a
decrease in menin may inhibit the binding of Pol II and/
or other transcription factors to the promoter, resulting in
repression of Cyclin D1 expression. Indeed, accompanying
the Cyclin D1 repression observed under reduced menin
expression, down-regulation of histone H3 lysine 4
trimethylation (H3K4me3; Supplementary Fig. S2) was found.
Simultaneously, the up-regulation of p18
(Supplementary Fig. S3) was found to be associated with lower menin
expression, although the detailed underlying mechanisms
are not yet clear.
Abundant, but Modulated Expression of Menin
in the Mammary Epithelium During Different
Lactation Stages Indicates a Potential Regulatory
Role of Menin in Epithelial Cell Survival
Menin was found to be abundantly expressed in the cell
layers of the mammary epithelium during different stages
of lactation, including the dry period (Fig. 6), indicating
its potential regulatory role in epithelial cell survival.
Notably, menin expression was slowly suppressed with
advancing lactation and reached a lower point around
the peak milk stage (supplementary Fig. S1, Fig. 7).
However, by the involution stage, menin expression was
slowly increasing. Provided that a single mammary gland
from an individual animal was used to track the
expression of MEN1/menin throughout the lactation period, a
statistically significant change would be observed. More
Fig. 6 Menin expression in mammary gland tissue of late
lactation was more abundant than peak lactation staged tissue.
Mammary gland sections from + 55 days in milk (DIM) at the peak
lactation stage and + 312 DIM in late lactation cows were assessed for
menin expression by immunohistochemistry staining with anti-menin
antibody. Normal rabbit IgG was used as a negative control.
Representative results are shown in (a), bar = 200 µm. Brown indicates
positive menin staining (brown in the nucleus and cytoplasm). The
right panel is a high-powered magnification of the black dashed area
in the left panel, with magnification of ×100, × 400, and × 1000,
respectively. (b) Quantification of the averaged percentages of
meninexpressed epithelial cells in randomly selected microscopic views
from Figure A. Different numbers of microscopic views were
randomly selected to quantify the mean density of positive staining
signals in mammary epithelial cells at peak lactation stage (+ 55, n = 9)
and late lactation stage (+ 312; n = 12), respectively. ** P < 0.01
importantly, the expression model of menin throughout the
lactation cycle in mammary glands was found to be
negatively correlated with the normal lactation curve, showing
that the milk yield continues to increase until peak
lactation on days of 60–90, but decline thereafter until
involution and/or the dry period.
The number of secretory mammary epithelial cells inside
the mammary gland ultimately determines the potential milk
yield of an animal. The mammary gland undergoes dramatic
functional and metabolic changes during the milking period.
For example, the number of epithelial cells slowly declines
after lactation begins [
]. The internal molecular
Late lactation/Dry period
cell growth derepression
extracellular matrix remodeling
Keratin filament (KRT5)
ubiquitin-related domain (SUMO2)
cell growth repression
extracellular matrix collapse
Keratin filament (KRT5)
ubiquitin-related domain (SUMO2)
Fig. 7 Hypothetical model of the menin-mediated effect on
mammary epithelial cell numbers and the lactation curve pattern of
mammary glands. The gradual decrease in menin expression after
the initiation of lactation in mammary glands causes epithelial cell
growth arrest in G1 stage through repression of Cyclin D1, also
inducing cell apoptosis. Menin expression continues to decrease until
reaching its lowest point at the peak lactation stage. However, in the
late lactation stage and/or dry period (no lactation), menin expression
levels are recovered, possibly related to differences in hormone
secreregulatory mechanism of this phenomenon has long been
a biological conundrum for the mammary biologist. Here,
we found that MEN1/menin is the factor that controls the
dynamic balance between cell proliferation and cell death in
the lactating mammary gland, thus modulating the lactation
curve pattern of the mammary gland (Fig. 7).
The decreasing expression of menin after lactation begins
may slowly result in growth arrest of mammary epithelial
cells through the cell cycle regulator Cyclin D1 and even
apoptosis, suggesting that MEN1/menin is one of the internal
regulatory molecular mechanisms causing the decline in the
number of epithelial cells. Indeed, gene expression profiling
of bovine mammary glands suggested that the onset of
lactation was accompanied by up-regulation of genes involved
in milk synthesis, and by inhibition of genes related to cell
15, 36, 37
]. Cyclin D1 was also found to be
significantly down-regulated in early lactation mammary
gland tissue compared to late pregnancy tissue (dry stage)
More specifically, with decreasing menin expression,
mammary epithelial cells exhibited increased early
apoptosis (Fig. 3c, Supplementary Figure S4) and
extracellular matrix remodeling activity (Fig. 2e). Genes showing
inhibited expression that involved in extracellular matrix
activity included extracellular matrix components
(keratin filament KRT5, laminin filament LAMB3), cell
adhesion molecule (cadherin DSG2), proteolysis and catabolic
tion (Li et al. 2017; Karnik et al. 2007). The growth of epithelial cells
becomes derepressed, and extracellular matrix degradation resulting
from decreased menin ceases. Epithelial cells proliferate again, and
the extracellular matrix undergoes remodeling, in preparation for the
next lactation cycle. “+” indicates relative menin expression levels;
grey-filled circles indicate G1-arrested mammary epithelial cells.
Upward arrows indicate increased expression or activity, while
downward arrows indicate decreased expression or activity
processes (ubiquitin-related domain, SUMO2;
oxidoreductase, BLVRA; hydrolase-like, MEST; peptidase, ECE1) and
calcium ion binding (S100 calcium-binding protein A2,
S100A2) (Fig. 2c). Interestingly, the role of menin in
regulating cell–cell adhesion was also observed in mice with
Men1 inactivation-induced lesions in the mammary glands
] and pancreatic islets [
], although the underlying
mechanism requires further investigation. Additionally, and
of greater interest, similar groups of gene have been shown
to be significantly suppressed in early lactation mammary
glands compared with the dry period before parturition
according to previous reports [
]. A study in mice found
that the communication between mammary epithelial cells
and their environment became weak during the lactation
]. These results demonstrated that in the period
before peak milk, the communications between epithelial
cells and their extracellular matrix were slowly weakened
17, 18, 36, 37, 42
], with increasing apoptosis of mammary
epithelial cells and collapse of extracellular matrix (Fig. 7);
these changes then slowly recover in the dry period because
of the expression restoration of MEN1/menin, in preparation
for the next lactation cycle. Thus, menin is at least one of the
internal regulators inside the mammary gland that modulates
epithelial cell numbers through the lactation cycle, although
other factors are yet to be revealed.
Regarding the modulation of menin expression in
mammary epithelial cells at different lactation stages, the
reproductive hormone prolactin could be the upstream
controller, which inhibits MEN1/menin expression [
4, 12, 43
In bovine mammary gland tissue, the different expression
levels of menin during the lactation period may be a
consequence of menin responding to the waves of reproductive
hormone secretion, thus modulating mammary epithelial
cell numbers. In addition to the role of menin in the
control of normal mammary cell growth and proliferation, we
found that menin can mediate the role of surrounding
hormones and energy in milk protein synthesis in epithelial cells
through the PI3K/Akt/mTOR pathway [
Considering these data together, we hypothesize that
MEN1/menin could be one of the primary mediators
controlling the growth of epithelial cells through the cell cycle
progression factor Cyclin D1, dynamically modulating the
lactation persistency and the lactation curve of normal
mammary glands. The discovery of this unknown role of menin
may shed light on the molecular mechanisms underlying
the long-term biological conundrum of declining milk yield
after peak lactation in mammary glands. There could be
other factors besides of MEN1/menin playing similar
regulatory role in mammary glands. This discovery would result
in new ideas for adjusting animal milk production and a
possible new mechanism in mammary biology.
Materials and Methods
All experiments were carried out according to the
Regulations for the Administration of Affairs Concerning
Experimental Animals published by the Ministry of Science and
Technology, China (2004) and were approved by the Animal
Care and Use Committee of Shandong Agricultural
University, Shandong, China.
Animals and Mammary Gland Tissue Collection
Fifteen healthy multiparous Holstein cows were biopsied
for the mammary gland samples at Holstein Cattle
Association Jiabao Farm in Shandong province. These cows were
not pregnant, with an average parity of 2.67 ± 2.06 and an
average weight of 602 ± 19.2 kg. Samples were obtained on
lactation days (calving day as day 1) of 4.6 ± 1.5, 55 ± 4.3,
163 ± 6.24, 312 ± 24.6 and during the dry period (36 ± 6.8
days after milking stopped), representing 5 different
lactation stages (3 animals for each stage) with a variable
developmental status of their mammary glands. Mammary
biopsies were collected using the Bard Magnum biopsy
system (Bard Peripheral Vascular, Inc., Tempe, AZ, US) as
]. The mammary tissue samples were
immediately frozen in liquid nitrogen and stored at − 80 °C for
subsequent analysis or fixed in a 4% formaldehyde
solution for immunohistochemistry. Cows were housed in a free
stall barn with constant access to water and feed. Diets were
formulated to meet all NRC (2001) recommendations for
early- lactation, mid-lactation and/or dry cows using the
Cornell-Penn-Miner system (CPM-Dairy, version 3.0.7) to
meet the metabolizable energy and protein requirements.
Feed was provided ad libitum, and the lactation cows were
milked three times daily.
Cell Culture and Transient Transfection Assays
Bovine mammary epithelial cells (MAC-T) were grown in
Dulbecco’s modified Eagle’s medium (DMEM),
containing 10% fetal bovine serum (FBS), penicillin (100 U/L) and
streptomycin (100 mg/L) in a 5% CO2 atmosphere at 37 °C.
The cells (5 × 105 cells/well) were pre-seeded in 6-well
culture plates in medium without antibiotics at a density of
70–80% confluence at 24 h prior to transfection.
siRNAs specific for the bovine MEN1 gene were designed
and synthesized (Ribobio, Guangzhou, China;
Supplementary Table S1) and used for MEN1/menin knockdown.
MAC-T cells were transfected with 50 nM target-specific
pool siRNAs per well using OPTI-MEM® I Medium
(Invitrogen, Carlsbad, CA, USA) and Lipofectamine 2000
(Invitrogen, Carlsbad, CA, USA) according to the
manufacturer’s protocol, and a non-specific scramble negative control
siRNA (Ribobio, Guangzhou, China) transfections were
conducted at the same time. The cells were processed for
protein and/or RNA isolation at 24 h post-transfection. All of
the experiments were performed at least three times for each
transfection. Preliminary experiments were conducted under
the same condition by using separate and/or combinations of
target siRNAs, so as to obtain the best knockdown efficiency.
RNA Isolation and Quantitative RealT‑ime RT‑PCR
Total RNAs were extracted from mammary tissues (about
20 ng) or MAC-T cells (5 × 105 cells) using an RNA
extraction kit (Tiangen Biotech, Beijing, China), followed by
treatment with DNaseI (Ambion, Austin, Texas, USA). The
quality of total RNA was assessed through agarose gel
electrophoresis and the calculation of OD260/OD280.
Oligo(dT)primed first-strand cDNA was employed for quantitative
real-time RT-PCR (qRT-PCR) using SYBR-Premix Ex Taq
II (TaKaRa, Dalian, China) and an Mx3000p cycler
(Stratagene, La Jolla, CA, USA). Each CT value was obtained from
the averaged CTs of triplicate reactions. The expression level
of target gene was normalized to its corresponding internal
control β-actin. The data were plotted as fold change over
their corresponding controls in MAC-T cells or certain
lactation stage in mammary gland tissues. The 2− ΔΔCT method
was used to calculate the relative abundance of mRNA. All
primer sequences are listed in Supplementary Table S2.
Total protein were extracted in RIPA lysis buffer
(containing 1% PMSF) (Beyotime, Nanjing, China) from MAC-T
cells after 24 h of transfection or mammary gland tissues.
The proteins (approximately 20 μg of total protein) were
separated on a 10% SDS–PAGE gel and transferred onto
nitrocellulose membranes using 200 mA of constant current.
The western blot was performed as per standard protocols.
Primary antibodies against bovine menin (Bethyl
Laboratories, Texas, USA), bovine cyclin D1 (Beyotime, Nanjing,
China), and β-actin (Beyotime, Nanjing, China) were used
at 1:1000 dilution. β-actin was used as the total protein
loading control. The HRP-conjugated secondary antibody
(Beyotime, Jiangsu, China) was diluted by 1000 as the
working solution. Chemiluminescence detection was performed
using BeyoECL Plus (Beyotime, Beijing, China). The data
are shown as the expression level normalized to their
corresponding negative controls. The results are representative of
three independent experiments that were used to determine
the statistical significance.
RNAs from three independent MEN1-specific siRNA
treatments of MAC-T cells collected at 24 h after transfection
were extracted and pooled in equal proportions for SE50
high-throughput sequencing to identify the differentially
expressed genes (DEG), as were their negative control
siRNA treatments. mRNA Sequencing was performed on
the Illumina HiSeq 2500 platform. Sequencing libraries were
generated using the NEBNext® Ultra™ RNA Library Prep
Kit for Illumina® (NEB, USA) following the manufacturer’s
recommendations and index codes were added to attribute
the sequences to each sample, followed by assessment of
library quality using the Agilent Bioanalyzer 2100 system.
Raw data (raw reads) in the fastq format were first processed
with in-house perl scripts. All downstream analyses were
based on high quality clean reads of 10 M. The index for
the reference genome was built using Bowtie v2.0.6, and
paired-end clean reads were aligned to the reference genome
using TopHat v2.0.9. Prior to differential gene expression
analysis, for each sequenced library, the read counts were
normalized and standardized with the edgeR program
package using the method of trimmed mean of M values (TMM).
Differential expression analyses of two conditions were
performed using the DEGSeq R package (1.12.0). The P values
were adjusted via the Benjamini and Hochberg method. A
corrected P-value of 1.3E-5, q-value of 0.001 and log2
(foldchange) of 0.3 were set as the threshold for significantly
differential expression. Gene Ontology (GO) enrichment
analysis of differentially expressed genes was implemented
with the GOseq R package, in which gene length bias was
corrected. GO terms with corrected P-values of less than
0.05 were considered significantly enriched with
differentially expressed genes.
Cell Proliferation and Cell Cycle Analysis
Equal number of MAC-T cells (1 × 106) were transfected
with MEN1-specific siRNAs for 24 h as well as the
corresponding negative control. The same amount of cells
(2.5 × 105) were harvested, fixed and analyzed in
Vindelov’s propidium iodide buffer for analysis of the
distribution of cell cycle phases (FACSCalibur, BD Biosciences).
All of the experiments were performed three times for each
Cell Apoptosis Assays
Apoptosis levels were detected in MEN1-specific
siRNAtreated MAC-T cells (2.5 × 105) at 24 h post-transfection,
as well as the untreated and scramble siRNA transfected
cells, using an annexin V-FITC Apoptosis kit (Biovision) for
immunofluorescence analysis, followed by cell analysis via
flow cytometry (FACSCalibur, BD Biosciences) employing
Cell Quest Pro software. Aliquots of the same cells (about
5 × 103) were also stained with annexin V (green) and
propidium iodide (red) and mounted in DAPI (blue)-containing
anti-fade mounting medium. Microscopy and
photomicrography were performed with AxioObserver Z1 (Zeiss,
Thornwood, NY, USA).
Chromatin Immunoprecipitation (ChIP)
Briefly, 5 × 106 normal MAC-T cells were subjected to
cross-linking with 1% formaldehyde, after which the cells
were lysed in 1 ml of SDS lysis buffer and sonicated with
a Bioruptor sonicator (UCD-200TM-EX, Diagenode,
Belgium) to shear the chromatin into 200 – 1,000 bp fragments.
The sonicated cell lysates (100 μl of the 1 ml supernatant)
and 2 μg of antibodies were used for each
immunoprecipitation. Anti-menin (Bethyl Laboratories, Montgomery,
TX, USA) and anti-rabbit IgG (Sangon Biotech, Shanghai,
China) were used for ChIP analyses. The precipitated DNA
was used as template for quantitative real-time PCR using
the SYBR-Premix Ex Taq II (TaKaRa, Dalian, China).
Primer pairs screening of 1200 bp promoter region of Cyclin
D1 gene were designed and synthesized, their detailed
information were listed in Supplementary Table S3. For
quantitative real-time PCR, CT values for each ChIP were obtained
from triplicate reactions. The results were representative
of three independent ChIP experiments, showing as the
percentage of input by quantifying the amount of chromatin
obtained from immunoprecipitation relative to the amount
in the input samples.
Tissues dissected from the mammary glands of cows were
fixed in 4% paraformaldehyde and embedded in paraffin.
Sections (5 mm thickness) of the tissues were then
subjected to standard immunohistochemistry staining.
Detection of menin was performed incubating the slides with a
rabbit polyclonal antibody (1:100; Bethyl Laboratories,
Texas, USA) for 2 h at 37 °C. After the slides were washed,
they were treated with HRP-conjugated goat anti-rabbit IgG
(1:50; Beyotime, Nanjing, China) for 1 h at 37 °C. Images
were captured using a microscope (Leica TCS SP2 AOBS,
Germany). Image- Pro Plus (IPP) 6.0 software was used
to quantify the mean density of positive-menin signals in
mammary epithelial cells by randomly selecting different
numbers of microscopic views.
The data are presented as the mean ± S.E.M. of at least
three independent experiments. Statistic differences among
groups were compared with one-way ANOVA and
difference between pair-designed experiments were compared
with Student’s t-tests by using SAS8.2 software (SAS
Institute Inc., Cary, USA). Significant differences were declared
when P-values were < 0.05 (*) or < 0.01 (**).
Acknowledgements Thanks go to the two anonymous reviewers and
the editor, whose comments and suggestions have greatly improved
our paper. We would like to thank Dr. Ying Yu (China Agricultural
University), Dr. Li Jiang (China Agricultural University) and Dr.
Jinming Huang (Research Center of Dairy Cow, Shandong Academy of
Agricultural Science) for their helpful discussions about the project
and manuscript; Dr. Nana Yang (Taishan Medical University) for her
nice support on the flow cytometry analysis; Dr. Fuchang Li (Shandong
Agricultural University) for providing the microscope and
photographing assistance; Dr. Hui Tang (Shandong Agricultural University) for his
nice support on statistical analysis; Ms. Zijuan Qin (Animal Husbandry
Laboratory platform of Shandong Agricultural University) for her kind
laboratory support on the routine experimental tests. This work was
financially supported by the National Natural Science Foundation
of China (31402054), the Natural Science Foundation of Shandong
(ZR2013CM013), the Key Project of Agricultural Fine Breeding of
Shandong Province (2016LZGC030), the Youth Innovation Fund of
Shandong Agricultural University (24036), the Modern Agricultural
Industry Technology System (CARS-36), Funds of Shandong
“Double Tops” Program (SYL2017YSTD08), the Support Program of Tai
Mountain Scholar Talent Team for Agricultural Breeding
(2014LZ0704) and the Modern Agricultural Industry Technology System of
Shandong Province (SDAIT-12-011-06).
Author Contributions K.S., X.L. and Z.X. designed and conceived
experiments; X.L., H.L. and Q.C. performed the experiments; K.S.,
Compliance with Ethical Standards
Conflict of Interest None declared.
Open Access This article is distributed under the terms of the Creative
Commons Attribution 4.0 International License
(http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided you give appropriate
credit to the original author(s) and the source, provide a link to the
Creative Commons license, and indicate if changes were made.
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