Background/Aims: Muscle cells are able to trans-differentiate into adipocytes with adipogenesis induction. MicroRNAs (miRNAs), a class of small non-coding RNAs, widely participate in the regulation of growth and development of cells. However, the expression and regulatory role of miRNAs in the trans-differentiation of muscle cell are largely unknown. Methods: C2C12 myoblasts were inducted to adipogenesis trans-differentiation and microarrays were used to assay the changes of expression profile of miRNAs. MiR-199a, a miRNA showed significant change in the trans-differentiation, was selected for the subsequent function study via over- expression and knock down. Results: Dozens of miRNAs showed different changes followed the adipogenesis trans-differentiation of C2C12 cells. In which, miR-199a was decreased in the adipogenic cells and miR-199a over-expression inhibited the trans-differentiation and decreased lipid accumulation in the cells. Moreover, Fatty acid transport protein 1 (Fatp1), a major regulator of trans-membrane transportation and the oxidative metabolism of free fatty acids, was showed to be a target of miR-199a by computational and luciferase reporter assays. Additionally, Fatp1 knock-down by small interfering RNA had similar inhibitory effects on the trans-differentiation in C2C12 cells. Conclusion: Our study reveals an important role for miR-199a in the regulation of adipogenic trans-differentiation in muscle cells via suppression of Fatp1 gene.

Previous studies have shown that myogenic cell lines are capable of trans-differentiating into adipocytes or adipocyte-like cells following treatment with an adipogenesis stimulant [1,2,3,4]. These trans-differentiated cells lose their myogenic specialty when they acquire an adipogenic capacity. Obviously, the adipogenic trans-differentiation has a different metabolism and molecular regulatory mechanism relative to the normal myogenic differentiation.

MicroRNAs (miRNAs) are a class of small non-coding RNAs, around 18-25 nt in length, that modulate gene expression by targeting 3' untranslated regions (UTRs) of genes at the post-transcriptional level [5,6]. In recent years, some miRNAs have been shown to play a central role in the growth and development of muscle and adipose tissues via the regulation of functional genes. For example, miR-1, miR-133 and miR-206 appear to be muscle-specific miRNAs [7,8,9], and are required for skeletal muscle formation, while Let-7 [10], miR-137 [11], miR-143 [12] and are essential for adipose formation. These miRNAs are regulated by myogenic or adipogenic transcriptions factors, and then control their target genes through feedback mechanism. However, the expression profile and functional role of miRNAs in the adipogenic trans-differentiation of muscle cells have not yet been elucidated.

Here, we compare the expression profile difference of miRNAs between adipogenic trans-differentiated C2C12 cells (ADCs), normal myogenic differentiated C2C12 cells (MDCs), and pre-differentiated C2C12 cells (PDCs). Functional studies using miR-199, which is reduced in the ADCs, show that miR-199a over-expression inhibits the trans-differentiation and decreases lipid accumulation in C2C12 cells. In addition, it is verified that fatty acid transport protein 1 (Fatp1) is a target of miR-199a and Fatp1 knock down similarly inhibits the trans-differentiation in C2C12 cells. These results therefore indicate miR-199a plays a key role in the adipogenic trans-differentiation of muscle cell by targets Fatp1 and the findings help to elucidate the molecular mechanism of trans-differentiation between muscle and adipose tissues.

Cell culture and induction of differentiation

C2C12 myoblast were cultured in Dulbecco's modified Eagle's medium (DMEM, GIBCO, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (GIBCO) at 37°C and 5% CO2. To induce normal myogenesis the growth medium was replaced with a "differentiation medium" (DMEM supplemented with 2% horse serum) after the cells had reached 70% confluence. Cells were maintained in this differentiation medium for several days, as indicated for each experiment. To induce adipogenesis tans-differentiation, C2C12 cells were transferred from their growth medium to DMEM-F12 medium (GIBCO) supplemented with 1 mg/mL insulin (Sigma, St. Louis, MO, USA), 1 µM Dexamethasone (Sigma) and 0.5 mM 3-isobutyl-1-methylxanthine (Sigma) when the cells had reached 70% confluence. After 2 days, cells were transferred to medium supplemented with only 1 mg/mL insulin for another 2 days. Subsequently, medium was changed every 2 days until the cells were well differentiated. All experiments were performed using three different cell clones.

Oil Red O staining and extraction assay

Adipogenesis in the cells were assessed by the Oil Red O method. Briefly, cells were washed three times with phosphate buffered saline (PBS, pH 7.2) and fixed in 4% paraformaldehyde for 30 min at ambient temperature. The fixed cells were washed three times in PBS again and incubated in staining solution (60% Oil Red O stock solution and 40% H2O) for 30 min at ambient temperature. Lastly, the cells were washed with deionized water, and the stained lipid droplets were dissolved with 100 % avantin for 10 min and the solution were collected for colorimetric analysis at 510 nm (cellular triglycerides assay).

Giemsa staining

During myogenic differentiation, multinucleated myotubes were visualized with the giemsa staining. Cells were washed twice with PBS, and then fixed in 4% paraformaldehyde for 30 min and then stained for 1 min with giemsa stain solution (suolaibao, Beijing, china) at ambient temperature. Cells were washed twice with deionized water and observed under an inverted microscope. The number of typical muscular tube was counted.

MicroRNA microarray assay

To compare expression difference of miRNAs between ADCs, MDCs and PDCs, commercial mouse microRNA microarrays which contain probes for 1,916 mouse mature microRNAs from the Sanger mirBase database v.20.0 (two probes for each miRNA on one chip) were used in this study. Microarray hybridizations were implemented in the LC Sciences Biotech Company (Hangzhou, china). The tagged miRNAs were purified and hybridized with the LC Sciences microRNA Microarray-Single following the instructions of the manufacturer's instructions. After the hybridization, the chips were subjected to a stringent wash and fluorescence data were collected by using an Axon laser scanner model 4000B (Axon Instruments) and the chips were scanned at a pixel size of 10 µM with Cy3 Gain at 460 and the Cy5 Gain at 470 scanning. Equal RNA from six individual cell samples with same treatment was mixed and each mixture sample was repeated two times.

RNA oligonucleotides and transfection

The special miR-199a mimic, miR-199a inhibitor, negative control (NC) (RiboBio, Guangzhou, China) were transfected into C2C12 cells for over-expression and knock down of miR-199a respectively. The oligonucleotides were severally transfected into C2C12 cells at the 2nd day of adipogenic differentiation. The exhaustive transfection procedure was in accordance with the manufacturer's instructions for the Lipofectamine 2000 cellular transfection reagent (Invitrogen, CA, USA).

Small interfering RNA (siRNA) against Fatp1 and transfection

A targeted Fatp1 gene siRNA and a nonspecific duplex (negative control) were designed and synthesized by the Biomics Biotechnology Company (Nantong, China). The siRNA was transfected into the C2C12 cells for silencing of endogenous Fatp1 gene. The sense strand of the siRNA was 5'-CAGUACAUAGGUGAAAUCUdTdT-3', the antisense strand was 5'- AGAUUUCACCUAUGUACUGdTdT-3'. Transfection was performed with the Lipofectamine 2000 reagent (Invitrogen) combined with 100 nM of anti-Fatp1 siRNA.

Target genes of miRNAs prediction, gene ontology and KEGG pathway analysis

Targets of the differentially expressed miRNAs were predicted by using the online software, TargetScan (V7.0, in conjunction with miRanda (, and PicTar ( The overlapping genes predicted by the software were further analyzed in terms of their Gene Ontology (GO) categories ( and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways (

Dual-luciferase reporter assay

The region of Fatp1 3ˋUTR flanking the miR-199a binding site was amplified from mouse genomic DNA using PCR and subcloned into the pGL3-promoter vector at the XbaI restriction site of the 3ˋUTR of the luciferase cDNA and the resultant plasmids were used as luciferase reporter vectors. For the dual-luciferase reporter analysis, HEK293 cells were seeded in 24-well plates. When the cell density reached 60% confluence, the cells were transfected with mixed DNA of the miR-199a mimic and individual luciferase reporters by using the calcium phosphate transfection method. A b-Gal plasmid was co-transfected as an internal control. At 48 h post-transfection, luciferase activities were measured with the Dual-Luciferase Reporter Assay System (Promega, CA, USA).

Real-time quantitative PCR for gene expression

Total RNA was extracted from cells with RNAiso Reagent (TaKaRa, Dalian, China) at appointed time and 1.0 µg of each sample was reverse-transcribed to cDNA with the PrimeScript™ RT reagent Kit (TaKaRa). PCR was performed using a Step One system (ABI, NY, USA). The 18S and GAPDH were used as reference genes with similar results and primers were purchased from the Sangon Biotech Company (Shanghai, China). The specific primer sequences are shown in Table 1.

Western blotting

The cells were collected at appointed time and lysed in the RIPA buffer (Beyotime, Beijing, China) and the lysates were analyzed using a standard western blot procedure. GAPDH antibody was used as a loading control. FATP1 and FABP4 antibody was obtained from Abgent (CA, USA). PPARγ, C/EBPα, Myogenin, MyoD, MYF5 antibodies horseradish peroxidase conjugated secondary antibodies were obtained from CST (MA, USA).

Statistical analysis

The SPSS statistical software package (Version 19.0, IBM SPSS, USA) was used for data analysis. All data were shown as means ± S.D. One-way variance (ANOVA) was used to determine significance between three or more groups. Student's t-test was used to determine significant differences between two groups. P < 0.05 or P < 0.01 was considered statistically significant.

Adipogenic trans-differentiation of C2C12 cells

Following adipogenic induction, the C2C12 cells gradually became rounded while accumulating intracellular lipid droplets (Fig. 1A, upside). In contrast, the C2C12 cells stretched and became spindle shape during myogenic differentiation and progressing to form myotubules (Fig. 1A, downside). Some adipogenesis regulators showed augmented protein (Fig. 1B and D) and mRNA (Fig. 1C) expression levels, including peroxisome proliferator-activated receptor (PPAR)γ‚ CCAAT-enhancer-binding protein α (C/EBPα), fatty acid synthase (FAS), and adipocyte fatty acid binding protein (FABP) 4 in ADCs 8 days post-differentiation compared with PDCs. During this same period, the expression levels of myogenic regulatory factors including myogenin, MyoD, and Myf5 were decreased to different degrees (Fig. 1B-D).

Expression differences of miRNAs in ADCs and MDCs

Commercial miRNA microarrays were used to detect differences in the miRNA expression profiles of ADCs, MDCs, and PDCs. A total of 57 miRNAs showed differential expression (Fig. 2A and B, microarray signal data > 500, One-Way ANOVA P < 0.05) among the three groups of cells. Compared with PDCs, myogenic differentiation caused a change in the expression of 26 miRNAs (microarray signal data > 500, P < 0.05), of which 13 were up-regulated and 13 were down-regulated. Compared with PDCs, ADCs showed a change in the expression of 31 miRNAs, including 15 that were up-regulated and 16 down-regulated. Clearly, some known muscle-specify miRNAs (i.e. miR-1a, miR-145a and miR-206) showed reduced expression when some known adipocytes-specify miRNAs (i.e. miR-103 and let-7 a/b) have augmented expression in ADCs. Moreover, the student-test assay indicated that there were 29 miRNAs (P < 0.05) have different expression between ADCs and MDCs and that also were confirmed by the qRT-PCR assay (Fig. 3). The KEEG pathway analysis indicated that most targets of these altered miRNAs belong to the insulin signaling pathway (P < 0.0001) which is closely involved in the proliferation and differentiation of fat cells and muscle cells.

Of all miRNAs showing differential expression between cell types, miR-199a was of particular interest because it has been shown to play a key role in the differentiation of adipocytes recently [13]. Similarly, our microarray data also indicated that both miR-199a and miR-199b significantly decreased in ADCs. This suggested that adipogenic trans-differentiation suppressed miR-199a/b expressions (Fig. 2C). But the qRT-PCR data showed that only miR-199a expression was impaired in ADCs compared with PDCs, and that miR-199b expression was not obviously different (Fig. 2D). Given the lower signal data of miR-199b in the microarray detection (microarray singal data < 500), we selected miR-199a for subsequent functional studies.

Over expression of miR-199a suppressed adipogenic differentiation in C2C12 cells

To explore the regulatory function of miR-199a in the adipogenesis trans-differentiation, artificial mimic and inhibitor were transfected into the cells at the initial of trans-differentiation (day 2) to over-express and knock-down intracellular mature miR-199a, respectively. As shown in Figure 4, there was a 40-folds increase and a ∼70% decrease in the expression level of miR-199a 48 h after the transfections, respectively (Fig. 4A). These changes could maintain for at least 8 days after adipogenic induction.

The miR-199a over-expression induced by mimic significantly attenuated lipid accumulation in C2C12 cells on day 8 of differentiation, as shown by the Oil Red O staining (Fig. 4B and C). The qRT-PCR data also showed that PPARγ, C/EBPα, FASn, Fatp1 and FABP4 mRNA levels were down-regulated to different extents by the elevated miR-199a level (Fig. 4D). However, the miR-199a inhibitor had a limited effect on the trans-differentiation and adipogenesis.

Fatp1 is a bona fide target of miR-199a in adipogenic trans-differentiated C2C12 cells

TargetScan online analysis point out there was an evolutionarily conserved miR-199a binding site in the Fatp1 3ˋUTR (Fig. 5A), suggesting that Fatp1 was a possible target for miR-199a. We also observed a negative correlation between Fatp1 and miR-199a expressions during the trans-differentiation of C2C12 cells. MiR-199a highly expression at the initial period of trans-differentiation and then decreased with the trans-differentiation progressed, however, the Fatp1 expression pattern was inversely correlated with that of miR-199a (Fig. 5B). As expected, both the mRNA (Fig. 5C) and protein (Fig. 5D) levels of Fatp1 were significantly decreased 48 h after the miR-199a mimic transfection. Furthermore, the dual-luciferase reporter assay showed co-transfection of Luc-Fatp1 with miR-199a mimic in 293T cells resulted in a 45% decrease in luciferase activity as compared to co-transfection using control miRNA, whereas the Fatp1 mutant did not lead to any significant changes in luciferase activity that suggested miR-199a directly regulated Fatp1 expression by binding the 3ˋUTR (Fig. 5A and E). Additionally, we found that miR-199a expression only weakly increased during the process of myogenic differentiation in C2C12 cells but mRNA level of Fatp1 was augmented simultaneously (Fig. 5F).

Fatp1 knock-down suppresses adipogenic differentiation in C2C12 cells

To simulate the suppression effect of miR-199a on Fatp1, a synthetic small interfering (si)RNA was transfected into C2C12 cells for knock-down (KD) endogenous Fatp1 expression. This impeded adipogenic trans-differentiation by down-regulating the expression of adipogenesis regulation factors (Fig. 6A) and decreasing the accumulation of intracellular triglycerides (Fig. 6B, C, D). However, there was no obvious feedback regulation effect on the expression of miR-199a (Fig. 6E). Additionally, we found Fatp1 KD also influenced the normal myogenic differentiation of C2C12 cells (Fig. 7). It was observed that Fatp1 KD accelerated the differentiation and increased expression of myogenesis factors to different extent.

Besides serves as a model of myoblast cells, the C2C12 cell line is also characterized as a mesenchymal progenitor cell that can differentiate into several cell types, including adipocytes. Teboul et al. first reported that adipogenic inducers, such as thiazolidinediones or fatty acids, prevented myogenin expression and specifically converted the differentiation pathway of myoblasts into that of adipoblasts [1]. However, they also found myotubes were insensitive to adipogenic induction compounds. Asakura et al. later reported that satellite cell-derived primary myoblasts and satellite cells on isolated muscle fibers readily differentiated into adipogenic lineages when the cells treated with adipogenic inducers [2]. At recent, it has been reported that high concentration of dexamethasone could promote the tendon stem cells differentiate into adipocyte by up-regulated the inhibitory molecule dickkopf1 (DKK1) [14]. However, most of details of molecular regulators involved in the trans-differentiation were unclear.

In the present study, we induced C2C12 cells to differentiate into adipocytes, and observed large numbers of lipid droplets and a remarkable increase in the expressions of adipogenic genes (PPARγ, C/EBPα, FAS and FABP4), as seen in true adipocytes [15]. However, myogenic regulators in the trans-differentiated cells were found to lose their function. Given the distinct changes we speculated that the molecular mechanism of adipogenic trans-differentiation in myoblasts was similar to that of adipocytes.

The role of miRNAs in adipogenic trans-differentiation has not been clarified, although some miRNAs were recognized as prerequisites for normal differentiation in myoblasts or adipocytes [8,16]. Similar to the changes in cellular phenotype, we observed significant changes in the expression of many miRNAs during the trans-differentiation. Some muscle-specific miRNAs, e.g., miR-1, miR-133, and miR-206 showed significant decreases in expression, while some adipose-specific miRNAs, e.g., miR-103 and Let-7, were increased. These results indicated that the expression and function of miRNAs, especially tissue-specific ones, adherence to cellular fate changes.

Recently, the miR-199 family has been shown to be widely expressed in multiple animal tissues [17,18] and to participate in a variety of biological events, including signal transduction, immune response, muscle cell differentiation, and cancer progression [19,20,21,22]. In our study, adipogenic trans-differentiation in myoblasts significantly reduced miR-199a expression levels. It was also shown that miR-199a over-expression substantially impede the trans-differentiation and adipogenesis. A similar study previously found that miR-199a over-expression in porcine preadipocytes significantly promoted cell proliferation while attenuating lipid deposition through controlling the expression of caveolin-1 [13].

Our results indicate that the influence of miR-199a on cells may occur, at least in part, via the targeted decrease of Fatp1 expression. Fatp1 was the most important member of the Fatp1 protein family, serving to transfer circulating free fatty acids, especially long chain and very long chain fatty acids, across the plasma membrane [23,24,25]. Fatp1 also acts as an acyl coenzyme A synthetase in the mitochondrial oxidation of fatty acids [26,27]. It demonstrated steady high expression level in animal muscle and adipose tissues, and was increasing during development. In vitro studies have found Fatp1 to be a pivotal regulator in adipocyte differentiation that affected lipid accumulation [28,29]. Therefore, its normal expression and function were essential to adipogenesis, and a deficiency could decrease the usefulness of free fatty acids and the formation of triglycerides, leading to a dysfunction of the adipogenesis system.

In summary, our data show that miR-199a plays an important role in the adipogenic trans-differentiation of C2C12 myoblasts, and confirm Fatp1 to be the target of miR-199a in these cells. MiR-199a over-expression inhibits the trans-differentiation by impairing Fatp1 expression, and affecting that of adipogenesis regulators. We propose that miR-199a and Fatp1 participate in the control of trans-differentiation between muscle cells and adipocytes, and, therefore, in maintaining the energy balance within the body.

This work was supported by the National Basic Research Program of China (Project Number 2012CB124702), the National Nature Science Foundation of China (Project Number 31302055 and Project Number 31470117), the Chongqing Fundamental Research Project (Project Number 14441 and 14403) and the earmarked fund for Modern Agro-industry Technology Research System (Cars-36).

The authors declare that they have no competing interests.

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R. Qi and D.long contributed equally to this work.

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