Long noncoding RNA ACART knockdown decreases 3T3-L1 preadipocyte proliferation and differentiation

Abstract Obesity is a main risk factor for diabetes and cardiovascular disorders and is closely linked to preadipocyte differentiation or adipogenesis. Peroxisome proliferator-activated receptor γ (PPARγ) is an indispensable transcription factor in adipogenesis. A newly identified long noncoding RNA, Acart, exerts a protective effect against cardiomyocyte injury by transactivating PPARγ signaling. However, the function of Acart in preadipocyte differentiation is unclear. To investigate the function of Acart in adipogenesis, a well-established preadipocyte, the 3T3-L1 cell line, was induced to differentiate, and Acart level was assessed during differentiation using quantitative real-time PCR. The biological role of Acart in adipogenesis was analyzed by assessing lipid droplet accumulation, PPARγ and CCAAT/enhancer-binding protein α (C/EBPα) expression, and 3T3-L1 cell proliferation and apoptosis after Acart silencing. We found that Acart level was promptly increased during preadipocyte differentiation in vitro. Acart was also significantly upregulated in obese mouse-derived subcutaneous, perirenal, and epididymal fat tissues compared with nonobese mouse-derived adipose tissues. Functionally, Acart depletion inhibited preadipocyte differentiation, as evidenced by a significant decrease in lipid accumulation and PPARγ and C/EBPα expression levels. Acart silencing also inhibited 3T3-L1 cell proliferation, whereas Acart overexpression accelerated 3T3-L1 cell proliferation and decreased cell apoptosis. Taken together, the current results reveal a novel function of Acart in regulating preadipocyte proliferation and differentiation.


Introduction
Epidemiological studies show that obesity has become a public health challenge worldwide, as there are approximately 2 billion obese people [1,2]. Obesity is closely correlated with an increased risk in multiple serious disorders, including diabetes, cardiovascular disorders, and cancers [3,4], and is closely associated with preadipocyte differentiation or adipogenesis [5]. Adipogenesis is pivotal to adipose tissue mass accumulation and obesity occurrence [6,7]. Although adipogenesis is a complex process involving many proteins and noncoding RNAs (ncRNAs), it is well known that peroxisome proliferatoractivated receptor γ (PPARγ) and C/EBPα are two critical factors in adipogenesis [8,9].
Long noncoding RNAs (lncRNAs) are large and diverse noncoding transcripts that are >200 nucleotides in length and do not have protein-coding potential [10]. Over the past decade, lncRNA has attracted widespread attention from all fields of biology, such as chromatin remodeling, cell differentiation, cancer biology, and metabolism [11,12]. With the advancements of next-generation sequencing and microarrays, great progress has been made in identifying differentially expressed lncRNAs during adipogenesis. The results from RNA-sequencing analysis revealed that 175 lncRNAs are significantly dysregulated more than 2-fold during adipocyte differentiation [13]. Zhao et al. identified 21 lncRNAs enriched in brown adipose tissue that are upregulated during brown adipocyte differentiation through a microarray analysis that covered 31,423 annotated lncRNAs [14]. They further demonstrated that the overexpression of lncRNA-Blnc1 accelerates brown adipocyte differentiation. Lo et al. showed that 68 lncRNAs are dysregulated in high-fat diet (HFD)-fed obese mice and that lncRNA-Leptin accelerates adipogenesis by maintaining leptin expression [15].
Emerging studies have demonstrated that many lncRNAs control adipogenesis by regulating PPARγ signaling, which is a critical factor for adipogenesis. For instance, the overexpression of lncRNA-Plnc1 accelerates preadipocyte differentiation by activating PPAR-γ signaling [16]. lncRNA-U90926 expression is reduced during preadipocyte differentiation. Forced expression of lncRNA-U90926 suppresses adipogenesis by inhibiting PPAR-γ transactivation [9]. lncRNA-Acart (hereafter named Acart) is a newly identified lncRNA in fibrotic cardiac tissue that attenuates cardiomyocyte injury by activating PPARγ signaling [17]. Based on the above findings, the biological role of Acart in adipogenesis was investigated.

Animal model and adipose tissues
Adult male C57BL/6 mice were purchased from the Shanghai Experimental Animal Center, housed at an optimal temperature and humidity, and given free access to water and food. The animal work was performed in accordance with the guidelines and regulations of the CCCA and the approval of the Animal Ethics Committee of Shanghai Shuguang Hospital (No. PZSHUTCM220124012). Obesity was achieved by feeding the mice an HFD (catalog number: D12492, Research Diets, NJ, USA) for 7 weeks (n = 5) [18]. Normal mice (n = 5) were fed a basal diet (BD, catalog number: D12450B, Research Diets). Mice were euthanized via isoflurane inhalation, and then, subcutaneous, perirenal, and epididymal fat was collected when the difference in body weight between the two groups was 30% [9].
Ethical approval: The research related to animal use has been complied with all the relevant national regulations and institutional policies for the care and use of animals and has been approved by Animal Ethics Committee of Shanghai Shuguang Hospital (No. PZSHUTCM220124012).

Cell culture
A murine preadipocyte cell line, 3T3-L1, was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). These cells were maintained in complete medium (Dulbecco's modified eagle's medium supplemented with 10% fetal bovine serum; Product number: SLM-243-B; Sigma-Aldrich, MO, USA) in an incubator with 5% CO 2 at 37°C, as described previously [19]. Two days after complete fusion, 3T3-L1 cells were induced to differentiate (Day 0, D0) using a previously described standard cocktail regimen: 0.5 mM IBMX (Product number: 15879; Sigma-Aldrich), 10 −6 M dexamethasone (Product number: D4902; Sigma-Aldrich), and 5 µg/mL insulin (Product number: 12643; Sigma-Aldrich) (designated as MDI) [20]. Two days after this induction, the medium was replaced with complete medium supplemented with insulin (5 µg/mL) for 48 h. Finally, the cells were maintained in complete medium without any inducer.

Oil Red O staining
After treatment with the specified reagents, 3T3-L1 cells (2 × 10 5 cells per well in six-well plates) were fixed with 4% PFA (Product number: P1110; Solarbio, Shanghai, China) and stained with Oil Red O (Product number: 08010-5; Solarbio) to assess lipid accumulation, as previously described [21]. After staining, cells were visualized and photographed using a microscope (Olympus, Tokyo, Japan).

qRT-PCR
Total RNA was extracted with TRIzol reagent (Product number: T9424; Sigma-Aldrich). Genomic DNA was degraded using DNase I (Product number: EN0521; Thermo Fisher Scientific) prior to reverse transcription. cDNA was synthesized using Moloney's murine leukemia virus reverse transcriptase (Product number: M1302; Sigma-Aldrich) and random primers (3 µg/µL; Product number: 48190011; Invitrogen, CA, USA). qRT-PCR was performed in triplicate on a ABI 7500-Fast Real-Time PCR System (Applied Biosystem, CA, USA) using a OneStep qPCR kit (Product number: 210210; Qiagen, Duesseldorf, Germany). The temperature protocol was 95°C for 15 min, followed by 39 cycles of 95°C for 15 s and 58.5°C for 15 s. Beta (β)-actin served as an internal control. The mRNA levels were analyzed using the 2 (−ΔΔCT) method [22]. The primer sequences are shown in Table 1. The specificity of qRT-PCR product was ascertained through gel electrophoresis and melt curve analysis.

Cell proliferation
Cell proliferation assays were performed with cell counting kit (CCK)-8 reagent (Product number: CA1210; Solarbio). Briefly, 24 h after transfection with sh-Acart or sh-NC, 3T3-L1 cells were plated into 96-well plates (4 × 10 3 cells per well) and incubated for 1, 2, 3, and 4 days. Ten microliters of CCK-8 was added to the wells for 60 min, and then, the absorbance was measured at 450 nm with a microplate reader (Bio-Rad, CA, USA).

Edu incorporation assay
Twenty-four hours after transfection with sh-Acart or sh-NC, 3T3-L1 cells (0.5 × 10 5 cells per well in 24-well plates) were induced to differentiate for 24 h and labeled with EdU (Product number: CA1170; Solarbio) and Hoechst (Product number: CA1170; Solarbio) for 1 h. The fluorescence of EdU and Hoechst was observed using a BX53 fluorescence microscope (Olympus).

Data analysis
Data are shown as the mean ± standard deviation from three independent experiments. Statistical analysis was carried out using two-tailed Student's t-test with SPSS 20.0 statistical software (IBM, NY, USA). p < 0.05 was considered statistically significant.

Acart level was upregulated during preadipocyte differentiation
Acart is an lncRNA with 2193 bp in length, mainly located in cytoplasm ( Figure A1(a-c)). In a previous study, Acart was identified to play an important role in protecting against cardiomyocyte injury by activating PPARγ signaling [17]. Given that PPARγ is an essential transcription factor in adipogenesis, we first investigated whether Acart was dysregulated during preadipocyte differentiation. To this end, the 3T3-L1 cell line, a well-established preadipocyte, was treated with MDI differentiation medium to simulate adipogenesis in vitro. As shown in Figure 1a, MDI treatment significantly increased lipid droplet accumulation in a time-dependent manner. To further validate 3T3-L1 preadipocyte differentiation, the expression of major adipogenesis markers was assessed. The results from qRT-PCR analysis revealed that the expression of Pparγ, C/ebpα, and Fabp4 exhibited a differentiation-dependent increase ( Figure 1b). Based on these results, Acart level was assessed in 3T3-L1 cells during differentiation. Figure 1c shows that Acart levels were significantly upregulated during MDIinduced 3T3-L1 preadipocyte differentiation. , and epididymal (c) adipose tissues were collected from HFD-fed mice and BD-fed mice, and Pparγ and C/ebpα mRNA levels were assessed using the qRT-PCR analysis. Subcutaneous (d), perirenal (e), and epididymal (f) adipose tissues were collected from HFD-fed mice and BD-fed mice, and Acart levels were assessed using the qRT-PCR analysis. ***p < 0.001.

Acart was increased in adipose tissues from obese mice
To investigate the association of Acart with obesity, subcutaneous, perirenal, and epididymal fat was obtained from HFD obese mice and BD normal mice. As expected, Pparγ and C/ebpα expression levels were higher in subcutaneous, perirenal, and epididymal adipose tissues from mice with HFD-induced obesity than from mice fed a BD (Figure 2a-c). More importantly, Acart level was also markedly increased in subcutaneous, perirenal, and epididymal adipose tissues in HFD-fed obese mice compared with BD-fed normal mice (Figure 2d and f). We did not detect Acart expression in muscular tissues ( Figure A2).

Acart depletion inhibited preadipocyte differentiation
To explore the biological role of Acart in preadipocyte differentiation, 3T3-L1 cells were treated with pLKO.1-sh-Acart to repress Acart level and then treated with MDI differentiation medium. The results from Oil Red O staining showed that Acart depletion resulted in a significant decrease in lipid droplet accumulation (Figure 3a). Figure 3b shows that the expression levels of four major adipogenesis markers (Pparγ, C/ebpα, Fabp4, and Adipoq) were decreased in Acart-deficient cells. The results from the western blot analysis revealed that PPARγ and C/EBPα protein expression levels were significantly decreased in Acart-deficient 3T3-L1 cells (Figure 3c-e).

Acart depletion inhibited preadipocyte proliferation
Given that preadipocyte proliferation is an essential precondition for cell differentiation [19] and that Acart level is promptly increased during preadipocyte differentiation, the role of Acart in regulating preadipocyte proliferation was further explored. The results from the EdU assay showed that the EdU-positive cell proportion was significantly decreased in Acart-deficient cells compared with the negative control cells (Figure 4a and b). Acart knockdown increased BAX expression and decreased BCL-2 expression in 3T3-L1 cells, indicating that Acart knockdown facilitated cell apoptosis (Figure 4c). The CCK-8 assay revealed that cell proliferation was significantly repressed from day 2 to day 4 after Acart depletion (Figure 4d). Conversely, Acart overexpression accelerated preadipocyte proliferation (Figure 5a and b) and inhibited cell apoptosis ( Figure  5c and d). Acart overexpression decreased BAX expression and increased BCL-2 expression in 3T3-L1 cells, indicating that Acart inhibited cell apoptosis (Figure 5e). The results from Oil Red O staining revealed that Acart overexpression resulted in a significant increase in lipid droplet accumulation (Figure 5f). These results demonstrate that Acart knockdown inhibits preadipocyte proliferation and differentiation.

Discussion
Adipocytes are a major component of adipose tissue and exert a critical role in homeostatically regulating whole body metabolism [23]. In addition to the primary function of controlling energy balance, adipocytes play an important role in regulating tissue metabolism [24], immune responses [25,26], insulin resistance [27], and cardiovascular disorders [23,28,29]. Preadipocyte differentiation is a complex multistep process involving many transcription factors and differentiation-related proteins and ncRNAs [8]. Here, we demonstrated that (1) Acart was upregulated during preadipocyte differentiation, (2) Acart was increased in adipose tissues from obese mice, (3) Acart silencing inhibited preadipocyte differentiation, and (4) Acart silencing inhibited preadipocyte proliferation. The current data identified the function of Acart in accelerating adipogenesis and indicated that Acart might exert an important role in adipogenesis. PPARγ and C/EBPα synergistically control preadipocyte differentiation and are frequently used as markers to indicate adipose differentiation [30]. As a transcription factor, PPARγ can initiate the transcription of lipid metabolism-related mRNAs, including those for perilipin1 [31], fatty acid synthase [32], and insulin-like growth factor-binding protein-2 [33]. The mechanisms underlying PPARγ signaling activation are constantly being revealed. For instance, the upregulation of lncRNA-Gm15290 in adipose tissue accelerates fat deposition by activating PPARγ [34].
Recently, transcriptomic analyses have identified thousands of abnormally expressed lncRNAs during adipogenesis, and functional analyses have revealed the biological role of lncRNAs in adipocyte development and differentiation. Through microarray analysis, Xiao et al. revealed that 677 lncRNAs are downregulated and 513 lncRNAs are upregulated in mesenteric white adipose tissues (mWATs) from fasted mice compared with the mWATs from control mice [20]. Functional studies further demonstrated that the downregulation of lncRNA-Fr332443 contributes to adipogenesis by regulating RUNX1 and the mitogen-activated protein kinase pathway [20]. lncRNA-Plnc1 could upregulate PPARγ2 transcription and expression by repressing the methylation of the PPARγ2 promoter and thus promote adipogenesis [16].
In a recent study, a newly identified lncRNA, Acart, exerted a protective effect against cardiomyocyte injury by transactivating PPARγ signaling. Given the key role of PPARγ in adipogenesis, we next investigated whether Acart regulates preadipocyte differentiation. As expected, Acart was increased during 3T3-L1 preadipocyte differentiation. Moreover, Acart was markedly increased in obese mouse-derived adipose tissues. Functionally, Acart knockdown significantly decreased 3T3-L1 preadipocyte proliferation and differentiation. There are major limitations of this study. First, the direct effect of Acart on regulating PPARγ signaling activation has not been validated in 3T3-L1 cells. Second, the underlying mechanism by which Acart accelerates 3T3-L1 cell proliferation and differentiation has not been identified. Generally speaking, lncRNAs enriched in the nucleus epigenetically control gene expression by regulating histone methyltransferase-or deacetylasemediated chromatin remodeling [35,36]. lncRNAs located in the cytoplasm commonly function as competing endogenous RNAs to control target gene expression [37,38]. It is essential to identify the subcellular localization of Acart in 3T3-L1 cells to explore the underlying mechanisms.
Funding information: Authors state no funding involved.
Conflict of interest: Authors state no conflict of interest.
Data availability statement: The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. Figure