The animals in this study were 24 female Tianfu black rabbits from a strain breed at the Sichuan Agricultural University in China that were 35 days of age. Rabbits were randomly divided into two groups and fed either a standard normal diet (SND) or a high-fat diet (HFD; a 10% lard was added to the standard normal diet) for four weeks. Animals had free access to water and were fed twice a day. Each rabbit was housed in a clean iron cage (600 × 600 × 500 mm) and kept in an environmentally controlled room. At the end of the trial, rabbits were screened for obesity using serum triglycerides (TG) concentration and body fat rate, and three rabbits from each group meeting the obesity requirements were randomly selected for sampling.
Blood Collection and Measurement
Blood samples from animals fasted overnight were collected the next morning from the auricular vein using vacutainer tubes. Then, blood samples were centrifuged at 4 °C for 5 minutes and the serum was transferred to clean frozen pipes and stored at -80 °C. Serum triglyceride concentrations were obtained by personnel of the Lilai Biological Company (Lilai, Chengdu, China).
Adipose tissue sample collection
Perirenal adipose tissue samples were taken immediately after rabbits were euthanized (Shock and bleed treatment). Tissue blocks were placed in 4 ml EP tubes and stored in a -80 °C freezer. All experimental protocols were performed under the direction of the Institutional Animal Care and Use Committee from the College of Animal Science and Technology, Sichuan Agricultural University, China (DKY-B2019202015).
Total RNA from samples was extracted using RNAiso Plus Reagent (Invitrogen, Hong Kong, China), following the guidelines of the manufacturer. Subsequently, the purity, concentration, and integrity of RNA were determined by Nanodrop (Thermo Fisher Scientific, Waltham, MA, USA), Qubit 2.0 fluorometer (Life Technologies, Carlsbad, CA, USA) and Agilent 2100 Bioanalyzer system (Agilent Technologies, Carlsbad, CA, USA), and only qualified RNA was used for the trial.
Small RNA library construction and sequencing
To identify changes in miRNAs from perirenal adipose tissue induced by HFD, we constructed six small RNA libraries (SND-1, SND-2, SND-3, HFD-1, HFD-2, HFD-3) from SND and HFD rabbits. The small RNA libraries were constructed using TruSeq Small RNA Sample Prep Kits (Illumina, San Diego, USA), following the manufacturer's instructions. Small RNA of sizes ranging from 15 to 30 nt were isolated using a 15% PAGE gel. The 5' and 3' adaptors were ligated sequentially to the small RNA using a T4 RNA ligase (Promega, USA) and then amplified by q-PCR. Lastly, the libraries were sequenced using the Illumina HiSeq platform, and then 50bp paired-end reads were generated.
Identification of miRNAs
SOAPnuke software was used to filter the sequencing reads, and after removing the 3' containing sequencing adaptors, reads larger than 18nt (clean reads) were retained. The miRBase was used as a reference database, and software miRDeep2 was used to predict novel miRNAs. The mapped sequences contained some known types of RNA, such as RFam data (tRNA, rRNA, snRNA, snoRNA), repeated sequences, and mRNA degraded fragments. Small RNA reads of 18nt or greater were aligned to rabbit reference genomes with Bowtie2, and comparisons between multiple databases (Rfam database, genebank database, and repeat database) were used to remove known types of RNA sequences and repeated sequences. Next, the filtered small RNA reads were compared to the miRbase database to identify known miRNAs that could be directly used for subsequent analyses. Software package miRDeep2 was used to identify novel miRNAs from unannotated reads. The edgeR data analysis package from R was used for differential expression analysis. The threshold was defined to be |log2(fold change)| ≥ 1 and FDR < 0.05. The known DE miRNAs in the six libraries were clustered using the R function Heatmap.
Target gene prediction and functional enrichment analysis
Software miRanda and RNAhybrid were used to predict the target genes of miRNAs, and the intersection of the predicted results was taken as the final outcome. Gene Ontology (GO) terms and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis of DE miRNAs target genes was performed using online software DAVID Bioinformatics Resources 6.7 (http://david.abcc.ncifcrf.gov/home.jsp).
Validation of DE miRNAs
Reverse transcription of miRNAs was performed using the Mir-XTM miRNA First-Strand Synthesis Kit (TakaRa, Dalian, China), following the manufacturer’s protocol. Subsequently, q-PCR was performed in triplicate using the TB GreenTM Premix Ex TaqTM II (TakaRa, Dalian, China) on a CFX96 instrument (Bio-Rad, USA), and relative expression levels of miRNAs were calculated by 2-ΔΔCT method.
SPSS Statistics 20.0 (SPSS Inc., Chicago, IL, USA) was used for statistical analysis. Groups were compared using Student’s t-test. Differences were considered to be statistically significant at P < 0.05.