Experiments were performed according to the Regulations for the Administration of Affairs Concerning Experimental Animals (Ministry of Science and Technology, China, revised in June 2004) and approved by the Institutional Animal Care and Use Committee at Zhejiang University, Zhejiang, China. Animals were allowed access to food and water ad libitum under controlled environmental conditions and were humanely sacrificed as necessary to ameliorate suffering.
Two multiparous dairy cows were used for miRNA library construction. The first was a 6-year-old cow that had been lactating for 2 months, which was used to make the lactation miRNA library, and the second was a 4-year-old non-lactating, non-pregnant cow, which was used to construct the non-lactation miRNA library.
In the microarray assay, two other multiparous cows were added to each period, and mixed RNA samples were made. The two additional lactation cows had been lactating for 3 and 4 months and were 4 and 5 years old, respectively. The two additional non-lactating, non-pregnant cows were 4 and 5 years old.
Bovine MG tissues were collected and immediately stored in liquid nitrogen until further use. Blocks of MG tissue were fixed in 4% formalin for 48 hours, processed and embedded into paraffin blocks according to routine procedures.
The paraffin-fixed blocks were serially sectioned into 8 μm coronal slices and stored at −20°C until further use. For routine histological studies, paraffin sections were stained with hematoxylin and eosin. Hematoxylin-eosin stained sections were analyzed by light microscopy using a Nikon fluorescence microscope (Nikon, Japan).
Alpha-casein was detected in frozen sections by immunofluorescence. Sections were fixed with 4% formaldehyde for 10 minutes. The slides were then rinsed 3 times in PBS for 5 minutes each and blocked for 60 minutes. The blocking solution was replaced by primary antibody solution (1:100, gene Tex, USA), and the samples were incubated overnight at 4°C. The next day, slides were rinsed 3 times in PBS for 5 minutes each. FITC-conjugated secondary antibody (1:200) with DAPI was added, and the slides were incubated for 1 hour at 37°C in the dark, followed by 3 rinses in PBS for 5 minutes each. The specimens were viewed under a fluorescence microscope (Nikon, Japan).
Total RNA isolation, small RNA library preparation and sequencing
Total RNA was extracted using a Qiagen miRNeasy Mini Kit (Qiagen, USA) according to the manufacturer’s protocol. Subsequently, the RNA samples were sent to LC Science (Houston, USA) to construct the small RNA libraries using an Illumina small RNA kit (Illumina, San Diego, USA) and to be sequenced using Genome Analyzer (Illumina, San Diego, USA).
Sequencing data analysis
Small RNA reads were processed using Illumina’s Genome Analyzer, and the ACGT101-miR program was then used to process the sequencing data. The mammalian miRbase (miRbase 17.0: http://www.mirbase.org/index.shtml) and the bovine mRNA Rfam, Repbase, genome and EST databases (http://www.ncbi.nlm.nih. gov/projects/genome/guide/cow/ and BTA 4.0: ftp://ftp.ensembl.org/pub/release-57/ fasta/bos_Tau-rus/dna/) were exploited. The sequencing data were first filtered into mRNA using Rfam and Repbase, and then mapped to miRbase. The mapped data were then aligned to genome and EST databases for annotation purposes. The remaining unmapped data were mapped to genome and EST data, secondary structures were predicted using UNAFold software , and IDEG 6 was used to identify significant differentially expressed miRNAs .
All sequencing data were categorized into three groups: (1) known miRNAs reported in miRbase; (2) conserved miRNAs sharing highly similar sequences corresponding to their precursors in other mammalian genome assemblies, and (3) bovine novel candidates where reads and the predicted secondary structures are not mapped to the miRNAs or pre-miRNAs in miRbase, but are mapped to the bta genome with extended sequences from the genome that form hairpins.
Total RNA was extracted using a Qiagen miRNeasy Mini Kit (Qiagen, USA). For each stage, equal quantities of total RNA isolated from three individual cows were pooled. A custom-designed microarray assay was performed to analyze miRNA expression patterns in lactating and non-lactating periods by LC Science (Houston, USA). The array included probes for 523 miRNA derived from the sequencing data and reported bovine miRNA (from miRbase) with 5S rRNA as a data normalization control. The probes were synthesized by in situ synthesis using PGR (photogenerated reagent) chemistry. Hybridization was performed overnight on a μParaflo microfluidic chip using a micro-circulation pump (Atactic Technologies) . Hybridization images were collected using a laser scanner (GenePix 4000B, Molecular Device) and digitized using Array-Pro image analysis software (Media Cybernetics). Data were analyzed by first subtracting the background and then normalizing the signals using a LOWESS (Locally weighted Regression) filter .
Target prediction and network construction
The starting point of the miRNA target prediction strategy was the utilization of known miRNAs listed in Additional file 1. TargetScan (Version 5.0) was used to predict putative targets with an established miRNA seed database and a bovine 3’EST database. EGAN software was used to depict the relationships between miRNAs, target genes and lactation. Due to limited data in cattle, data from human orthologs were also included for these targets in EGAN software .
Quantitative RT-PCR assay
The gene expression assay and differentially expressed miRNAs identified using deep sequencing were validated using real-time PCR. Total RNA were extracted from the MG tissues in both periods separately using Trizol reagent (Invitrogen, USA). The RNA was divided into two portions, one for genetic testing and the other for miRNA detection. Genetic testing started with 500 ng of total RNA, and this RNA was reverse transcribed to cDNA using a SYBR® PrimeScript® RT-PCR Kit (TAKARA, Japan). For miRNA detection, 2 μg of total RNA was reverse transcribed to cDNA with a specific stem-loop primer using M-MLV (Invitrogen, USA), with incubation for 60 minutes at 42°C, followed by heating for 10 minutes at 95°C and storage at 4°C. These cDNA were then used as templates in a SYBR® Premix Ex Taq™ kit (TAKARA, Japan) with specific primers (Additional file 8). Real-time PCR was performed on an ABI7500 system (Applied Biosystem, USA). The reaction mixtures were incubated in a 96-well plate at 95°C for 30 seconds, followed by 40 cycles of 95°C for 5 seconds and 60°C for 34 seconds. All reactions were run in triplicate. GAPDH was used as a gene assay control and bovine S18 rRNA as a miRNA control. Fold changes were determined by the threshold cycle (CT). Fold changes of miRNA expression were calculated using the 2−ΔCt method, where ΔCt = (Ct target − Ct control) Sample.
Cell culture and transfection
The cells used in this study were from the Mac-T cell line, which was donated by Dr. Zhao (University of Vermont, Burlington, USA). Cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM, Gibco, USA) supplemented with 10% (V/V) fetal bovine serum (FBS, Gibco, USA), 100 U/mL penicillin and 100 mg/mL streptomycin. The cells were maintained at 37°C with 5% CO2 and subcultured every other day.
Transfection of Mac-T cells with mimics and inhibitors
Cells were seeded in a 24-well plate at concentration of 1×105 cells/ml/well on the day before the transfection. Mimics of miR-125b, miR-141, miR-181, miR-199a, miR-484 and miR-500 and the antisense inhibitor miR-141 were transfected by Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer’s protocol. The transfection efficiency was examined using FAM-conjugated siRNA. The mimics were RNA duplexes, the inhibitors were single-stranded, and the negative controls (NC) and inhibitor negative controls (INC) for all miRNA mimics and inhibitors were designed by Invitrogen and had no homology to any bovine genome sequences (Additional file 9). The culture medium was changed 6 hours after the transfection of 20 pmol/L of mimics or inhibitors. All transfection data are representative of three independently repeated transfections and each 3-well group of cells were treated as one experimental unit.
Western blotting analysis
Proteins were extracted using a protein extraction kit according to the manufacturer’s instructions (Kegen, China). Equal amounts of protein lysate were separated by SDS- polyacrylamide gel electrophoresis (PAGE) and then electrophoretically transferred to polyclonal difluoride membranes. Each protein was incubated with a specific antibody and detected with an electrogenerated chemiluminescence (ECL) kit. Beta-actin was used as a loading control. Antibodies for STAT5 and β-actin were manufactured by Boster (China), and the HK2 antibody was purchased from Santa Cruz (USA). The intensity of the protein fragments was quantified using Imagpro-Plus software. All data are from three independently repeated experiments.
All data were analyzed using SPSS software (V16.0, SPSS Inc., USA). Values in the texts and figures represent the results of at least three separate experiments. Group comparisons were performed using ANOVA with the Student’s t-test. Differences were considered statistically significant at P<0.05.