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) or low hair density (<10000/cm2) 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 (high density and high density, low density and low density 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[51,52]. 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
Small RNA fragments measuring 18–30 nt were isolated and purified from the total RNA using 15 % denaturing polyacrylamide gel electrophoresis (PAGE). Subsequently, 3′ and 5′ RNA adaptors were ligated to the RNA pool using T4 RNA ligase, and the samples were used as templates for cDNA synthesis. The cDNAs were amplified using the appropriate number of PCR cycles needed to produce the sequencing libraries, which were subsequently subjected to the proprietary Solexa sequencing by synthesis method using the BGISEQ-500 platform at Shenzhen Huada Biotech Co., Ltd. [53, 54] (Shenzhen, China).
The raw reads produced from BGISEQ-500 sequencing were filtered to remove low-quality reads, and reads that passed the quality filter were trimmed to remove the 5′ and 3′ adaptor sequences. Similarly, the reads with a poly (A) sequences were excluded. The length distribution of the clean reads was calculated. The remaining reads were analysed using BLAST with Bowtie-1.0.0 software, Rfam [55] and Repbase to discard messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), small nuclear ribonucleic acid (snRNA), small nucleolar RNA (snoRNA) and repeat sequences. The remaining sequences were used for miRNA identification and compared with the mature miRNAs and pre-miRNAs from Oryctolagus cuniculus listed in miRBase 21.0 [56], with permission of one mismatch. Subsequently, the miRDeep 2 software [57] was used to predict the novel miRNAs by exploring the secondary structure, Dicer cleavage sites and minimum free energy of the unannotated small RNA tags that mapped to the Oryctolagus cuniculus genome. The sequences remaining after the identification of conserved miRNAs were aligned to the Oryctolagus cuniculus genome to identify new Oryctolagus cuniculus miRNAs. Certain target sequences around the small RNAs were used to explore the secondary structures and folding energy (218 kcal/mol).
Transcripts per million (TPMs) were calculated to standardize the expression levels of small RNA [58], which potentially avoided the effect of different sequencing quantities on quantitative accuracy. Based on the assumption that RNA sequencing is a random process, namely, each sequence is uniformly randomly amplified from its own sample [59], the expression of each transcript is presumed to exhibit a binomial distribution. Using the model described above, DEGseq calculated differential expression based on MA-plot [60, 61]. Assuming that C1 and C2 are the total reads in the comparison of two samples, they exhibit a binomial distribution, where Textit {M}= log2 C1 - log2 C2, A= (log2 C1 + log2 C2)/2. Under the condition of random sampling, the distribution of M obeys A=a and approximates a normal distribution. 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.
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. Twenty-four 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. Twenty microliters of a 5 g/L thiazolyl blue tetrazolium bromide (MTT) solution (Solarbio, Beijing, China) were added to each well and incubated for 4 h. Next, dimethyl sulfoxide (DMSO, Solarbio, Beijing, China) was added to each well and incubated at 37 °C. Optical density (OD) values were recorded at 490 nm using an enzyme labelling instrument (BioTek Elx-808, USA) after 10 min of oscillation. Within a certain cell number range, the amount of MTT crystallization is proportional to the number of cells. Using to the measured OD value, we calculated the number of living cells. A larger OD value indicates higher cell proliferation activity. For the flow cytometry analysis, DPCs were transfected with the indicated adenovirus. The cells were digested with trypsin and fixed with 70 % cold ethanol for at least 18 h. Cells were then centrifuged at 500 g for 5 min, the supernatant was discarded, and added 0.5 mL of propidium iodide (PI; BD Biosciences, Cat: 550825) was incubated with the cells for 15 min at 37 °C in the dark. The cells were then analysed using flow cytometry (BD Accuri C6, BD). The results were analysed with the ModFit LT 5.0 software.
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). 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
Total RNA was extracted from tissues or cells with the RNAiso reagent (TaKaRa, Japan), according to the manufacturer’s instructions. The integrity and quality of the total RNA were evaluated using a 2100 Bioanalyzer RNA Nano chip device (Agilent, Santa Clara, CA, USA) and agarose gel electrophoresis, respectively, and the concentration was measured with an ND-1000 spectrophotometer (NanoDrop, Wilmington, DE).
For quantitative RT-PCR of mRNA, 1 μg of total RNA of was used to synthesize cDNAs with a Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostics GmbH Mannheim, Germany). Quantitative RT-PCR was performed to determine the expression levels of target mRNAs, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as the reference gene. All quantitative PCR primers (Additional file 10) were designed using Primer Premier 5 software and synthesised by SANGON Biological Engineering Co., Ltd. (Shanghai, China). PCR amplification was performed using the Fast Start Universal SYBR Green Master Mix (Roche Diagnostics GmbH Mannheim, Germany). The volume of each reaction was 20 µL, including 2 µL of cDNAs, 10 µL of SYBR Premix Ex TaqTM (2×), 0.5 μL of the PCR Forward Primer (10 μM), 0.5 µL of the PCR Reverse Primer (10 μM), 0.4 µL of ROX Reference Dye II (50×) and 6.6 µL of ddH2O. The number of cycles set for the linear amplification of the cDNAs was 40. All samples were analysed in duplicate, and the standard curves were generated using pooled cDNAs from the samples being assayed.
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. Three biological replicates with three technical replicates were conducted for each qRT-PCR. The relative expression levels of mRNAs and miRNAs were calculated using the arithmetic formula 2−△△Ct [65].
Western immunoblotting.
Tissues or cells were lysed in RIPA buffer on ice for 30 min. The supernatant was centrifuged at 12,000 g for 30 min at 4 °C, and protein concentrations were determined using a BCA Protein Assay Kit (Kangwei, China). The extracted proteins (50 ng/sample) were solubilized in 40 millimoles of SDS-loading buffer (Solarbio, China) and then resolved by electrophoresis (Bio-Rad, Richmond, USA) on 12.5 % SDS-PAGE gels prior to being electrophoretic transfer to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, USA). The standard markers for protein molecular masses were purchased from Thermo (USA). The membranes were blocked with 5 % skimmed milk in 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.