Animal and sample collection
The Rex rabbits used in this study were purchased from Taishan Rabbit Farm (Shandong, China). Ten adult female Rex rabbits with a high (>14000/cm2, HD) or low hair density (<10000/cm2, LD) were chosen and divided into 2 groups. An adult male rabbit was selected to mate with the female rabbits within each group. The first generation of offspring (F1 generation) was obtained and then inbred with F1 in the same nest (HD and HD, LD and LD inbreeding). Three rabbits each with high and low hair densities were selected from the second generation of offspring (F2 generation) 30 days after birth. Selected animals were electrically stunned (120 V, pulsed direct current, 50 Hz for 5 s) and euthanized by exsanguination of the carotid artery and before skin was harvested from the experimental rabbits. The DPCs were separated and cultured, methods according to references[51]. After the cells had established a monolayer (approximately 12 days), the total RNA was extracted, tested for quality.
Construction of the small RNA libraries, sequencing analysis, miRNA identification and prediction of new miRNAs in DPCs
The methods of construction small RNA libraries according to references[52]. Solexa sequencing by synthesis method using the BGISEQ-500 platform at Shenzhen Huada Biotech Co., Ltd. (Shenzhen, China) [53,54]. The raw reads produced from sequencing were filtered to remove low-quality reads, and the clean reads were analysed using BLAST with Bowtie-1.0.0 software, Rfam [55]. The clean reads were used for miRNA identification and compared with the mature miRNAs and pre-miRNAs from Oryctolagus cuniculus listed in miRBase 21.0 [56]. Subsequently, the miRDeep 2 software was used to predict the novel miRNAs by exploring the secondary structure[57], minimum free energy and dicer cleavage sites of the unannotated clean reads that mapped to the Oryctolagus cuniculus genome. After the identification of conserved miRNAs, the clean reads were aligned to the Oryctolagus cuniculus genome for identify new Oryctolagus cuniculus miRNAs.
In order to eliminate the effect of different sequencing quantities on quantitative accuracy, transcripts per million (TPMs) were calculated to standardize the expression levels of small RNA [58]. Based on the assumption that RNA sequencing is a random process, the expression of each transcript is presumed to exhibit a binomial distribution[59]. DEGseq calculated differential expression based on MA-plot [60, 61], and the P-value of each miRNA was corrected by performing multiple hypothesis tests using Q-values. When the difference in coincidence was more than two-fold and the Q-value was less than or equal to 0.001, the miRNAs were considered significantly differentially expressed (|log2(HD/LD)|>1.00 and Q-value≤0.001).
The miRNAs were aligned to the EST unigenes of Oryctolagus cuniculus and the target genes were predicted using the miRanda algorithm to obtain a better understanding of the potential functions of the significantly differentially expressed miRNAs in Rex rabbits with different hair densities [62]. An enrichment analysis of the predicted target genes was conducted with GO terms and KEGG pathways [63].
Construction and identification of adenovirus vectors overexpressing and silencing ocu-miR-205
HBAD-GFP (HANBIO adenovirus-green fluorescent protein; empty vector), HBAD-ocu-miR-205-GFP (overexpression), HBAD-ocu-miR-205-5p-sponge-GFP (silencing) adenoviruses were synthesized and constructed by Hanheng Biotechnology Co., Ltd. (Shanghai, China). The infective titres of HBAD-GFP, HBAD-ocu-miR-205-GFP and HBAD-ocu-miR-205-5p-sponge-GFP were 1.26*1010 PFU/mL, 1.58*1010 PFU/mL and 1.26*1010 PFU/mL, respectively.
Third-generation DPCs displaying good growth conditions were inoculated into a disposable 6-well plate. The cell density was approximately 1.0*105 cells/mL. Prior to the infection, the virus was subjected to 10-fold gradient dilution. Generally, the MOI (multiplicity of infection) was controlled in the range of 10-1000. HBAD-GFP, HBAD-ocu-miR-205-GFP and HBAD-ocu-miR-205-GFP were individually transfected into Rex rabbit DPCs at an MOI 200, and a negative control was established using cells undergoing normal culture.
Fifty microliters of the purified adenovirus were injected into the skin of each Rex rabbit with microinjector at a concentration of 5.0*108 - 1.0 * 109 virus particles per Rex rabbit after shaving the middle part of the back of 100 3-month-old Rex rabbits with similar body weights and good health. 24 h after transfection, one Rex rabbit from each group was randomly selected, euthanized, and frozen sections were prepared from the locally injected skin. The adenovirus-transfected skin was observed under a positive fluorescence microscope (Nikon ECLIPSE 80i, Japan).
Assessment of the proliferation, cell cycle and apoptosis of DPCs
DPCs were plated in a 96-well plate at a density of 104 cells/well, cultured in basal medium for 24 h, and then transfected with the indicated adenoviruses. The methods assessment of the proliferation according to references[51]. Third-generation DPCs were plated in a disposable 6-well plate at a density of 104 cells/mL, with 2 mL of the cell suspension plated in each well. After a 24-h incubation to allow cells to adhere, the culture medium was removed. After treatment and culture for a certain time, the cells were digested with a trypsin digestion solution lacking EDTA (Solarbio, Beijing, China). The methods assessment of the cell cycle according to references[51]. For apoptosis assessment, cells were centrifuged and collected into a 1.5 mL centrifugal tube, and then washed with PBS. After centrifugation, 500 μL of 10X Annexin V Binding Buffer was added to re-suspended the cells, followed by the labelling of F-actin. Cells were incubated with the FITC Annexin V and Propidium Iodide Staining Solution for 15 min at 4 °C and then analysed using flow cytometry. The percentages of early apoptotic cells (Q4), late apoptotic cells (Q2) and total apoptotic cells (Q2 + Q4) in each sample were calculated.
Total RNA extraction and Real-time PCR analysis
The methods of total RNA extraction and real-time PCR of mRNA was according to references[64]. All quantitative PCR primers were designed using Primer Premier 5 software (Additional file 11). For the quantitative RT-PCR of miRNAs, 1 μg of total RNA was reverse transcribed with Bulge-Loop miRNA-specific reverse transcription primers (RiboBio, China), and quantitative PCR was performed using Fast Start Universal SYBR Green Master Mix (Roche Diagnostics GmbH Mannheim, Germany) and Bulge-Loop primers (RiboBio, Guangzhou, China) on the 7500 Fast System 1.4 system with small nuclear RNA U6 as the normalisation control. The volume of each reaction was 20 µL, including 2 µL of cDNAs, 10 µL of SYBR Green Master (2X), 0.8 μL of the Bulge-LoopTM miRNA Forward Primer (5 µM), 0.8 µL of the Bulge-LoopTM Reverse Primer (5 µM), 0.4 µL of ROX Reference Dye II (50×) and 6.0 µL of ddH2O. PCR was performed under the following conditions: 10 min of template denaturation at 95 °C, followed by 40 cycles of 95 °C for 2 s, 60 °C for 20 s, and 70 °C for 10 s. Melting curves (70 °C-95 °C) for each sample were analysed after each run to confirm the specificity of amplification reactions. The relative expression levels of mRNAs and miRNAs were calculated using the arithmetic formula 2−△△Ct [65].
Western immunoblotting.
The methods of total protein extraction and SDS-PAGE were according to references[64]. The membranes were blocked with 5 % skimmed milk in phosphate-buffered saline (PBS; Solarbio, China) at 4 °C overnight and incubated with primary antibodies (tubulin AT819, Beyotime, China; phospho-CTNNB1-S552 pAb, Abcam, US; phospho-GSK3B-S9 pAb, Abcam, US; phospho-AKT1-S473 pAb, Abcam, US; or NOG polyclonal antibody, Abcam, US). The membranes were then rinsed with Tris-buffered saline containing Tween (TBST; Solarbio, China), and subjected to detection with a 1:3000 dilution of a horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG antibody (Beyotime, China) at 37 °C for 1 h. Proteins were visualized using BeyoECL reagents (Beyotime, China). The intensity of the bands was quantified with a Pro Plus 6.0 Biological Image Analysis System. The levels of phospho-CTNNB1, phospho-GSK3B, phospho-AKT1 and NOG were normalized to the internal control beta-tubulin, and the relative expression levels were calculated.
Statistical analysis
All data were analysed with SAS software (SAS version 8e; SAS Institute, Cary, NC, USA). A one-way ANOVA was used to evaluate the differences in mean values among various groups. The data are presented as the means and R-MSE. P < 0.05 was regarded as statistically significant.