PDGFRα Regulated by miR-34a and FoxO1 Promotes Adipogenesis in Porcine Intramuscular Preadipocytes through Erk Signaling Pathway

Suitable intramuscular fat (IMF) content improves porcine meat quality. The vital genes regulating IMF deposition are necessary for the selection and breeding of an IMF trait. However, the effect and mechanism of PDGFRα on IMF deposition are still unclear. Here, PDGFRα is moderately expressed in porcine longissimus dorsi muscle (LD), whereas it highly expressed in white adipose tissue (WAT). Moreover, PDGFRα-positive cells were located in the gaps of LD fibers which there were IMF adipocytes. Compared with 180-day-old and lean-type pigs, the levels of PDGFRα were much higher in one-day-old and fat-type pigs. Meanwhile the levels of PDGFRα gradually decreased during IMF preadipocyte differentiation. Furthermore, PDGFRα promoted adipogenic differentiation through activating Erk signaling pathway. Based on PDGFRα upstream regulation analysis, we found that the knockdown of FoxO1 repressed lipogenesis by downregulating PDGFRα, and miR-34a inhibited adipogenesis through targeting PDGFRα. Collectively, PDGFRα is a positive regulator of IMF deposition. Therefore, we suggest that PDGFRα is a possible target to improve meat quality.


Introduction
The content of intramuscular fat (IMF) is considered as a crucial indicator of porcine meat quality [1]. Increasing the IMF content can improve the flavor and eating quality of meat [2]. The number of IMF progenitors is associated with the IMF deposition [3]. It is well-known that adipocytes are derived from mesenchymal stem cells (MSCs), which commit into adipocyte lineage and finally give rise to preadipocytes [4][5][6]. The preadipocytes, which have lost the ability to differentiate to other cell lineages, are positive for mesenchymal stem cell markers and negative for blood and endothelial cell markers [7]. Then, preadipocytes go through terminal differentiation to mature adipocytes. The cell surface factors CD24, CD34 and Sca1 are used to isolate and analyze adipocyte precursors from white adipose tissue (WAT) through fluorescence-activated cell sorting (FACS) in mice or humans [5,8]. Otherwise, the biomarker of IMF adipocytes is still unclear.
Platelet-derived growth factor receptor α (PDGFRα) is important for embryonic organogenesis and development by effecting the differentiation and function of specialized mesenchymal cells [9]. In the human heart, PDGFRα positive cells are cardiac progenitors, which contribute predominantly to the mesenchymal compartments and vascular [10]. Because PDGFRα expressed in adipogenic stromal cells of adipose tissues, it has been implicated in the adipocyte lineage [11]. In skeletal muscle, the fat and muscle do not originate from the same progenitor cell [12]. Pericytes are thought to act like MSCs [13,14], which express PDGFRα and may contribute to fat deposition in skeletal muscle [15]. In contrast, skeletal muscle satellite cells are committed solely to myogenesis [16]. Another group reported that the PDGFRα-positive mesenchymal progenitors were located in the muscle interstitium, which are the major contributors to IMF deposition in skeletal muscle of mice [17]. Therefore, PDGFRα may be a potential cell surface biomarker of IMF preadipocyte progenitors. However, the effect and regulatory mechanism of PDGFRα on IMF deposition is still unclear.
MiRNAs (MicroRNAs) are non-coding RNAs, approximately 18-22 nucleotides in length, which regulate gene expression at the post-transcriptional level [18]. PDGFRα expression can be inhibited by some miRNAs in tumors. For example, miR-34c inhibits lung cancer proliferation, migration and invasion by targeting PDGFRα/β [19]; miR-34a affects the growth of pulmonary artery smooth muscle cells in human by targeting PDGFRα [20]. PDGFRα can also be regulated by miRNAs in nontumor cells [21]. Therefore, we presume that miR-34a may affect PDGFRα expression by targeting it during porcine IMF preadipocyte differentiation.
The FoxO transcription factors have a wide range of biological functions, including the regulation of cell proliferation, apoptosis, and differentiation [22]. FoxO family members are widely expressed in the mammalian organizations. FoxO proteins are responsible for maintaining the basal expression levels of PDGFRα, as well as the accumulation of PDGFRα under serum starvation condition [23]. Depletion of endogenous FoxO proteins in neuroblastoma cells results in a disregulated differentiation phenotype, which can be rescued by PDGFRα expression. PDGFRα is a critical downstream target gene of the FoxO1 proteins [24]. However, the relationship between PDGFRα and FoxO1 needs further clarification in porcine IMF deposition.
In this study, we elucidated the effect and regulatory mechanism of PDGFRα on pig IMF deposition. Therefore, we suggested that PDGFRα could serve as a positive regulator associated with meat quality for pig selection and breeding of IMF trait.

Porcine Tissue Expression Profile of PDGFRα
To elucidate the tissue expressions of PDGFRα in pigs, the levels of its mRNA and protein were detected in various tissues including heart, liver, spleen, kidney, LD and WAT. The results showed that the levels of PDGFRα, both mRNA and protein, were abundant in WAT, moderate in LD and the least in the liver ( Figure 1A-C). muscle, the fat and muscle do not originate from the same progenitor cell [12]. Pericytes are thought to act like MSCs [13,14], which express PDGFRα and may contribute to fat deposition in skeletal muscle [15]. In contrast, skeletal muscle satellite cells are committed solely to myogenesis [16]. Another group reported that the PDGFRα-positive mesenchymal progenitors were located in the muscle interstitium, which are the major contributors to IMF deposition in skeletal muscle of mice [17]. Therefore, PDGFRα may be a potential cell surface biomarker of IMF preadipocyte progenitors. However, the effect and regulatory mechanism of PDGFRα on IMF deposition is still unclear. MiRNAs (MicroRNAs) are non-coding RNAs, approximately 18-22 nucleotides in length, which regulate gene expression at the post-transcriptional level [18]. PDGFRα expression can be inhibited by some miRNAs in tumors. For example, miR-34c inhibits lung cancer proliferation, migration and invasion by targeting PDGFRα/β [19]; miR-34a affects the growth of pulmonary artery smooth muscle cells in human by targeting PDGFRα [20]. PDGFRα can also be regulated by miRNAs in nontumor cells [21]. Therefore, we presume that miR-34a may affect PDGFRα expression by targeting it during porcine IMF preadipocyte differentiation.
The FoxO transcription factors have a wide range of biological functions, including the regulation of cell proliferation, apoptosis, and differentiation [22]. FoxO family members are widely expressed in the mammalian organizations. FoxO proteins are responsible for maintaining the basal expression levels of PDGFRα, as well as the accumulation of PDGFRα under serum starvation condition [23]. Depletion of endogenous FoxO proteins in neuroblastoma cells results in a disregulated differentiation phenotype, which can be rescued by PDGFRα expression. PDGFRα is a critical downstream target gene of the FoxO1 proteins [24]. However, the relationship between PDGFRα and FoxO1 needs further clarification in porcine IMF deposition.
In this study, we elucidated the effect and regulatory mechanism of PDGFRα on pig IMF deposition. Therefore, we suggested that PDGFRα could serve as a positive regulator associated with meat quality for pig selection and breeding of IMF trait.

Porcine Tissue Expression Profile of PDGFRα
To elucidate the tissue expressions of PDGFRα in pigs, the levels of its mRNA and protein were detected in various tissues including heart, liver, spleen, kidney, LD and WAT. The results showed that the levels of PDGFRα, both mRNA and protein, were abundant in WAT, moderate in LD and the least in the liver ( Figure 1A-C).

Identification of PDGFRα-Positive Cells in Porcine LD
To study a PDGFRα-positive cell and its localization in porcine LD, immunofluorescent staining was performed with a PDGFRα antibody. The results showed that PDGFRα-positive cells were in LD ( Figure 2A) and the number of PDGFRα-positive cells was significantly more numerous in fat-type pigs than in lean-type pigs ( Figure 2B). Interestingly, we further found that PDGFRα-positive cells were located in the gaps of LD fibers in which there were IMF adipocytes ( Figure 2C). It was clear that the IMF content was greater in LD of fat-type pigs than that of lean-type pigs ( Figure 2D). Furthermore, the results indicated that PDGFRα was located at mature adipocyte membrane ( Figure 2E). Therefore, the above results indicated that PDGFRα may be a vital biomarker of pig IMF deposition.

Identification of PDGFRα-Positive Cells in Porcine LD
To study a PDGFRα-positive cell and its localization in porcine LD, immunofluorescent staining was performed with a PDGFRα antibody. The results showed that PDGFRα-positive cells were in LD ( Figure 2A) and the number of PDGFRα-positive cells was significantly more numerous in fat-type pigs than in lean-type pigs ( Figure 2B). Interestingly, we further found that PDGFRα-positive cells were located in the gaps of LD fibers in which there were IMF adipocytes ( Figure 2C). It was clear that the IMF content was greater in LD of fat-type pigs than that of lean-type pigs ( Figure 2D). Furthermore, the results indicated that PDGFRα was located at mature adipocyte membrane ( Figure 2E). Therefore, the above results indicated that PDGFRα may be a vital biomarker of pig IMF deposition.

Comparison on the Levels of PDGFRα in Different Ages and Types of Pigs
Because the different day-old and types of pigs is implicated in meat quality, levels of PDGFRα mRNA and protein were detected in LD using real time qPCR and western blot. As shown in Figure  3A, the mRNA expression of PDGFRα was significantly higher in 1-day-old pigs than that in 180-day-old pigs. Likewise, the protein level was also higher in 1-day-old pigs than that in 180-day-old pigs ( Figure 3B,C). Moreover, PDGFRα mRNA and protein expressions of fat-type pigs were more abundant than that of lean-type pigs ( Figure 3D-F).

Comparison on the Levels of PDGFRα in Different Ages and Types of Pigs
Because the different day-old and types of pigs is implicated in meat quality, levels of PDGFRα mRNA and protein were detected in LD using real time qPCR and western blot. As shown in Figure 3A, the mRNA expression of PDGFRα was significantly higher in 1-day-old pigs than that in 180-day-old pigs. Likewise, the protein level was also higher in 1-day-old pigs than that in 180-day-old pigs ( Figure 3B,C). Moreover, PDGFRα mRNA and protein expressions of fat-type pigs were more abundant than that of lean-type pigs ( Figure 3D-F).

Time Course Expression of PDGFRα during Porcine IMF Preadipocyte Differentiation
To verify whether PDGFRα is a potential molecular regulator of IMF deposition, porcine IMF preadipocytes were isolated and cultured using differential adhesion method, and induced to adipogenic differentiation. The expression profile of FABP4 confirmed that IMF preadipocytes differentiated into mature adipocytes ( Figure 4B,C). As expected, the levels of PDGFRα gradually decreased during IMF preadipocytes differentiation ( Figure 4A-C).

Time Course Expression of PDGFRα during Porcine IMF Preadipocyte Differentiation
To verify whether PDGFRα is a potential molecular regulator of IMF deposition, porcine IMF preadipocytes were isolated and cultured using differential adhesion method, and induced to adipogenic differentiation. The expression profile of FABP4 confirmed that IMF preadipocytes differentiated into mature adipocytes ( Figure 4B,C). As expected, the levels of PDGFRα gradually decreased during IMF preadipocytes differentiation ( Figure 4A-C).

Knockdown of Modulated-FoxO1 PDGFRα Inhibits Adipogenesis through Erk Signaling Pathway
To explore the effect and mechanism of PDGFRα on IMF deposition, the knockdown experiments of PDGFRα and FoxO1 were performed in porcine IMF preadipocytes. The results showed that knockdown of PDGFRα significantly downregulated the mRNA levels of PDGFRα, PPARg and FABP4 ( Figure 5A). Likewise, knockdown of FoxO1 markedly reduced the mRNA levels of PDGFRα, PPARg and FABP4 ( Figure 5B). It implied that FoxO1 was an upstream regulator of PDGFRα. Oil Red O assay revealed that shPDGFRα and shFoxO1 inhibited adipogenesis, respectively ( Figure 5C). To determine whether Erk signaling pathway is involved in lipogenesis regulated by PDGFRα, the levels of Erk1/2 and p-Erk1/2 were examined. The results showed that PDGFRα knockdown repressed Erk1/2 activation by downregulating the levels of p-Erk1/2 ( Figure  5D,E-H). Based on above results, we suggested that PDGFRα, which was regulated by FoxO1, promoted lipogenesis through Erk signaling pathway in porcine IMF preadipocyte differentiation.

Knockdown of Modulated-FoxO1 PDGFRα Inhibits Adipogenesis through Erk Signaling Pathway
To explore the effect and mechanism of PDGFRα on IMF deposition, the knockdown experiments of PDGFRα and FoxO1 were performed in porcine IMF preadipocytes. The results showed that knockdown of PDGFRα significantly downregulated the mRNA levels of PDGFRα, PPARg and FABP4 ( Figure 5A). Likewise, knockdown of FoxO1 markedly reduced the mRNA levels of PDGFRα, PPARg and FABP4 ( Figure 5B). It implied that FoxO1 was an upstream regulator of PDGFRα. Oil Red O assay revealed that shPDGFRα and shFoxO1 inhibited adipogenesis, respectively ( Figure 5C). To determine whether Erk signaling pathway is involved in lipogenesis regulated by PDGFRα, the levels of Erk1/2 and p-Erk1/2 were examined. The results showed that PDGFRα knockdown repressed Erk1/2 activation by downregulating the levels of p-Erk1/2 ( Figure 5D,E-H). Based on above results, we suggested that PDGFRα, which was regulated by FoxO1, promoted lipogenesis through Erk signaling pathway in porcine IMF preadipocyte differentiation.

miR-34a Represses Lipogenesis through Targeting PDGFRα
Using mRNA TargetScan analysis, we found that PDGFRα may be targeted by miR-34a. Therefore, the experiment on PDGFRα regulated via miR-34a was performed. Overexpression efficiency of miR-34a mimics was high enough to carry out subsequent experiments ( Figure 6A). The results showed that overexpression of miR-34a dramatically suppressed mRNA levels of PDGFRα and lipogenesis genes, including PPARg and FABP4 ( Figure 6B), which was consistent with the protein level ( Figure 6D,E-I). Furthermore, Oil Red O assay revealed that preadipocyte treated with miR-34a mimics had fewer lipids ( Figure 6C). Taken together, miR-34a represses lipogenesis in IMF preadipocytes through targeting PDGFRα.

miR-34a Represses Lipogenesis through Targeting PDGFRα
Using mRNA TargetScan analysis, we found that PDGFRα may be targeted by miR-34a. Therefore, the experiment on PDGFRα regulated via miR-34a was performed. Overexpression efficiency of miR-34a mimics was high enough to carry out subsequent experiments ( Figure 6A). The results showed that overexpression of miR-34a dramatically suppressed mRNA levels of PDGFRα and lipogenesis genes, including PPARg and FABP4 ( Figure 6B), which was consistent with the protein level ( Figure 6D,E-I). Furthermore, Oil Red O assay revealed that preadipocyte treated with miR-34a mimics had fewer lipids ( Figure 6C). Taken together, miR-34a represses lipogenesis in IMF preadipocytes through targeting PDGFRα.

Discussion
Interestingly, PDGFRα moderately expressed in porcine LD, but did not express in myotubes [3] implying that PDGFRα-positive preadipocytes existed in the LD of pigs. Because PDGFRα is an important biomarker of preadipocytes from white adipose tissues [8,25], it is possible that its expression levels are much higher in SAT than in the other tissues. PDGFRα-positive cells are the origin of vascular and mesenchymal compartments in the human heart [10], and we also observed that PDGFRα was relatively high in the porcine heart. In addition, we found that PDGFRα expressed in the LD, WAT, heart, spleen and kidney of the pig, but could hardly be detected in the liver, which may be a reason why obese pigs resist fatty liver disease [26].
Abundant PDGFRα expression is a characteristic of undifferentiated MSCs [27]. In addition, it was observed that PDGFRα was downregulated after differentiation of mesenchymal progenitors [28]. Consistently, our results indicated that the levels of PDGFRα gradually decreased during IMF preadipocyte differentiation in vitro. Many genes are decreased during preadipocyte differentiation,

Discussion
Interestingly, PDGFRα moderately expressed in porcine LD, but did not express in myotubes [3] implying that PDGFRα-positive preadipocytes existed in the LD of pigs. Because PDGFRα is an important biomarker of preadipocytes from white adipose tissues [8,25], it is possible that its expression levels are much higher in SAT than in the other tissues. PDGFRα-positive cells are the origin of vascular and mesenchymal compartments in the human heart [10], and we also observed that PDGFRα was relatively high in the porcine heart. In addition, we found that PDGFRα expressed in the LD, WAT, heart, spleen and kidney of the pig, but could hardly be detected in the liver, which may be a reason why obese pigs resist fatty liver disease [26].
Abundant PDGFRα expression is a characteristic of undifferentiated MSCs [27]. In addition, it was observed that PDGFRα was downregulated after differentiation of mesenchymal progenitors [28].
Consistently, our results indicated that the levels of PDGFRα gradually decreased during IMF preadipocyte differentiation in vitro. Many genes are decreased during preadipocyte differentiation, however, they all promote adipogenic differentiation, such as KLF9, KLF4, C/EBPβ, and so on. In our study, the expression of PDGFRα was higher at day 0, and it promotes adipogenic differentiation at early phase. It is wondered whether this occurred in vivo as well. Compared with piglets, there was more IMF in the LD of adult pigs [29], but the expression of PDGFRα of adult pigs was fewer than that of piglets. We also found that the level of PDGFRα is highest in fat-type piglets. Therefore, we thought that PDGFRα-positive preadipocytes differentiated into mature adipocytes during IMF deposition with the decrease of PDGFRα expression.
Bamei is a local pig breed in China, which is a representative fat-type breed with good IMF content. In contrast, the Yorkshire is an introduced breed, which is regarded as lean-type pig breed [30,31]. Previous studies have shown that fat-type breed pigs are more inclined to IMF deposition than a lean-type pig breed [32] However, whether the number of PDGFRα-positive cells is the main cause of the IMF deposition differences between breeds has not been understood. In this study, we found that both PDGFRα expression levels and the number of PDGFRα-positive cells were significantly greater in fat-type porcine LD than in lean-type porcine LD, suggesting that the PDGFRα may be an indicator of IMF deposition in pigs.
Using PDGFRα shRNA to knockdown PDGFRα, we found that the total Erk1/2 protein level did not change, but the p-Erk1/2 reduced, indicating PDGFRα-regulated preadipocyte differentiation through the activation of Erk1/2. The Erk signaling pathway plays a pivotal role in many vital cellular processes, including proliferation and differentiation [33]. Erk1/2 facilitates the early stage of adipogenic differentiation, but needs to be turned off at the adipocyte maturation phase, suggesting a dual role of Erk1/2 in adipogenesis [34]. Our results showed that the knockdown of PDGFRα inhibited the activity of p-Erk1/2. The findings suggested that PDGFRα mediated the lipogenesis through the Erk signaling pathway in porcine IMF preadipocyte.
To further study the upstream regulation of PDGFRα and the knockdown of FoxO1 using shRNA, the results demonstrated that the knockdown of FoxO1 markedly reduced the levels of PDGFRα. FoxO1 is always considered as a negative regulator of adipogenesis [35]. However, other research considered that FoxO1 promoted adipogenesis [36]. In our study, we observed that FoxO1 promoted lipogenesis during porcine intramuscular preadipocyte differentiation by downregulating PDGFRα. The results may be due to other biological processes, of which FoxO1 contributes. It was reported that silencing FoxO1 suppressed adipocyte differentiation and LD growth via potently reducing autophagy activity [37]. In addition, using mRNA TargetScan analysis, we found that PDGFRα may be targeted by miR-34a. So we transfect miR-34a mimics into porcine IMF preadipocyte. Interestingly, the levels of both PDGFRα and p-Erk1/2 were significantly decreased. Therefore, miR-34a may directly target PDGFRα and repressed IMF deposition through inhibiting the Erk signaling pathway.
IMF is localized in the interstitial space of muscle tissue [38,39]. In the study, we found that PDGFRα may be a membrane protein of porcine IMF adipocytes and a biomarker on IMF content. Based on these results, we suggest that PDGFRα may be a candidate gene associated with meat quality for pig selecting and breeding of IMF trait.

Animal and Sample Collection
In this study, all experimental procedures were performed according to the Guide for Northwest A&F University Animal Care Committee. The experimental protocol was approved by the Departmental Animal Ethics Committee of Northwest A&F University (14-233, 10 December 2014).
Fat-type breed Bamei pigs (180 days old) were obtained from Huzhu prefecture of Qinghai province. Lean-type breed Yorkshire adult pigs (180 days old) and piglets (one day old) were provided by the experimental farm of Northwest A&F University. All pigs were killed at a slaughterhouse under the guidelines of Northwest A&F University Animal Care Committee. The heart, liver, spleen, kidney, LD and SAT were dissected, and rinsed with PBS. Samples for western blot and real-time qPCR were frozen in liquid nitrogen until analysis. For the frozen section, the samples were fixed with 4% paraformaldehyde and kept at room temperature until use.

Cell Isolation and Culture
The LD of piglets were separated sterilely, visible connective tissue was removed, then finely minced. Preadipocytes were isolated based on previously studies. In brief, muscle tissues were digested in a digestion buffer consisting of 0.2% collagenase I (270 U/mg; Gibco, Carlsbad, CA, USA) and medium/F12 (DMEM/F12) (Gibco BRL Co., LTD, San Francisco, CA, USA) in a shaking water bath for 1.5 h at 37 • C. The digest sample was filtered aseptically through 70 and 200 µm nylon mesh filters to isolate cells. Then the filtered cells were washed three times with DMEM/F12 by centrifugation at 1500 rpm for 5 min. Cells were seeded at a density of 2.5 × 10 5 cells per 35-mm culture dish in DMEM/F12 medium with 10% fetal bovine serum (Hyclone, Logan, UT, USA) supplement with penicillin (100 U/mL) and streptomycin (100 U/mL). After 1 h, cells were rinsed with DMEM/F12 medium to wipe off unadhered cells. At day 2, and after cells reach to 100% confluence, cells were cultured with DMEM/F12, supplemented with 5 µg/mL (872 nM) insulin, 1 µM dexamethasone and 0.5 mM isobutyl methylxanthine (IBMX, Sigma-Aldrich, St. Louis, MO, USA), for 2 days to induce differentiate. Then the medium was changed by DMEM/F12 with 10% FBS and 5 µg/mL insulin for another 4 days to maintain differentiation. At day 0, 3 and 6, cells were harvested for further analysis.

Real-Time Quantitative PCR
To detect the expression of miRNA and genes associated with adipogenic differentiation, the total RNA was extracted with Trizol reagent (TakaRa, Otsu, Japan). The concentration of total RNA was measured by the NanoDrop 2000 (Thermo, Waltham, MA, USA). Then we used reverse transcription kits (TakaRa, Otsu, Japan) to synthesize cDNA. For miRNA analysis, specific reverse transcription primers and procedures were used, whereas the normal process was performed for mRNA analysis. In real-time quantitative PCR, every reaction performed in triplicate using SYBR green kits on a Bio-Rad iQTM5 system. The expressions of all genes were normalized to GAPDH, but U6 small RNA was internal reference when examined the level of miR-34a. The primer sequences used for qPCR were shown in Table 1. Table 1. Primer sequences used in this study.

Western Blots
The various tissue samples of the pigs and Cells were split by radio immunoprecipitation assay (RIPA) buffer (Beyotime, China) add with protease inhibitor (Pierce, WA, USA). The total protein sample was pointed into and separated in the SDS-polyacrylamide gel. Then transferred it into a PVDF membrane (Millipore, Bedford, MA, USA). Nest, the membrane was blocked in 5% defatted milk for 2 h. After that, the membrane was incubated with primary antibodies at 4 • C overnight followed by a secondary antibody at room temperature for 1.5 h. Protein bands were exposure by chemiluminescence reagents (Millipore, Bedford, MA, USA) and quantified using the Image Lab Image Document. Following primary antibodies were used: PDGFRα (1:300; Boster, Wuhan, China), FABP4 (1:500; Santa Cruz Biotechnology, Dallas, TX, USA), β-actin (1:1000; Santa Cruz Biotechnology, Dallas, TX, USA), β-tubulin (1:500; Santa Cruz Biotechnology, Dallas, TX, USA). The secondary antibodies were anti-rabbit, anti-goat and anti-mouse antibodies (Santa Cruz Biotechnology, Dallas, TX, USA). The targeted proteins were detected using the Gel Doc XR System (Bio-Rad, Hercules, CA, USA) as the instructions of the manufacturer.

Frozen Section and HE Staining
Fixed tissues were dehydrated in 30% sucrose and sectioned (10 µm) through sliding microtome (leica, Wetzlar, German). Hematoxylin-eosin staining was realized as previously reported (Godwin, 2011). Then, the sections were observed and took pictures with microscope (Olympus, New York, NY, USA) in 100× magnification.

Immunohistochemistry
Frozen sections were treated with 3% H 2 O 2 for 10 min to eliminate endogenous oxidase activities and washed three times with PBS. Then, sections were treated with 0.5% TritonX-100/TBS at room temperature for 10 min, and washed three times using distilled water. Antigens were unmasked by high-temperature antigen retrieval (10 min boiling in 0.05 mM Tris/EDTA buffer (pH = 9) and cooling slowly at room temperature. They were then washed three times with TBS. The 5% BSA/TBS as a blocking solution was incubated for 2 h at room temperature. Sections were incubated with primary antibodies of PDGFRα (1:100), and diluted in blocking solution at 4 • C overnight. They were then washed three times with TBST and incubated with anti-rabbit red fluorescent secondary antibody (1:1000) (life Technologies, Carlsbad, CA, USA) for 1 h. Then sections were washed with TBST three times for 5 min each. For nuclear visualization, DAPI (4 ,6-diamidino-2-phenylindole; Roche, Basel, Switzerland) was incubated 10 min, then the section was rinsed with TBS. After treatment, the sections were observed under a fluorescence microscope (Nikon, Tokyo, Japan).

Oil Red O and BODITY Staining
After being fixed in 4% paraformaldehyde solution, incubated with 0.5% Oil Red O for 30 min, and washed three times with PBS, the myoblast cells were visualized by phase-contrast microscopy (IS-Elements software, Nikon ECLIPSE, Tokyo, Japan). Oil Red O dissolved in lipid droplets was extracted with 100% isopropanol and its relative concentrations were determined by measuring the absorbance at 510 nm.
Frozen sections were treated with 0.5% TritonX-100/TBS at room temperature for 10 min and washed three times with TBS. They were then stained with BODITY (1 µg/mL; Life Technologies, Carlsbad, CA, USA) for 10 min; the sections were washed with TBST three times for 5 min each. For nuclear visualization, DAPI (4 ,6-diamidino-2-phenylindole; Roche) was incubated for 10 min, then the section was rinsed with TBS. After treatment, the sections were observed under fluorescence microscope (Nikon, Tokyo, Japan).

Vector Construction Interference
Vectors were constructed with lentiviral plasmid pLentiHI (Invitrogen, Carlsbad, CA, USA) and the inserted shRNAs were designed by online Invitrogen RNAi Designer (Available online: https://rnaidesigner.invitrogen.com/rnaiexpress/). PDGFRα and FoxO1 shRNA vectors were constructed. They were annealed and inserted into pLentiHI plasmids at BamHI and XhoI sites, and confirmed by sequencing.

Transfection of miRNA Mimics
Preadipocytes were seeded in 12-well or 6-well plates, and miR-34a mimics or negative control (NC) (GenePharma, Shanghai, China) were transfected into cells at 80% density in 50 nM using X-tremeGENE siRNA Transfection Reagent (Roche) and Opti-MEM (Gibco) culture medium according to the manufacturer's protocol and the culture medium was changed to fresh medium after 24 h. When the cells grew to confluence after transfection, adipogenic differentiation was initiated by switching to differentiation medium.

Statistical Analysis
Statistical analyses were performed using SPSS18.0 software (SPSS Inc., Chicago, IL, USA). Data represented mean ± SEM. Multiple comparisons were performed by one-way ANOVA followed by Dunnett's tests. p < 0.05 was considered to be significant.

Conclusions
Taken together, our findings indicated that PDGFRα-positive cells were porcine IMF adipocyte and PDGFRα contributed to IMF deposition. Mechanically, PDGFRα regulated by miR-34a and FoxO1 promotes adipogenensis in porcine intramuscular preadipocytes through activating Erk signaling pathway.