A total of 3 three-years-old male alpacas were obtained from an alpaca breeding base in Shanxi, China. Animal care and skin sample collection were in accordance with the International Guiding Principles for Biomedical Research Involving Animals (http://www.encyclopedia.com/science/encyclopedias-almanacs-transcripts-and-maps/international-guiding-principles-biomedical-research-involving-animals) and approved by the Animal Experimentation Ethics Committee of Shanxi Agricultural University (Taigu, China). Two skin samples were collected from the ear and back (n=2/site) of each alpaca: One biopsy sample was selected for RNA extraction, and the other biopsy sample was selected for protein extraction; all samples were soaked in liquid nitrogen.

Alpaca fibroblasts and 293T cells were stored in liquid nitrogen at the Alpaca Biological Laboratory in Shanxi Agricultural University (Taigu, China) and the primary alpaca fibroblasts were cultured in Alpaca Biological Laboratory, Shanxi Agricultural University. 293T cells were obtained from Shanghai Bioleaf Biotech Co., Ltd. (Shanghai, China). Alpaca fibroblasts and 293T cells were cultured in 37°C, 5% CO₂, in Alpaca fibroblasts medium (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and Dulbecco's modified Eagle's medium (Thermo Fisher Scientific, Inc.), respectively.

The pmirGLO dual-luciferase miRNA target expression vector and the negative control (empty vector) were purchased from Promega Corporation (Madison, WI, USA). The pcDNA6.2-GW/EmGFP-let-7b overexpression vector based on the let-7b mature sequence; the negative control (empty vector) did not contain the let-7b sequence. The vectors were obtained from a previous deep sequencing analysis on alpaca skin (15) and were purchased from Invitrogen (Thermo Fisher Scientific, Inc., Waltham, MA, USA).

Total RNA was isolated from the six skin samples (diameter=8 mm; three samples each from the ear skin and the back skin) and the transfected and untransfected alpaca fibroblast cells by using TRIzol LS Reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols. The specific transfection method is mentioned below in ‘Alpaca fibroblast culture and transfection of the let-7b eukaryotic expression vector’. Total RNA concentration was determined with a NanoDrop 1000 spectrophotometer (NanoDrop Technologies; Thermo Fisher Scientific, Inc.), and the integrity of total RNA was confirmed by 1% agarose gel electrophoresis and ethidium bromide staining for 5 min at room temperature, then rinse with water at room temperature. cDNA was synthesized using a PrimeScript II First-Strand cDNA Synthesis kit (Takara Biotechnology Co., Ltd., Dalian, China) according to the manufacturer's protocols. The PCR product of EDA was 202 bp, as confirmed by 1% agarose gel and 2000 DNA Marker; the EDA gene was sequenced by the Beijing Genomics Institute (Shenzhen, China). Gene sequencing analysis revealed that the alpaca EDA gene was homologous with other species [published on National Center for Biotechnology Information (NCBI), Bethesda, MD, USA] by NCBI-BLAST https://blast.ncbi.nlm.nih.gov/Blast.cgi; accession nos. XM_015250693.1 XM_014566996.1, XM_010978765.1, XM_010973676.1, XM_008155957.1, XM_008155955.1, XM_004871764.2, XM_004871761.2, XM_012643114.1 and XM_012643097.1. Then qPCR was conducted with SYBR Premix Ex Taq II (Perfect Real Time; Takara Biotechnology Co., Ltd.) according to the manufacturer's protocols. The primer sequences used were: EDA, forward, 5′-GTGGACGGCACCTACTTCATCTAT-3′, reverse: 5′-GCACCATCTTCACAGCGATCTTCT-3′; EDA expression was normalized to 18S rRNA. The reaction conditions constituted pre-denaturation: 95°C for 10 min; denaturation: 95°C for 15 sec; annealing: 63°C for 30 sec; extension 72°C 30 sec; 40 cycles; 95°C 15 sec, 60°C 1 min and 95°C, 15 sec. and the relative expression level was calculated using the 2^−ΔΔCq method (16).

Total protein content from the six skin samples (diameter=8 mm; three samples each from the ear skin and the back skin), the transfected and untransfected alpaca fibroblasts were extracted with a protein extraction kit (radioimmunoprecipitation lysis buffer, Beyotime Institute of Biotechnology, Haimen, China) according to the manufacturer's protocols. The specific transfection method is mentioned below in ‘Alpaca fibroblast culture and transfection of the let-7b eukaryotic expression vector’. The protein concentrations were determined using a NanoDrop 1000 spectrophotometer (NanoDrop Technologies; Thermo Fisher Scientific, Inc.). Equal amounts (300 µg/lane) of protein from alpaca skin samples and equal amounts (500 µg/lane) of protein from cells were separated by 10% SDS-PAGE and transferred onto polyvinylidene fluoride membranes (constant pressure 100 V, 1 h). The membranes were blocked with 5% nonfat milk (w/v) in TBST (TBS with 0.1% Tween 20) at room temperature for 1 h. Membranes were incubated with primary antibodies against EDA (1:200, ab125233, Abcam, Cambridge, UK) and anti β-actin antibody (1:1,500; CW0096 M, CWBIO, Beijing, China) at 4°C overnight. Following washing in TBST, membranes were incubated with the goat anti-rabbit horseradish-peroxidase (HRP) secondary antibody (1:8,000 in 3% TBST; bs-0295G-HRP, BIOSS, Beijing, China) and the goat anti-mouse (HRP) secondary antibody (1:5,000 in 3% TBST; bs-0296G-HRP, BIOSS) for 1 h at 37°C. Membranes were washed with TBST and protein bands were visualized with an Enhanced Chemiluminescence Western Blot kit (CWBIO). EDA signal intensity was quantified using ImageJ (ImageJ Launcher 1.4.3.67, National Institutes of Health, Bethesda, MD, USA) and Quantity One software (Quantity One 4.6.2.70, Bio-Rad Laboratories Inc, Hercules, CA, USA); relative expression levels were normalized to β-actin.

The 3′-untranslated region (UTR) of EDA from Alpaca back and ear skin samples was amplified by PCR using the following primers to construct the dual-luciferase vector pmirGLO-EDA-3′-UTR: Forward (SacI restriction site underlined) 5′-CGAGCTCGACCCTGAATCCGTACTTGG-3′, reverse (XbaI restriction site underlined) 5′-GCTCTAGAGCAAATCCCTCTGTCCTATCCTC-3′. The reaction conditions were: Pre-denaturation: 94°C, 2 min; denaturation: 94°C, 30 sec; annealing 53°C, 30 sec; extension 72°C, 30 sec; 35 cycles; end extension: 72°C, 2 min. A 2×Taq MasterMix (CWBIO) was used, and composed of Es Taq DNA Polymerase, Mg2 +, dNTPs and PCR stabilizer and enhancer. The amplified product (469 bp) was connected to the T-Vector pMD19 (Takara Biotechnology Co., Ltd.), and then the EDA-3′-UTR-T-Vector pMD19 and the pmirGLO vector (Promega Corporation) were double digested by the SacI and XbaI. Finally, the two digested products were ligated with T4 ligase. The dual-luciferase recombinant vector was constructed. The empty pmirGLO vector (368.7 µg/µl) and let-7b negative control (349.6 ng/ul) served as negative controls. Untransduced 293T cells were plated in 24-well plates to ~60% confluence and transfected with the pmirGLO-EDA-3′-UTR (422.3 ng/µl; pmirGLO-EDA) vector and the pcDNA6.2-GW/EmGFP-let-7b (348.4 ng/µl) overexpression vector using the Attractene Transfection Reagent (Qiagen, Shanghai, China). Dual-luciferase assays (Promega Corporation) were performed 48 h post-transfection according to the manufacturer's protocols, and luciferase activity was determined using a GloMax96 Microplate Luminometer (Promega Corporation). Firefly and Renilla luciferase served as the experimental and control, respectively. The firefly/Renilla ratios were normalized to a negative control and that of an empty plasmid control.

Alpaca fibroblasts were cultured in six-well plates at 37°C and 5% CO₂ to ~60% confluence; the cells were transfected with 1.2 µg of the let-7b expression vector (treatment group) by using the Attractene Transfection Reagent (Qiagen) according to the manufacturer's protocols. Untransfected cells (cell control) incubated in Attractene Transfection Reagent (Qiagen) and cells transfected with let-7b negative control expression vectors served as negative controls. Following 48 h incubation, fluorescence from the GFP was observed under a fluorescence microscope (Leica Microsystems GmbH). The cells were collected for total RNA and protein extraction for the analysis of EDA expression by RT-qPCR and western blotting, respectively as aforementioned.

Alpaca fibroblasts were seeded onto slides in 24-well plates at 37°C and 5% CO₂ to ~60% confluence. The cells were transfected with 0.4 µg of the let-7b expression vector (treatment group) by using the Attractene Transfection Reagent (Qiagen) according to the manufacturer's protocols, and cells incubated in Attractene Transfection Reagent (Qiagen) served as an untransfected control. Following 48 h incubation (37°C, 5% CO₂), the fibroblasts were processed for immunocytochemistry as previously described (17). Cells were fixed with 4% paraformaldehyde for 30 min at room temperature, incubated in 3% hydrogen peroxide at 37°C for 10 min, blocked with 5% bovine serum albumin (Beijing Solarbio Science & Technology, Beijing, China) at 37°C for 30 min and incubated with anti-EDA rabbit polyclonal antibody (1:30, ab198022, Abcam Cambridge, UK) at 4°C for 14 h; cells only for the negative staining control were incubated in PBS in similar conditions. HRP-conjugated goat anti-rabbit immunoglobulin G (SP Rabbit HRP kit, CWBIO) was used as a secondary antibody and cells were incubated at 37°C for 30 min. 3–3′diaminobenzidine (DAB) was used with the SP Rabbit HRP kit (CWBIO), which served as a positive control, containing DAB-A (20×) and DAB-B (1×) reagents; DAB produces a brown precipitate following catalysis by HRP. Cells of the -positive control group were incubated in DAB ~3 min in the dark at room temperature and subsequently counterstained with hematoxylin for ~5 min at room temperature. The slides were mounted, cover-slipped and evaluated under a Leitz DMRB microscope (Leica Microsystems GmbH), five fields were examined per slide.

Data are presented as the mean ± standard deviation of three replicates. Data were analyzed using SPSS v17.0 software (SPSS, Inc., Chicago, IL, USA). Multiple comparison analyses were performed by one-way analysis of variance followed by a Newman-Keuls post hoc test. P<0.05 was considered to indicate a statistically significant difference.

RT-qPCR results demonstrated that the amplification curves of EDA and 18S rRNA were smooth; the inflection points were clear with marked repeatability, and the melting curves revealed a single peak, indicating that there was no nonspecific amplification during the amplification process. EDA mRNA expression levels in ear skin were relatively 2.425 higher compared with expression in the back skin (Fig. 1).

EDA protein expression levels were detected in alpaca ear and back skin by western blot. The molecular size of the EDA protein was 41 kDa, whereas that of the β-actin protein was 43 kDa (Fig. 2A). The relative protein expression levels in the ear and back skin were 0.9254±0.0473 and 0.6145±0.0582, respectively. EDA protein expression in alpaca ear skin was 1.506-fold higher compared with expression in back skin (Fig. 2B).

Our previous study reported that let-7b may be involved in regulating hair growth and was differentially expressed in alpaca skin from various parts of the body with different fiber qualities (6). Previous bioinformatics predictions using the publicly available algorithm from TargetScan release 4.1 (www.targetscan.org) indicated that EDA may be a target of let-7b (Fig. 3) (6). In the present study, a dual-luciferase recombinant vector that contained the 3′-UTR of EDA was constructed by combining target prediction algorithms. The firefly/Renilla ratio collected from the experimental group (cells transfected with pmirGLO-EDA-3′-UTR and let-7b overexpression vector) significantly decreased, whereas of the control groups (cells transfected with pmirGLO and let-7b negative controls) did not change (Fig. 4).

Alpaca fibroblasts were transfected with the let-7b overexpression and negative control vectors, and cultured under standard conditions, to further evaluate let-7b regulation of EDA. Relative EDA mRNA expression levels were determined by RT-qPCR (Fig. 5), and protein expression levels were detected through western blot analysis (Fig. 6). The results demonstrated that let-7b overexpression resulted in decreased EDA expression at both the mRNA and protein level. let-7b transfected cells exhibited a 1.914-fold reduction in EDA mRNA quantity compared with the control groups, whereas the EDA protein expression level was 1.589-fold lower in the let-7 overexpressing cells, compared with the control groups.

EDA expression in fibroblast cells. Alpaca fibroblasts were transfected with the let-7b overexpression vector. Positive reactivity was noted as predominantly fibroblastic cytoplasmic staining, and the negative staining control cells exhibited no specific immunostaining of fibroblasts as EDA was not expressed (Fig. 7). Immunoreactivity staining was barely detected in the let-7b overexpression vector-transfected group, which was consistent with the results of RT-qPCR and western blotting.