Animals and sample collection
Experimental animals were provided by the Datong Pig Breeding Farm (Shanxi, China). Three 90-day-old male Mashen pigs and Large White pigs were selected for slaughter. The biceps femoris, psoas, subcutaneous fat, spleen, liver, intestine, and stomach were collected from each animal and stored at − 80 °C.
Porcine preadipocyte culture and adipogenic differentiation
A seven-day-old male Mashen pig was used for isolation of porcine preadipocytes based on previously established methods . Subcutaneous adipose tissue (SAT) was digested using 0.1% type-I solution (Sigma) for 2 h at 37 °C, followed by centrifugation at 1000×g for 8 min. Afterwards, the resulting mixture was filtered through 100 μm and 40 μm mesh filters and centrifuged for another 8 min at 1000×g to obtain the porcine preadipocytes. After resuscitation, cells were cultured in DMEM (Gibco, USA) containing 10% fetal bovine serum (FBS; Gibco, USA) and 1% penicillin/streptomycin. For adipogenic differentiation, cells were treated with 1 μM dexamethasone (DEX), 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 10 mg/mL insulin, and 100 μM indomethacin for 2 days. The cells were then transferred to medium supplemented with 10% FBS and 10 mg/mL insulin for 2 days. Subsequently, they were maintained in 10% FBS for another 4 days.
RNA interference and overexpression
To inhibit expression of lncMYOZ2, a customized siRNA preparation was used. This reagent contains a mixture of three synthetic siRNAs designed to target the intronic region of lncMYOZ2, with sequences (5′–3′) AGATGAACAACTGCTCTAA, AAGCTATGGTAACTAATCC, and GGAATGAAGCAATCTGACAG. A single siRNA was used to target MYOZ2 (GACAAAGAAGATCTGACAA). The cDNA for lncMYOZ2 was obtained by gene synthesis (GeneCreate Inc., China) with flanking 5′-BamHI and 3′-XhoI sites and cloned into pCMV-N-FLAG using these restriction sites. For transfection, porcine preadipocytes were inoculated into 12-well plates and transfected with siRNA or plasmids using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA), following the manufacturer’s instructions.
RNA isolation and qPCR
Total RNA was extracted using the Trizol reagent (Life Technologies, USA) and transcribed into cDNA using a PrimeScript first strand cDNA synthesis kit (Takara Bio, Japan). Quantitation of mRNA levels by qPCR was performed on a real-time PCR system using SYBR Premix Ex Taq II (Takara Bio, Japan). The mean of the triplicate cycle thresholds (Ct) of the target gene was normalized to the mean of the triplicate Ct of the reference (18 s RNA) gene, using the 2−ΔΔCt method, to yield relative gene expression levels. Primer sequences are listed in Supplementary Fig. S1.
Cytoplasmic and nuclear fractionation
Cytoplasmic and nuclear fractions were isolated using the PARIS Kit (Thermo Fisher Scientific, USA) according to the manufacturer’s instructions. The RNA was extracted and reverse-transcribed into cDNA, then nuclear and cytoplasmic expression of lncMYZO2 were detected by qPCR. The positive controls were GAPDH (cytoplasmic) and U6 (nuclear).
Sequence analysis and positional information comparison of lncMYZO2 were performed using the NCBI (https://www.ncbi.nlm.nih.gov/) and Ensembl (http://asia.ensembl.org/index.html) websites. The CPC tool (http://cpc.cbi.pku.edu.cn/) was used to predict the protein-coding properties of lncMYOZ2.
Cells were extracted using a protein lysis buffer supplemented with protease inhibitor cocktail (Boster, China). Protein concentration was determined using a BCA kit (Boster, China). Then, samples were loaded at 20–30 μg/lane and separated on a 10% precast SDS–PAGE gel, followed by transfer onto PVDF membrane (Millipore, USA). After blocking with 5% skimmed milk powder (Sigma–Aldrich, USA) for 3 h, the membrane was incubated overnight at 4 °C with the appropriate primary antibody: against PPARγ (A0270, Abclonal, China), β-actin (AC038), FABP4 (A0232), CEBP-α (A0904), MYOZ2 (A6468), or AHCY (10,757, Proteintech, China). Then, IRDye® 800CW rabbit secondary antibodies (926-32,211, LI-COR, USA) were used to detect the primary antibodies, and the membrane was imaged using a far-infrared light scanning system (LI-COR, USA).
Oil red O (ORO) staining
Eight days after adipogenic induction, cells were washed twice with phosphate buffered saline (PBS) and fixed in 4% paraformaldehyde for 30 min at room temperature. For staining, the cells were washed again with PBS and stained with freshly diluted ORO (Solarbio, China) for 15 min. After an additional wash with PBS, the cells were then imaged using a microscope (Leica, Germany). For quantitation of lipids, the dye retained in the cells (after washing) was eluted into isopropanol and the OD value at 510 nm was measured using a microplate reader (BioTEK, USA).
RNA pull-down and RNA immunoprecipitation (RIP) assay
The sense and antisense strands of lncMYOZ2 were used as template for an in vitro transcription kit (GeneCreate, China) to obtain the target RNA. Following in vitro transcription, the biotin-labeled probes were incubated using a Pierce RNA 3′-End Desthiobiotinylation Kit (Thermo Fisher Scientific, USA). The biotin-labeled sequence were incubated with streptavidin coupled dynabeads (Invitrogen, USA) for 1 h at RT, and then with the lysate of porcine preadipocyte overnight at 4 °C, after which were separated by elution using 1D-SDS–PAGE and silver staining. Differentially expressed range in candidate region betwween two samples (sense and antisense) were then analyzed by mass spectrometry to assess the protein composition, which these proteins were then subjected to GO and KEGG enrichment analysis [17,18,19].
The RIP assay was performed using a RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, Bedford, MA, USA), in accordance with the manufacturer’s instructions. Briefly, cell lysates were incubated with magnetic beads conjugated with negative control (IgG) or anti-AHCY antibody. The immunoprecipitated RNAs were then extracted and analyzed by qPCR to confirm the enrichment of binding targets.
Genomic DNA was extracted from porcine preadipocytes using the phenol/chloroform extraction method, and bisulfite conversion was carried out with the EZ DNA Methylation Kit (ZYMO, USA) as per the manufacturer’s instructions. The promoter sequence of the pig MYOZ2 gene was obtained from the NCBI website, and the MethPrimer (http://www.urogene.org/methprimer/) website was used to predict the methylation sites of MYOZ2. Thus, bisulfite-sequencing PCR (BSP) primers (F: GTTGGTGTTTTATTGTATGGTTGATT; R: ACAATCCCACTCCTAAACATCTATC) were designed and synthesized. The bisulfite-treated DNA was then amplified by PCR followed by cloning and sequencing. The sequencing results were analyzed using QUMA (http://quma.cdb.riken.jp/). The results are presented using a black solid circle to indicate that a site is methylated; a white circle to indicate that a site is unmethylated; and “×” to indicate a vacancy.
All experiments were carried out with three biological replicates, and statistical analyses were performed using SPSS 22.0. Comparisons between two samples were made using the Student’s t-test, where P < 0.05 (*) and P < 0.01 (**) indicate statistically significant differences. Comparisons between three or more samples were performed using one-way analysis of variance (ANOVA), and Duncan’s method was used for multiple comparisons. Different capital letters indicate P < 0.05 (*), where the difference is statistically significant.