The porcine S-V cells derived from subcutaneous adipose tissue were placed in culture flasks for clone expansion. As shown in the Figure 1(A), the proliferation continued from days 1 to 5 till confluence of the cells. Differentiation was initiated at day 7 and terminated at day 17 (Figure 1(B)). The detectable mRNA expression of nine adipocyte-specific marker genes in the terminal differentiation adipocytes (Figure 1(C)) and evidence of the morphological characteristics after ORO staining (Figure 1(B), right), confirmed that the porcine preadipocytes had differentiated to bona fide adipocytes.

We obtained high transfection efficiency (~90% Figure 2(A)) as measured by the uptake of the FAM-labeled delivery control at a concentration of 100 nM in Lipofectamine 2000 (2:1, v/v). The transfected cells continued to exhibit normal viability when compared with the control groups (P = 0.139, Figure 2(B)). These results show that lipid-mediated miRNA transfection of fully differentiated porcine adipocytes took place high efficiency and with no detectable cytotoxicity, making them suitable for use in the subsequent analysis.

To investigate the potential functions of miRNAs in the lipid metabolism of porcine adipocytes, we performed over-expression and knockdown experiments by direct transfection of short double-stranded RNAs (miRNA mimics) and their OMe-modified antisense oligonucleotides (miRNA inhibitors). We next investigated the influence of miRNA on phenotypes of pig mature adipocytes via adipogenesis (deposition of TG) and adipolysis (TG are broken down to glycerol and FFA). Four high confidence standard curves (R² > 0.99, Figure 3) were obtained for concentration calculation.

As shown in Figure 4(A), a lower TG concentration in cell lysates (P < 0.001), and higher glycerol and FFA concentrations both in cell lysates and in culture medium (P < 0.001) were found in miR-27a mimic group compared with the concentrations in the negative control. As expected, the opposite results were observed when miR-27a inhibitor groups were compared with the control group. For the miR-143 mimic group (Figure 4(B)), in contrast to the results for miR-27a, a higher TG concentration in cell lysates (P < 0.001) and lower glycerol and FFA concentrations both in cell lysates and in the culture medium (P < 0.001) were found when this group was compared with the negative control. In addition, the results observed in the miR-143 inhibitor groups were the opposite of those for the mimic group.

These results indicated that miR-27a suppressed adipocyte differentiation, while miR-143 promoted adipocyte differentiation, confirming previous reports in human and rodent [7,8]. During adipogenesis, the abundance of miR-27a was inversely correlated with that of adipogenic marker genes, such as PPARγ and adiponectin, and the abundance of miR-27a in the mature adipocyte fraction of obese mice was down-regulated compared with that of the leaner mice [7]. It has been reported that miR-143 is up-regulated after the induction of differentiation in human preadipocytes and in mouse 3T3-L1 cells [8,9], and the higher abundance of miR-143 has also been detected in the mesenteric fat of high-fat diet-induced obese mice compared with the normal controls [15]. Furthermore, we found that there is good correlation of the concentrations of glycerol (r = 0.981, P = 5.47 × 10⁻⁴) and FFA (r = 0.975, P = 1.02 × 10⁻⁵) between the cell lysates and the culture medium (Figure 5), which provides a more comprehensive index for lipid metabolism.

Seven-day-old Taihu piglets were killed by exsanguination in a manner approved by the Sicuan Agricultural University Institutional Animal Care and Use Committee. Stromal-vascular cells (S-V cells) were isolated according to published protocols [16,17] with the following modifications: Subcutaneous adipose tissue was collected from the neck and back of the piglets and rinsed with serum-free medium (DMEM/F-12 medium supplemented with 15 mM NaHCO₃, 100 U penicillin/mL and streptomycin). The tissue mass was cut with scissors into fine pieces and digested with type-IV collagenase (DMEM/F-12 + 20 g/L BSA+1 g/L IV type collagenase) at 37 °C in a shaking water bath for 1 h, Then, DMEM/F-12 medium containing 10% fetal bovine serum (FBS) was added to stop digestion. The solution was filtered through sterile nylon mesh (100 μm pore size) to remove undigested tissues. The filtrate was centrifuged at 800 × g for 8 min to separate the floating adipocyte cells from the pellet of S-V cells. The S-V cells were then incubated with erythrocyte lysis buffer (0.154 M NH₄Cl, 10 mM KHCO₃, 0.1 mM EDTA) at room temperature for 10 min [18], followed by centrifugation at 500 × g for 5 min. The S-V cell pellet was washed with DMEM/F-12, centrifuged, and resuspended in plating medium (20% FBS, DMEM/F-12). Finally, cells were seeded in culture plates at a density of 5 × 10⁴ cells/cm² and cultured at 37 °C in a humidified atmosphere containing 5% CO₂. The medium was changed every second day.

For cell growth curve studies, preadipocytes were seeded in 96-well culture plates at a density of 10⁴/cm², and then 100 μL DMEM/F-12 medium containing 10% FBS was added to each well. At various points, medium was removed, we added 20 μL of the reagent into each well of the 96 well assay plate containing the cells in 100 μL of culture medium, and then cultured the cells for 1.5 h at 37 °C in a humidified, 5% CO₂ atmosphere. Afterwards, record the absorbance at 490 nm using a 96 well plate reader. The viable cell number is proportional to the absorbance.

Cultured preadipocytes were maintained in plating medium until confluence. Then, to induce differentiation, the cultures were exposed to medium containing 10% FBS, 5 μg/mL insulin, 0.5 mM 3-isobutyl-1-methylxanthine, 0.25 M dexamethasone and antibiotics in DMEM/F-12 for 48 h. This medium was then replaced with a lipid-filling medium (10% FBS, 5 μg/mL insulin, DMEM/F-12) to permit the adipocytes to accumulate TG. To qualitatively assess cell differentiation by microscopy, fully differentiated porcine adipocytes were fixed in 10 % formalin and stained with 0.3% Oil Red O (ORO) for lipid. In addition, the mRNA abundance of nine adipocyte-specific marker genes in porcine adipocyte was determined using a quantitative PCR (q-PCR) approach. The primers that were used for q-PCR are listed in Table 1.

Mimic and inhibitor oligonucleotides of ssc-miR-143-5p and ssc-miR-27a-3p (miRBase IDs: MIMAT0002148 and MIMAT0017374 respectively) and a negative control (with no sequence similarities to any reported porcine gene sequence) were synthesized by Ribobio (China). The 5-FAM fluorescence-labeled delivery control (Ribobio) was used to measure the transfection efficiency of porcine adipocytes. Mature adipocytes could be used for transfection in day 10, prior to miRNA transfection, the cell culture medium was replaced by a Serum-Reduced Medium, Opti-MEM I (Invitrogen, Carlsbad, CA, USA). The transfection was carried out using the lipid carrier Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Oligonucleotides (100 nM) prepared with lipid carrier (2:1, v/v) were subjected to the transfection in a 100 μL of Opti-MEM medium for each well of the standard 96-well plates. The adipocytes were incubated with the oligonucleotides/Lipofectamine complex for 6 h and then switching the Opti-MEM I medium to a growth medium. The cells were cultured for 3 days at 37 °C, and then subjected to the following analyses. In order to determine the optimal transfection conditions, the adipocyte viability was assessed at 24 h after transfection [19] using the MTT method.

The concentrations of TGs in the lysates of adipocytes, and of glycerol and FFAs in both the lysates of adipocytes and in the culture medium were measured using the commercial kits (Applygen Technologies, Beijing, China) according to the manufacturer’s instructions. The concentrations of the TGs, glycerol and FFAs were normalized to the protein content (μM/mg protein) using a bicinchoninic acid (BCA) assay kit (Pierce Chemical, Rockford, IL, USA).

The statistical significance of variations between the two variables was calculated by Student’s t-test (two-tailed). The Pearson’s correlation was used to determine the correlation of the concentrations of glycerol and FFA between the cell lysates and the culture medium. Values are mean ± S.D. (n = 3).