Animal selection and skin tissue preparation
Subo Merino sheep is a subtype of the Merino breed of sheep in China that is known for its high survival rate and excellent quality and yield of wool. A sheep herd located at Kechuang Animal Husbandry Breeding Center, Xinjiang, China, was selected for testing. From this flock, twenty healthy ewes were artificially inseminated with fresh sperm from the same ram. Insemination occurred on embryonic day 0 (E0). Previous reports have described the methods used for embryonic skin tissue collection on E65, E85, E105, and E135 [48, 49]. The method for the collection of skin tissues on postnatal days 7 and 30 (D7 and D30) has previously been described [48, 49]. For each of the six developmental stages represented by groups, three biological replicates were generated. All eighteen skin tissue samples were stored at − 80°C.
miRNA sequencing and data analysis
We isolated total RNA from tissues using TRIzol reagent (Invitrogen, USA) and tested the integrity of the samples using an Agilent 2100 Bioanalyzer (Agilent Technologies Inc., USA). Following the guide, paired-end libraries were synthesized using the QIAseq miRNA Library Kit (Qiagen, Germany). After purification and enrichment with PCR, the final cDNA library was quantified using a Qubit® 2.0 fluorometer from Life Technologies (USA). On the Illumina HiSeq Xten (Illumina, USA), clusters were generated by cBot with the library diluted to 10 pM. Our previous article describes the details of DE-miRNA sequence analysis [48].
Cell culture and transfection
Primary sheep dermal fibroblasts were cultured. SDFs were prepared from newborn lambs on postnatal day 7 (local anesthesia before surgery). The surface hair was removed with a blade, the skin was wiped with cotton balls soaked in 75% alcohol. Then, use Lidocaine aerosol (Lishuka, A1004157, Shanghai Sine Pharmaceutical Laboratories Co., Ltd., China) to spray on the exposed skin surface, and wait for 2 minutes, and collect 2 cm2 of skin tissue with Scalpel and scissors. Then, 100 IU/mL penicillin and 0.1 mg/mL streptomycin (2:100) were added to phosphate-buffered saline (PBS) at 4°C. In brief, skin tissues were incubated in 0.25% trypsin at 4°C overnight. The tissue blocks were cut into 0.8 mm2 tissue blocks with ophthalmic scissors, broken up with a pipette, and placed in a petri dish that that contained sterile medium (Dulbecco’s modified Eagle’s medium, DMEM) (Invitrogen, Carlsbad, CA, USA) basal medium supplemented with double antibodies and 10% fetal bovine serum (FBS) (Invitrogen, Carlsbad, CA, USA)). The cells were incubated at 37°C with 5% CO2, and then the medium was changed every three days to observe cell growth in the culture dish. If fibroblasts were freed, the tissue blocks could be washed away. Subsequently, adherent fibroblasts were reseeded in new culture plates with EMEM supplemented with 10% FBS, 100 IU/mL penicillin and 0.1 mg/mL streptomycin. The cells were passaged when the cell confluence reached more than 80%.
HEK-293T cells were provided by the Key Laboratory of Livestock and Poultry Multi-omics of MARA (Ministry of Agriculture and Rural Affairs). HEK-293T cells were cultured at 37°C in DMEM (Invitrogen, Carlsbad, CA, USA) supplemented with 10% FBS (Invitrogen, Carlsbad, CA, USA), 1.5 mM l-glutamine (Invitrogen, Carlsbad, CA, USA), 100 U/mL penicillin (Invitrogen, Carlsbad, CA, USA), and 100 mg/mL streptomycin (Invitrogen, Carlsbad, CA, USA) in a humidified incubator in an atmosphere containing 5% CO2 (Thermo, Waltham, MA, USA). None of the cell lines used in this paper were listed in the database of commonly misidentified cell lines that is maintained by ICLAC. All the cell lines were free of mycoplasma contamination. Adherent cells were passaged daily with 0.05% trypsin-EDTA (Invitrogen, Carlsbad, CA, USA).
RT‒qPCR
Total RNA was extracted using TRIzol reagent (Invitrogen, USA). Poly-A tails were added to the miRNAs according to the protocol of the Poly (A) Tailing Kit (Ambion, USA). The PrimeScript™ RT Reagent Kit with gDNA Eraser (Takara, Japan) and gene-specific primers or random primers were used to generate cDNA. RT‒qPCR was performed with a CFX96™ Real-Time System (Bio-Rad, USA) using SYBR® Green (Takara, Japan) and miRcute Plus miRNA qPCR Kit (SYBR Green) (TIANGEN, China). Glyceraldehyde phosphate dehydrogenase (GAPDH) and U6 snRNA were used as endogenous controls for mRNA and miRNA, respectively. The thermal cycling conditions used for mRNA RT‒qPCR were 95°C for 30 sec, followed by 40 cycles of 95°C for 5 sec and 60°C for 30 sec. The thermal cycling conditions used for miRNA RT‒qPCR were 95℃ for 15 min, followed by 45 cycles of 94℃ for 20 sec and 60℃ for 34 sec. The specificity of the SYBR green PCR signal was confirmed by melting curve analysis. There were three biological and technical replicates. The comparative Ct method was used to calculate the relative expression of the target RNAs. The primers were synthesized by Shanghai Sangong Biology Co., Ltd. The primer sequences are displayed in Tables S1 and S2. 2−ΔΔCt indicated the change in the expression of the gene of interest between the experimental group and the control group.
Western blotting analysis
Total protein was extracted from SDFs using radioimmunoprecipitation assay lysis buffer (Beyotime Biotechnology, China). The protein concentration was determined by a bicinchoninic acid (BCA) kit (Thermo Fisher Scientific, USA). The proteins were separated by polyacrylamide gel electrophoresis. Next, the separated proteins were transferred onto polyvinylidene fluoride membranes (Millipore, Germany), which were blocked in 5% BSA at room temperature for 1 h. The membranes were incubated overnight at 4°C with the following primary antibodies (Abclonal): anti-Notch1 rabbit mAb (A19090, 1: 10000), anti-TGFβ2 rabbit pAb (A3640, 1: 10000), anti-Cyclin D1 rabbit pAb (A11022, 1: 10000), anti-Bax rabbit pAb (A15646, 1: 10000), anti-p53 rabbit pAb (A11232, 1: 10000), anti-CDKN1A/p21CIP1 rabbit pAb (A1483, 1: 10000), and anti-GAPDH (A19056, 1: 10000). After 3 washes, the membranes were incubated with diluted horseradish peroxidase (HRP)-labeled goat anti-rabbit immunoglobulin G (IgG) (AS014, 1: 10000, Abclonal) for 1 h at room temperature. Then, the membranes were visualized with enhanced chemiluminescence substrates (BL520A; Biosharp). ImageJ 1.48u software (Bethesda, USA) was used for protein quantification analysis, and GAPDH was used as the internal reference.
Prediction and enrichment analysis of target genes
miR-23b and miR-133 target genes were predicted through the online websites TargetScan (http://www.targetscan.org), RNAhybrid (https://bibiserv.cebitec.uni-biele), PicTar (https://pictar.mdc-berlin.de), starBase (http://starbase.sysu.edu.cn/) and RNA22 (http://cbcsrv.watson.ibm.com/rna22.html).
GO and KEGG functional analyses of target genes were performed using Database for Annotation, Visualization, and Integrated Discovery (DAVID) (https://david.ncifcrf.gov/). GO is a database established by the Gene Ontology Consortium that is used for the functional annotation and classification of target genes. KEGG functional analysis was used to perform functional annotation and classification of pathways in the KEGG database for the target genes. The method of the KEGG pathway functional enrichment analysis was similar to that of the GO functional enrichment analysis. Based on the false discovery rate (FDR), we determined the GO terms and metabolic pathways that were significantly associated with the gene lists. FDR < 0.05 was applied to significant target genes associated with GO terms or KEGG pathways.
Luciferase reporter assay
Total RNA was extracted from sheep skin according to the instructions of the TRIzol kit (Invitrogen, USA). Extracted RNA was then reverse transcribed into complementary deoxyribose nucleic acid (cDNA) according to the instructions of the One StepPrimeScript® miRNA cDNA Synthesis Kit (TaKaRa, Japan). The 3'UTR fragments of NOTCH1 and TGFβ2 that contained putative oar-miR-23b/oar-miR-133 binding sites were amplified from sheep genomic DNA using forward and reverse primers containing XhoI and NotI restriction sequences, respectively. The amplified fragments were cloned at the XhoI and NotI sites downstream of the CV40 promoter-driven Renilla luciferase cassette in psiCHECK2 (Promega). The dual luciferase reporter vector carrying the wild-type (WT) 3'UTR of NOTCH1/TGFβ2 and the 3’UTR sequences with mutations (MUT) in the miR-23b and miR-133 binding sites were separately constructed. The primers and sequences used for the construction of wild-type and mutant vectors are shown in Table S3. HEK-293T cells with good growth were selected and seeded in 24-well plates at a density of 1×104 cells/well, followed by culture in an incubator at 37°C with 5% CO2 and saturated humidity. When the cells grew to approximately 50% confluence, cell transfection was performed according to the instructions of the Lipofectamine 3000 transfection kit (Invitrogen, USA). miRNA-23b-mimic (pro-miR-23b), psiCHECK2-NOTCH1-WT1 or psiCHECK2-NOTCH1-MUT1, psiCHECK2-TGFβ2-WT1 or psiCHECK2-TGFβ2-MUT1 and miRNA-NC (negative control) were transfected into HEK-293T cells. Similarly, miR-133 mimic (pro-miR-133), psiCHECK2-NOTCH1-WT2 or psiCHECK2-NOTCH1-MUT2, psiCHECK2-TGFβ2-WT2 or psiCHECK2-TGFβ2-MUT2 and miRNA-NC (negative control) were transfected into HEK-293T cells. The medium was replaced with fresh medium 6 h after transfection. Twenty-four hours after transfection, the relative luciferase activities were determined using the Dual-Glo Luciferase Assay System (Promega, USA). The assay was performed in triplicate for three independent trials.
Cell transfection
SDFs were cultured at 37°C in DMEM (Invitrogen, Carlsbad, CA, USA) supplemented with 10% FBS (Invitrogen, Carlsbad, CA, USA), 1.5 mM L-glutamine (Invitrogen, Carlsbad, CA, USA), 100 U/mL penicillin (Invitrogen, Carlsbad, CA, USA), and 100 mg/mL streptomycin (Invitrogen, Carlsbad, CA, USA) in a humidified incubator in an atmosphere containing 5% CO2 (Thermo, Waltham, MA, USA). When the cells grew to approximately 50% confluence, they were transfected with 200 nM synthetic oar-miR-23b inhibitor (anti-miR-23b) and oar-miR-133 inhibitor (anti-miR-133), oar-miR-23b mimic (pro-miR-23b) and oar-miR-133 mimic (pro-miR-133), or miRNA negative controls (mimic-NC and inhibitor-NC) using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA). The cells were harvested 48 h after transfection and used for further analyses.
Cell proliferation assay
A CCK-8 assay (Beyotime Institute of Biotechnology) was used to analyze the cell proliferation rate. Oar-miR-23b mimic, oar-miR-23b inhibitor, mimic-NC, inhibitor-NC, oar-miR-133-mimic, and oar-miR-133 inhibitor were transfected. Twenty-four hours after the SDFs were transfected, they were digested with EDTA-0.25% trypsin, and 600 µL/well (approximately 2×103 cells/mL) was seeded in a 24-well plate for culture, and 4 time points were established for each group: 24 h, 48 h, 72 h and 96 h. After being cultured for 24, 48, 72 h and 96 h, 60 µL of CCK-8 solution was administered to each well. Then, after being cultured for another 2 h, the supernatants were transferred to a 96-well plate (100 µL/well), and six replicates were established for each sample. Then, the absorbance was measured at 450 nm with a microplate reader, and the cell proliferation curve was generated according to the OD values.
Cell apoptosis analysis by Annexin V-FITC staining
SDFs in the logarithmic phase of growth and in good growth condition were collected, seeded in 6-well plates at a concentration of 5×105 cells/well, and cultured overnight at 37℃ in a 5% CO2 incubator. The cells were treated according to the following groups: oar-miR-23b mimic; oar-miR-23b inhibitor; mimic-NC; inhibitor-NC; oar-miR-133-mimic; and oar-miR-133 inhibitor. After 48 h of treatment, the instructions of the Annexin V-FITC/PI apoptosis detection kit were followed, and flow cytometry was used for analysis.
Transwell cell migration assay
SDFs were harvested at 48 h posttransfection (oar-miR-23b mimic, oar-miR-23b inhibitor, mimic-NC, inhibitor-NC, oar-miR-133 mimic, or oar-miR-133 inhibitor), and the cells were counted. Transwell chambers (Corning, NY, USA) were then placed in 24-well plates. A 200 µL cell suspension (3×105 cells/mL) was added to the Transwell chamber. The cells were incubated for 24 h in a humidified incubator (37°C/5% CO2). SDFs were then fixed with cold 70% ethanol and incubated at room temperature for 1 h. Then, the cells were stained with 0.5% crystal violet dye, photographed and counted (10ⅹ10, 3 pictures/group).
Flow cytometry analysis
SDFs were harvested at 48 h posttransfection (oar-miR-23b mimic, oar-miR-23b inhibitor, mimic-NC, inhibitor-NC, oar-miR-133 mimic, or oar-miR-133 inhibitor). The cells were subsequently resuspended in precooled PBS and fixed overnight at 4°C in precooled 70% ethanol. SDFs were washed three times with PBS, and then, the cells were centrifuged for 5 min at 1,500 rpm and resuspended in 200 µL of PBS. To each sample, 10 µL of RNase (10 mg/mL) was added and then incubated at 37°C for 30 minutes. Then, 10 µL of RNaseA (400 µg/ml) was added to these cells and incubated for 30 min at 4°C in the dark. Finally, cell cycle progression was analyzed by flow cytometry.
Statistical analysis
Statistical analyses were performed using GraphPad Prism 7 software (GraphPad Software, USA). The experimental results are expressed as the mean ± standard deviation (mean ± SD). The t test was used to analyze the differences between the two groups. One-way ANOVA was performed to analyze the differences among different groups, followed by the post hoc test (least significant difference). Significant differences are denoted by * P < 0.05, and extremely significant differences are indicated by ** P < 0.01. The plots were generated using GraphPad Prism software (version 7).