AABArchives Animal BreedingAABArch. Anim. Breed.2363-9822Copernicus PublicationsGöttingen, Germany10.5194/aab-60-199-2017Identification and expression analysis of miR-144-5p and miR-130b-5p in dairy cattleLiZhixiongWangHongliangChenLingZhaiMengxingChenSiLiNaLiuXiaolinliuxiaolin@nwsuaf.edu.cnCollege of Animal Science and Technology, Northwest A&F University, Shaanxi Key Laboratory of Molecular Biology for Agriculture, Yangling, Shaanxi 712100, P.R. ChinaCollege of Life Science and Technology, Southwest University for Nationalities, Chengdu 610041, P.R. ChinaState Key Laboratory for Molecular Biology of Special Economic Animals, Institute of Special Animal and Plant Sciences, Chinese Academy of Agricultural Sciences, Changchun 130112, P.R. ChinaXiaolin Liu (liuxiaolin@nwsuaf.edu.cn)19July20176031992049October201610May201713June2017This work is licensed under the Creative Commons Attribution 3.0 Unported License. To view a copy of this licence, visit https://creativecommons.org/licenses/by/3.0/This article is available from https://aab.copernicus.org/articles/60/199/2017/aab-60-199-2017.htmlThe full text article is available as a PDF file from https://aab.copernicus.org/articles/60/199/2017/aab-60-199-2017.pdf
MicroRNAs (miRNAs) can coordinate the main pathways involved in
innate and adaptive immune responses by regulating gene expression. To
explore the resistance to mastitis in cows, miR-144-5p and
miR-130b-5p were identified in bovine mammary gland tissue and
14 potential target genes belonging to the chemokine signaling pathway,
the arginine and proline metabolism pathway and the mRNA surveillance pathway were
predicted. Subsequently, we estimated the relative expression of
miR-144-5p and miR-130b-5p in cow mammary tissues by
using stem-loop quantitative real-time polymerase chain reaction. The results
showed that the relative expression of miR-144-5p and
miR-130b-5p in the mastitis-infected mammary tissues (n=5) was
significantly downregulated 0.14-fold (p<0.01) and upregulated 3.34-fold (p<0.01), respectively, compared to healthy tissues (n=5). Our
findings reveal that miR-144-5p and miR-130b-5p may have
important roles in resistance to mastitis in dairy cattle.
Introduction
Bovine mastitis, defined as “an inflammation of the mammary gland”, is a prevalent and complex infectious disease affected by
genetics and pathogens that can result in significant dairy cattle losses (Nash et al., 2003). Mastitis can be caused by many
bacteria, including Staphylococcus aureus and Escherichia coli. The primary defense against pathogens relies on
the appropriate expression of antigen-presenting molecules triggering the release of effector molecules in the innate immune
system. The immune system, as the central host determinant for dictating the outcome of intramammary infection, can defend against
in-breaking pathogens as the first line once the pathogens penetrate the physical barrier (Bannerman et al., 2009).
Recent studies have shown that microRNAs (miRNAs) play important roles in
regulating and modulating innate and adaptive immune responses (Zhou et al.,
2012; Gu et al., 2012). Mature miRNAs are a class of small non-coding RNA molecules that are ∼22 nucleotides
(nt) long processed from
∼ 70 nt long precursor miRNAs (pre-miRNAs) that form hairpin
secondary structures and are evolutionarily conserved (Bartel et al., 2004;
Cullen et al., 2004; Kim et al., 2005). miRNAs are post-transcriptional
regulators that inhibit the translation or induce the degradation of protein-coding
protein mRNAs that contain complementary sequences to miRNAs (Berezikov,
2011; Bartel et al., 2009).
The primer sequences of the stem-loop qPCR experiments.
Note: the underlined letters are the sequences from miR-144-5p and miR-130b-5p.
miR-144 plays a crucial role in hemoglobin synthesis during primitive erythropoiesis and is associated with anemia
severity in sickle-cell diseases (Fu et al., 2009; Sangokoya et al., 2010). miR-130b inhibits cell proliferation and
invasion in pancreatic cancer through targeting STAT3 and targets DICER1 for aggression in endometrial cancer
(Zhao et al., 2013; Li et al., 2013). miR-130b is associated with poor prognosis in colorectal cancer and is a prognostic
marker (Colangelo et al., 2013). Our previous research showed two differentially expressed miRNAs matched to bta-mir-144 and
bta-mir-130b that were detected in the peripheral blood of healthy and mastitis-infected dairy cattle (Li et al., 2014a). However, our
knowledge of the differential expression of the two miRNAs in cattle mastitis resistance remains largely limited. The two
miRNAs may have important roles in the development of the immune system against pathological changes in mammary tissue in dairy
cattle.
The aims of this study were (1) to investigate whether miR-144-5p
and miR-130b-5p are present in bovine tissues, (2) to predict the
target genes miR-144-5p and miR-130b-5p and (3) to analyze
whether miR-144-5p and miR-130b-5p are differentially
expressed in healthy and mastitis-infected mammary tissues.
Materials and methodsAnimal samples
Samples were collected from five healthy and five mastitis-infected Chinese Holstein cows of first lactation from a commercial
bovine slaughter farm. The selection of mastitis-infected cows was carried out as previously described (Li et al., 2014b). A part of
the 10 mammary tissue samples was collected and stored in liquid nitrogen for RNA isolation; others were used for the
identification of the pathogen. All 10 mammary tissue samples were used for analysis of the expression profile of
miR-144-5p and miR-130b-5p. The liver, heart, lung, kidney and spleen from five healthy samples were used to analyze
the expression pattern. This study was approved by the Northwest A&F University Animal Care and Use Committee.
RNA extraction and cDNA synthesis
Total RNA was extracted using Trizol Reagent (Invitrogen, USA) following the manufacturer protocol. RNA purity was verified by
measuring the absorbance at 260 and 280 nm with an ND-1000 spectrophotometer (NanoDrop Technologies, USA). First-strand cDNA
synthesis was performed in a 20 µL volume using a PrimeScript RT reagent kit (Takara, Japan) following the
manufacturer protocol with a specific stem-loop primer. The primers for the reverse transcription polymerase chain reaction
(RT-PCR) are shown in Table 1.
To identify miR-144-5p and miR-130b-5p expressed in the mammary tissue of dairy cattle, primers were designed
according to the sequences previously detected (Li et al., 2014). PCR was performed in a total volume of 25 µL
containing 50 ng of cDNA, 2.5 µL 10 × PCR buffer, 2.1 mMMgCl2, 0.1 mM dNTPs,
0.25 mM of each primer, 0.2 µL Easy Taq DNA polymerase and ddH2O run for 32 cycles at 95∘
for 40 s, 60∘ for 30 s and 72∘ for 30 s, followed by incubation at 72∘ for
10 min. PCR products were ligated into the T-Vector pMD19 (Takara, Japan) after gel extraction and then transformed into
competent E. coli DH5α. Finally, 10 randomly selected positive clones were sequenced. The experiment was repeated
twice to confirm the result.
Sequence analysis
Sequence alignment was performed to verify miR-144-5p and miR-130b-5p using DNAman (version 6.0) software. To
obtain the potential target genes of miR-144-5p and miR-130b-5p, the prediction of the target gene was performed
with
MIREAP software. The predicted target genes were classified by KEGG functional annotations; the identified pathways were actively
regulated by miR-144-5p and miR-130b-5p in healthy and mastitis-infected dairy cattle.
(a) Sequencing chromatograms for miR-144-5p.
(b) Sequencing chromatograms for miR-130b-5p.
(c) The bta-mir-144 sequence comparison with bta-miR-144 and
miR-144-5p. (d) The bta-mir-130b sequence comparison with
bta-miR-130b and miR-130b-5p.
Quantitative analysis of miRNAs
Stem-loop quantitative real-time polymerase chain reaction (stem-loop qPCR)
was used to analyze miRNAs according to Chen et al. (2005). The stem-loop
qPCR was performed in the Bio-Rad CFX96 Real-Time PCR Detection System using
the SYBR Green PCR kit (Takara, Japan) according to the manufacturer
instructions. 18S rRNA was used as the reference gene in the stem-loop qPCR
detection of bovine miRNAs, and all reactions were run in triplicate. The
relative expression level of miR-144-5p and miR-130b-5p was
calculated according to the method of Livak and Schmittgen (2001). The
primers for the qPCR are shown in Table 1.
Statistical analysis
The value of the relative quantity was presented as fold change. The means of
two groups were compared by a Student's paired-samples t test. The analysis
was performed with SPSS software (version 20.0); p<0.05 was regarded as
significant.
ResultsIdentification of <italic>miR-144-5p</italic> and <italic>miR-130b-5p</italic>
RT-PCR and sequencing were used to identify miR-144-5p and
miR-130b-5p with specific primers (Fig. 1a and b). The miRNAs of
bta-miR-144 and bta-miR-130b from miRBase
(http://www.mirbase.org/) are matched to the 3′ of
bta-mir-144 and bta-mir-130b. The cloned miRNAs are totally
matched to the 5′ of bta-mir-144 and bta-mir-130b (Fig. 1c and d),
so we called them miR-144-5p and miR-130b-5p.
Target genes of miR-144-5p and miR-130b-5p.
miRNA nameGene nameKEGG pathway namemiR-144-5pCXCL2Chemokine signaling pathwayCRKGNB5NOS2Arginine and proline metabolismARG1miR-130b-5pAMD1Arginine and proline metabolismSAT2ARG1GLS2PPP2R2BmRNA surveillance pathwayPPP2CBSMG1SAP18CHUKChemokine signaling pathwayCXCL2CXCL6GNAT2CCL11NRASBRAFTarget gene prediction of <italic>miR-144-5p</italic> and <italic>miR-130b-5p</italic>
Twenty potential target genes of miR-144-5p and miR-130b-5p
were predicted using MIREAP software (Table 2). Ten target genes of
miR-144-5p and miR-130b-5p belong to the chemokine signaling
pathway, which plays an important role in inflammatory responses and cancer
(Coussens and Werb, 2002; Charo and Ransohoff, 2006; Baggiolini and
Loetscher, 2000). Six target genes of miR-144-5p and
miR-130b-5p belong to the arginine and proline metabolism pathway, which
is closely involved in conceptus metabolism, growth and development. Four
target genes of miR-130b-5p belong to the mRNA surveillance pathway,
which ensures the viability and quality of mRNA.
Relative expression of miR-144-5p. (a) Relative
expression of miR-144-5p in healthy and mastitis-infected mammary
gland tissues using qPCR. H-MG denotes healthy mammary gland and M-MG denotes
mastitis-infected mammary gland. (b) Relative expression of
miR-144-5p in a variety of healthy tissues using qPCR. H denotes
the healthy cow group. The vertical bar represents the standard error.
Relative expression of miR-130b-5p. (a) Relative
expression of miR-130b-5p in healthy and mastitis-infected mammary
gland tissues using qPCR. H-MG denotes healthy mammary gland and M-MG denotes
mastitis-infected mammary gland. (b) Relative expression of
miR-130b-5p in a variety of healthy tissues using qPCR. H denotes
the healthy cow group. The vertical bar represents the standard error.
Expression of <italic>miR-144-5p</italic> and <italic>miR-130b-5p</italic> in healthy and mastitic cow tissues
The expression of miR-144-5p and miR-130b-5p in healthy and
mastitis-infected mammary tissues was investigated using stem-loop qPCR.
A lower expression of miR-144-5p was observed in the
mastitis-infected mammary tissues compared to that in the healthy samples
(Fig. 2a; p<0.01). The expression of miR-144-5p in mammary
glands was higher than that in other tissues, including the heart, liver, spleen,
lung and kidney in the healthy cows (Fig. 2b). The expression of the
miR-130b-5p was much higher in the mastitis-infected mammary gland
tissues compared to that in the healthy samples (Fig. 3a; p<0.01). The
expression of miR-130b-5p in mammary glands was higher than that
in other tissues, including the liver, heart, lung, kidney and spleen in the
healthy cows (Fig. 3b). The findings suggest that miR-144-5p and
miR-130b-5p are highly correlated with mastitis.
Discussion
Through sequencing alignment, bta-miR-144 and bta-miR-130b from
miRBase are actually miR-144-3p and miR-130b-5p; the cloned
miRNAs in our study are miR-144-5p and miR-130b-5p. In the
present study, miR-144-5p and miR-130b-5p were identified
in mammary, heart, liver, spleen, lung and kidney tissues at different
expression levels, which may indicate functional differences.
Twenty potential target genes of miR-144-5p and miR-130b-5p
were predicted, and 10 of them belonged to the chemokine signaling pathway, which
plays an important role in orchestrating leukocyte migration under normal
conditions and during inflammatory responses (Mellado et al., 2001), such as
CXCR4 (Lapteva et al., 2005; Balkwill, 2004) and CXCR2
(Acosta et al., 2008). Another important pathway including six target genes
is arginine and proline metabolism, which is known to be closely involved in
conceptus metabolism (Wu et al., 2008), growth and development. It is also a potential treatment for intrauterine growth restriction (Wu et al.,
2009), which is a significant problem in both human medicine and animal
agriculture. Four of them belonged to the mRNA surveillance pathway, which
assesses the quality of mRNAs to ensure that they are suitable for
translation (Vasudevan et al., 2002). mRNA surveillance facilitates the
detection and destruction of mRNAs that contain premature termination codons
(Wagner and Lykke-Andersen, 2002). Whether these genes are regulated directly
by miR-144-5p and miR-130b-5p needs to be confirmed in a further
study.
Conclusions
In summary, the differential expression of miR-144-5p and
miR-130b-5p in healthy and mastitis-infected mammary glands
indicates that miR-144-5p and miR-130b-5p may play
important roles in inflammation response. There could be
a relationship between miR-144-5p and miR-130b-5p and
mastitis in Chinese Holstein cattle. Our findings suggest that the
differential expression of the post-transcriptional regulators
miR-144-5p and miR-130b-5p may bind to complementary
sequences of target mRNAs, resulting in different translational
bovine repression. miR-144-5p and miR-130b-5p likely
play critical roles in mastitis resistance in dairy cattle.
The original data for the paper are available upon request from the corresponding author.
The authors declare that they have no conflict of interest.
Acknowledgements
This study was supported by the National 863 Program of China (2008AAl02144), the“13115” Sci-Tech Innovation Program of Shaanxi Province
(2008ZDKG-11)
and the Xi'an city science and technology project (NC09049-2). Comments from
anonymous reviewers have greatly improved the paper. Edited by: Steffen Maak Reviewed by: three
anonymous referees
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