miR-669a-5p promotes adipogenic differentiation and induces browning in preadipocytes

ABSTRACT Obesity is a major global health issue that contributes to the occurrence of metabolic disorders. Based on this fact, understanding the underlying mechanisms and to uncover promising therapeutic approaches for obesity have attracted intense investigation. Brown adipose tissue (BAT) can help burns excess calories. Therefore, promoting White adipose tissue (WAT) browning and BAT activation is an attractive strategy for obesity treatment. MicroRNAs (miRNAs) are small, non-coding RNAs, which are involved in regulation of adipogenic processes and metabolic functions. Evidence is accumulating that miRNAs are important regulators for both brown adipocyte differentiation and white adipocyte browning. Here we report that the expression of miR-669a-5p increases during the adipogenic differentiation of 3T3-L1 and C3H10T1/2 adipocytes. miR-669a-5p supplementation promotes adipogenic differentiation and causes browning of 3T3-L1 and C3H10T1/2 cells. Moreover, the expression of miR-669a-5p is upregulated in iWAT of mice exposed to cold. These data demonstrate that miR-669a-5p plays a role in regulating adipocyte differentiation and fat browning. Abbreviations: Acadl: long-chain acyl-Coenzyme A dehydrogenase; Acadm: medium-chain acyl-Coenzyme A dehydrogenase; Acadvl: very long-chain acyl-Coenzyme A dehydrogenase, very long chain; Aco2: mitochondrial aconitase 2; BAT: brown adipose tissue; Bmper: BMP-binding endothelial regulator; Cpt1-b:carnitine palmitoyltransferase 1b; Cpt2: carnitine palmitoyltransferase 2; Crat: carnitine acetyltransferase; Cs: citrate synthase; C2MC: Chromosome 2 miRNA cluster; DMEM: Dulbecco’s modified Eagle medium; eWAT: epididymal white adipose tissue; ETC: electron transport chain; FAO: fatty acid oxidation; Fabp4:fatty acid binding protein 4; FBS: fetal bovine serum; Hadha: hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit alpha; Hadhb: hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit beta; HFD: high fat diet; Idh3a: isocitrate dehydrogenase 3 alpha; iWAT: inguinal subcutaneous white adipose tissue; Lpl: lipoprotein lipase; Mdh2: malate dehydrogenase 2; NBCS: NewBorn Calf Serum; mt-Nd1: mitochondrial NADH dehydrogenase 1; Ndufb8:ubiquinone oxidoreductase subunit B8; Nrf1: nuclear respiratory factor 1; Pgc1α: peroxisome proliferative activated receptor gamma coactivator 1 alpha; Pgc1b: peroxisome proliferative activated receptor, gamma, coactivator 1 beta; Pparγ: peroxisome proliferator activated receptor gamma; Prdm16: PR domain containing 16; Rgs4: regulator of G-protein signaling 4; Sdhb: succinate dehydrogenase complex, subunit B; Sdhc: succinate dehydrogenase complex, subunit C; Sdhd: succinate dehydrogenase complex, subunit D; Sh3d21: SH3 domain containing 21; Sfmbt2: Scm-like with four mbt domains 2; TG: triglyceride; TCA: tricarboxylic acid cycle; Tfam: transcription factor A, mitochondrial; TMRE: tetramethylrhodamine, methyl ester; Ucp1: uncoupling protein 1; Uqcrc2: ubiquinol cytochrome c reductase core protein 2; WAT: White adipose tissue


Introduction
Obesity is the single biggest risk factor in a multitude of metabolic diseases, including certain cancers, diabetes, and cardiovascular disease [1,2]. The dramatic rise in obesity has put a serious strain on public health networks across the globe [3]. Despite nutritional interventions and physical education programmes, the prevalence of obesity is still increasing and ~600 million people worldwide are expected to be obese by 2025, according to World Health Organization (WHO) estimations [4]. Therefore, a thorough understanding of the mechanisms that regulate adipogenesis could have clinical relevance in preventing and treating obesity and the associated metabolic syndromes.
White adipose tissue (WAT) and brown adipose tissue (BAT) are the two principal types of adipose tissues in mammals. WAT primarily serves to store extra energy as triglycerides, while BAT is specialized to burn lipids for heat generation and energy expenditure as a defence against cold and obesity [5]. The development of WAT browning and BAT activation is dramatically enhanced during adaptation to cold or in response to treatment with β3-selective adrenergic agonists [6,7]. Accordingly, promoting BAT-like features in white adipose and BAT function is an attractive therapeutic approach to combat obesity [8].
MicroRNAs (miRNAs) are a family of 21-25 nucleotide small RNAs that negatively regulate gene expression at the post-transcriptional level. miRNAs exhibit temporally and spatially regulated expression patterns during diverse developmental and physiological processes [9][10][11]. Several miRNAs have recently emerged as important regulators of either brown adipocyte differentiation or white adipocyte browning. The miR-193b-365 cluster was the first reported miRNAs that sustain brown adipocyte differentiation by repressing the myogenic potential of preadipocytes [12]. Overexpression of the miR17-92 cluster in C3H10T1/2 cells enhances the thermogenic capacity of adipocytes, and there is a significant reduction in adiposity in adipose tissue-specific miR17-92 cluster transgenic mice as well [13]. Several miRNAs also negatively regulate brown fat development. miR-133 directly targets and negatively regulates Prdm16, and inhibition of miR-133 promotes differentiation of precursors from BAT and WAT to mature brown adipocytes [14]. A study reported that decreased miR-494-3p expression during browning regulates mitochondrial biogenesis and thermogenesis through PGC1-α in beige adipocytes [15]. Despite the fact that some miRNAs have been identified as central regulators of the brown adipogenic programme, the whole miRNA regulatory network is still not complete, and further studies are required to fully understand the regulatory roles of miRNAs in brown adipogenesis and to develop therapeutic approaches to treat obesity and obesity-related diseases.
Preadipocytes are often used as models for elucidating the underlying mechanisms of adipogenesis, which involves cell proliferation and differentiation. To identify the miRNAs related to lipid metabolism, we analysed RNA isolated from 3T3-L1 preadipocytes at different time points of differentiation, from preadipocytes to mature. microRNA sequencing data showed that most members of the miR 297-466-467-699 cluster are upregulated during 3T3-L1 adipocyte differentiation, indicating that the miR 297-466-467-699 cluster likely regulates adipocyte differentiation and lipid metabolism ( Figure 1). In addition, a previous study has shown that miR-669a-5p has a close relationship with progenitor cell differentiation [16], so we focused on miR-669a-5p to explore its role in preadipocytes.
miR-669a-5p is a member of the miR 297-466-467-699 cluster located in intron 10 of the Sfmbt2 gene on mouse chromosome 2 (hence Sfmbt2 miRNA cluster is also called Chromosome 2 miRNA cluster, C2MC). C2MC is one of the largest clusters of miRNAs, containing 72 miRNA precursor sequences [17], and these C2MC sequences show a high degree of similarity [18]. However, little is known about the biological functions of the C2MC biological functions. A small number of manuscripts have shown that C2MC miRNA members play important roles in cell differentiation and apoptosis [16,[19][20][21][22], as well as in development and the immune response [17,[23][24][25].
In this report, we determine the role of miR-669a-5p in adipogenesis using a mouse 3T3-L1 embryonic fibroblastic cell line and a C3H10T1/2 mesenchymal stem cell line. We describe the expression profiles of miR-669a-5p during adipogenic differentiation and demonstrate that miR-669a-5p enhances adipogenic differentiation and induces a brown adipocyte-like phenotype in 3T3-L1 and C3H10T1/2 preadipocytes. It is the first study that demonstrates the function of miR-669a-5p in adiposeness. It is also the first study to show that Sfmbt2 and C2MC are cotranscripted during adipogenic differentiation in 3T3-L1 and C3H10T1/2 cells, indicating that Sfmbt2 and C2MC may act in concert to regulate adipogenic differentiation.

The expression of miR-669a-5p is increased during differentiation of 3T3-L1 cells
To identify miRNAs involved in the adipogenesis process, we performed small RNA deep sequencing in 3T3-L1 cells undergoing differentiation. miRNA-Seq analysis indicates C2MC may regulate lipid metabolism: Nearly 90% of the members of the miR 297-466-467-699 cluster are upregulated in mature 3T3-L1 adipocytes (day 10) compared to 3T3-L1 preadipocytes (day 2) (Figure 1). miR-669a-5p is a member of C2MC, and a previous study has shown miR-669a-5p plays a role in the process of progenitor cells differentiation [16], but whether it impacts preadipocytes differentiation and adipogenesis has not been characterized.
To determine the role of miR-669a-5p in preadipocytes differentiation and adiposenesis, we first detected the levels of miR-669a-5p during 3T3-L1 white preadipocytes differentiation. Cells were collected at the indicated time points of differentiation (day 0, day 2, day 4, day 6, day 8 and day 10 after adipogenic treatment). miR-669a-5p was significantly increased during the adipogenic differentiation process (Figure 2 (a)). Differentiation of the cells was verified by measuring the mRNA expression of the adipogenic markers peroxisome proliferator activated receptor gamma (Pparγ) and fatty acid binding protein 4 (Fabp4) (Figure 2(b) and (c)). Taken together, these data indicate that miR-669a-5p may play a role in adipocyte differentiation.
The effect on adipogenesis of a miR-669a-5p inhibitor was also examined in the 3T3-L1 cell. Transfection of the miR-669a-5p inhibitor decreased miR-669a-5p expression ( Figure S1A). However, lipid droplet formation and adipogenic markers expression did not show a significant change following the miR-669a-5p inhibitor transfection (Figure S1B-F).

miR-669a-5p promotes adipogenic differentiation of C3H10T1/2 cells
To further demonstrate the role of miR-669a-5p in adipogenesis, we tested miR-669a-5p expression levels at different time-points during adipogenesis of the brown preadipocyte cell line C3H10T1/2. During the differentiation process of this cell line, a thermogenic programme is induced and the levels of brown fatrelated genes (also called thermogenic genes) such as uncoupling protein 1(Ucp1) and peroxisome proliferative activated receptor gamma coactivator 1 alpha (Pgc-1a) are increased. The levels of miR-669a-5p and the brown fat-related genes Pparγ and Pgc-1α were increased in C3H10T1/2 cells after the adipogenic differentiation treatment (Figure 4(a-c)). In addition, the adipogenic differentiation was accelerated by supplementing a miR-669a-5p mimic, as evidenced by the Oil Red O staining, measurement of the intracellular triglyceride (TG) content and Western blot analysis of brown fat-related genes (PPARγ, PGC-1α and UCP1) (Figure 4(d-h)). Consistent with the gene expression data, miR-669a-5p increased the mitochondrial content as shown by mtDNA quantification and Mito Tracker staining (Figure 4(i) and (j)). The mitochondrial membrane potential was also measured by TMRM, the results showed miR-669a-5p increased the mitochondrial membrane potential of C3H10T1/2 cells (Figure 4(k)). Also consistent with 3T3-L1 data, the miR-669a-5p inhibitor had no effect on lipid droplet formation and brown fat-related genes expression during adipogenic differentiation of C3H10T1/2 cells ( Figure S2). These results show that miR-669a-5p stimulates adipogenic differentiation in 3T3-L1 cells and C3H10T1/2 cells, and the effects of the miR-669a-5p inhibitor were not observed in either cell line.

miR-669a-5p induces a brown phenotype in 3T3-L1 adipocytes
Given the prominent effect of miR-669a-5p on the development of the thermogenic programme in brown adipocytes C3H10T1/2 (Figure 4), we asked whether miR-669a-5p also has similar effects in the white adipocyte 3T3-L1 cells. We tested the expression of thermogenic genes in differentiated 3T3-L1 transfected with the miR-669a-5p mimic. As shown in Figure 5, the miR-669a-5p mimic significantly enhanced the expression of the thermogenic marker genes Pgc-1α and Ucp1 in 3T3-L1 cells ( Figure 5(a-c)), suggesting that miR-669a-5p induces a brown phenotype in 3T3-L1 adipocytes.

miR-669a-5p promotes mitochondrial biogenesis 3T3-L1 cells
To illustrate the mechanisms by which miR-669a-5p controls adipogenesis, we conducted a transcriptome study by RNA-seq and quantified the mRNA changes during differentiation of 3T3-L1 cells transfected with the miR-669a-5p mimic transfection. Increased mitochondrial biogenesis is one of the characteristics in the adipocyte browning process. Our mRNA-seq data show that the transcripts regulating mitochondriogenesis are enhanced in the presence of the miR-669a-5p mimic ( Figure S3).
We next performed RT-qPCR to validate the transcript level of those key genes related to mitochondria. We found that miR-669a-5p treatment increased the expression of genes (Pgc-1a, Pgc-1β, Tfam) known to be involved in mitochondriogenesis (Figure 6(a)). A miR-669a-5p mimic treatment also led to an increased expression of the mitochondrial free fatty acid transporter machinery (Cpt1b, Cpt2, and Crat) ( Figure 6(b)). In addition, the expression of all components of the β-oxidation machinery and most genes of the tricarboxylic acid cycle (TCA) were upregulated by the miR-669a-5p mimic treatment (Figure 6(c) and (d)). Since the electron transport chain (ETC) is the We further tested the mitochondrial content in differentiated 3T3-L1 cells transfected with the miR-669a-5p mimic. We found that the miR-669a-5p mimic markedly increased mitochondrial content indicated by mtDNA quantification and Mito Tracker staining (figure 6(f) and (g)). To illustrate the mitochondrial activity, the mitochondrial membrane potential was measured by TMRM. The results showed miR-669a-5p increased the mitochondrial membrane potential of 3T3-L1 cells (Figure 6(h)).
Taken together, our results reveal that miR-669a-5p positively regulates the bioenergetics of 3T3-L1 cells by acquisition of a mature brown fat phenotype.

The expression of miR-669a-5p is upregulated in iWAT of mice exposed to cold
Since miR-669a-5p promotes adipogenesis in preadipocytes, we used diet-induced obesity (DIO) mice as an obesity model to investigate the function of miR-669a-5p in vivo. After being fed with a high-fat diet for 2 months (from week 8 to week 16), the body weight and body fat ratio of DIO mice were significantly increased compared with littermate controls ( Figure  S4A). There were no significant expression changes of miR-669a-5p in both Epididymal white adipose tissue (eWAT) and BAT of DIO mice. However, we found a declining trend of miR-669a-5p expression in eWAT of DIO mice ( Figure S4B), which indicates miR-669a-5p may participate in the process of WAT browning.
Given the impact of miR-669a-5p on the development of a brown fat-like thermogenic programme in white adipocytes (Figures 5,6), we studied whether the expression of miR-669a-5p is altered in a cold-induced WAT browning mice model which is a more widely used animal model for the research of WAT browning. Inguinal subcutaneous white adipose tissue (iWAT) is a typical adipocyte tissue that undergoes WAT browning process when mice are exposed to cold. After exposure to cold, we found a stronger induction of Ucp1 and Pgc-1α expression in iWAT (Figure 7(a,b)). More importantly, we found miR-669a-5p was increased in the iWAT of mice subjected to cold exposure, suggesting that miR-669a-5p participates in the process of WAT browning in vivo (Figure 7(c)).

Discussion
Obesity is characterized by increased lipid storage in adipocytes and an increased number of adipocytes. Adipocytes are derived from a pool of existing preadipocytes, which are ready to differentiate in response to an appropriate signal. This process has produced intense research into the mechanisms regulating adipocyte development [26]. Mesenchymal stem cell-line C3H10T1/2 and mouse 3T3-L1 preadipocytes are  often used as models for elucidating the underlying mechanisms of adipogenesis, which involves cell proliferation and differentiation [27][28][29][30].
Recent data indicate that microRNAs play a key role in modulating cell differentiation and metabolism of many tissue types in vivo, including adipocytes [12]. MicroRNAs that selectively promote energy expenditure through BAT thermogenesis or browning of WAT are of significant interest as potential therapeutics for anti-obesity treatments due to the ability of BAT and browning WAT to metabolize vast amounts of glucose and lipids [7,8].
In this study, we demonstrate the function of miR-669a-5p in adipogenesis. We show that the expression of miR-669a-5p is increased during adipogenic differentiation in 3T3-L1 and C3H10T1/2 cells. Meanwhile, we further show that miR-669a-5p is able to stimulate adipogenesis and induces a brown phenotype in 3T3-L1 and C3H10T1/2 cells. In line with the in vitro data, the expression of miR-669a-5p is upregulated in iWAT when mice are exposed to cold. Our data indicate that miR-669a-5p participates in the process of adipose tissue browning both in vivo and vitro. However, a miR-669a-5p inhibitor has no effect on adipogenic differentiation in 3T3-L1 and C3H10T1/2 cells. This result indicates that the function of miR-669a-5p in adipogenesis is not indispensable. As the sequences of C2MC show high degrees of similarity, the function of miR-669a-5p may be compensated by other C2MC members which have similar sequences with miR-669a-5p.
According to our previous studies and mRNA-seq data, we also tried to find the target of miR-669a-5p in the process of adipocyte differentiation. We have tested some genes, such as Fst, Vdr, Ctbp2, ect. (data not shown). However, we have not found the target of miR-669a-5p. It will be interesting to find the underlying mechanisms in the future.
Previous studies have shown that clustered miRNAs often coexpressed with their host genes [31][32][33], with this the expression pattern serving to regulate similar biological functions and signalling pathways. We found that the expression of Sfmbt2 and C2MC were increased during adipogenic differentiation in 3T3-L1 and C3H10T1/2, indicating Sfmbt2 and C2MC are cotranscripted ( Figure S5 and S6, Figures 2(a) and 4  (a)). The coexpression patterns and high sequence similarity of miRNA family members suggest that Sfmbt2 and C2MC may act in concert to regulate adipogenic differentiation in 3T3-L1 and C3H10T1/2 cells. In the present study, we focus on the function of miR-669a-5p. Further studies are needed to verify the function of the other miR 297-466-467-699 cluster members and to elucidate the role of the Sfmbt2 gene in the process of adipogenesis. As well, our findings about miR-669a-5p need more mechanistic investigations and verifications through in vitro and vivo experiments. In summary, we have demonstrated for the first time that the expression of miR-669a-5p is increased during adipogenic differentiation of 3T3-L1 preadipocytes and C3H10T1/2 cells. In addition, miR-669a-5p is able to stimulate adipogenesis and induce a brown phenotype in 3T3-L1 and C3H10T1/2 cells.

RNA sequencing
Small RNA sequencing libraries were generated using NEBNext®Multiplex Small RNA Library Prep Set for Illumina® (NEB) and the clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq SR Cluster Kit v3-cBot-HS (Illumia) according to the manufacturer's instructions. After cluster generation, the library preparations were sequenced on an Illumina Hiseq 2500/2000 platform and 50 bp single-end reads were generated. mRNA sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB) following manufacturer's recommendations. The library preparations were sequenced on an Illumina Novaseq platform and 150 bp paired-end reads were generated. Gene Ontology (GO) enrichment analysis of differentially expressed genes was implemented by the clusterProfiler R package, in which gene length bias was corrected. GO terms with corrected p value less than 0.05 were considered significantly enriched by differential expressed genes.

Animal studies
Male C57BL/6 mice were kept under constant temperature and humidity in a 12 h controlled dark/light cycle. 8-week-old male C57BL6/J mice were fed with a standard chow diet (10 kcal% fat, D12450J, Research Diets) or high fat diet (HFD, 60 kcal% fat, D12492, Research Diets) for 8 weeks. Mice were then euthanized and different adipose tissues were harvested for analysis. For cold-exposure experiments, 8-week-old male mice were exposed to 6°C for 72 h. All of the experiments were approved by the by the Animal Ethics Committee of the Kunming Institute of Zoology, Chinese Academy of Science.

Cell transfection
Mimic and inhibitor oligonucleotides of miR-669a-5p were synthesized by Ribobio. The transfection was carried out using the riboFECT TM Transfection Kit (C10511-05, Ribobio). According to the manufacturer's instructions, cells grown in standard 6-well plates were added with a mixture of mimic (200 nM) or inhibitor (100 nM). Transfection was carried out two times during the cell differentiation.3T3-L1 preadipocytes were transfected with mimic control/inhibitor control or mimic miR-669a-5p (100 nM)/inhibitor miR-669a-5p (200 nM) on day 0 and day 4 after differentiation, cells were harvested on day 8 for analysis. C3H10T1/2 preadipocytes were transfected with mimic control/inhibitor control or mimic miR-669a-5p (100 nM)/ inhibitor miR-669a-5p (200 nM) on day 0 and day 3 after adipogenic differentiation. The cells were collected on day 6 for analysis

Oil Red O staining and measurement of intracellular triglyceride (TG) content
Accumulation of lipids was assessed by oil red O staining. Cells were washed with PBS and fixed with 4% paraformaldehyde for 1 h at room temperature. 60% isopropanol was then added to the cells for 2 min. Cells were then incubated with freshly prepared oil red O working solution for 30 min at 37°C and observed under the microscope. For Oil Red O quantitative analysis, the intracellular absorbed Oil Red O was extracted with 100% isopropanol, and absorbance was measured at 510 nm wave length. Intracellular TG contents were measured using a commercial assay kit (E1013, APPLYGEN), TG concentrations were calculated based upon a standard curve made from TG standards and normalized to total cellular protein content.

Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR)
Total RNA was isolated from the samples using RNAiso Plus (9109, Takara). The expression levels of miRNA were analysed using an All-in-One™ miRNA qRT-PCR Detection Kit (QP015, GeneCopoeia). For mRNA expression analysis, total RNA was transcribed to complementary DNA using PrimeScript RT reagent Kit with gDNA Eraser (RR047A, Takara) following the manufacturer's instructions. Expression of selected genes was analysed using TransStart® Top Green qPCR SuperMix (AQ132, TransGen Biotech). Quantitative real-time polymerase chain reaction (qPCR) was performed on the ABI-7900HT system (Applied Biosystems). The relative expression of target genes was measured using the 2 −ΔΔCt method, and the amount of target gene was normalized to the endogenous control gene U6 or β-actin. Sequences of primers are shown in Table S1.

Total DNA isolation and mtDNA quantification
Total DNA was isolated from cells using the Blood/ Cell/tissue genomic DNA extraction kit (DP304, TIANGEN). The mtDNA copy number quantification was performed by real-time PCR. Amplification of the mitochondrial mt-Nd1 gene was compared with that of reference nuclear Lpl. Primers used are shown in Table S1.

Mitochondrial labelling
Mitochondrial mass was measured by fluorescence levels on staining with MitoTracker Red CMXRos (M7512, Invitrogen). Cells were incubated with 50 nM MitoTracker Red CMXRos for 10 min at 37°C. Cells were then fixed with 3.7% formaldehyde. The images were captured with a ZEISS LSM800 confocal microscope.

Mitochondrial membrane potential measurement
To measure the mitochondrial membrane potential, adipocytes were stained with cell growth medium containing 100 nM tetramethylrhodamine, methyl ester (TMRM, I34361, Invitrogen). Cells were then incubated for 30 min at 37°C. The images were captured with a ZEISS LSM800 confocal microscope.

Statistical analysis
All results are presented as mean ± standard errors of the means (SEM) based on at least three separate experiments. Differences between two groups were analysed by Student's t-test using GraphPad Prism 5.0. p < 0.05 was considered statistically significant.