Abstract
To elucidate the currently unknown mechanisms responsible for the diverse biological aspects between two-dimensional (2D) and three-dimensional (3D) cultured 3T3-L1 preadipocytes, RNA-sequencing analyses were performed. During a 7-day culture period, 2D- and 3D-cultured 3T3-L1 cells were subjected to lipid staining by BODIPY, qPCR for adipogenesis related genes, including peroxisome proliferator-activated receptor γ (Pparγ), CCAAT/enhancer-binding protein alpha (Cebpa), Ap2 (fatty acid-binding protein 4; Fabp4), leptin, and AdipoQ (adiponectin), and RNA-sequencing analysis. Differentially expressed genes (DEGs) were detected by next-generation RNA sequencing (RNA-seq) and validated by a quantitative reverse transcription–polymerase chain reaction (qRT–PCR). Bioinformatic analyses were performed on DEGs using a Gene Ontology (GO) enrichment analysis and an Ingenuity Pathway Analysis (IPA). Significant spontaneous adipogenesis was observed in 3D 3T3-L1 spheroids, but not in 2D-cultured cells. The mRNA expression of Pparγ, Cebpa, and Ap2 among the five genes tested were significantly higher in 3D spheroids than in 2D-cultured cells, thus providing support for this conclusion. RNA analysis demonstrated that a total of 826 upregulated and 725 downregulated genes were identified as DEGs. GO enrichment analysis and IPA found 50 possible upstream regulators, and among these, 6 regulators—transforming growth factor β1 (TGFβ1), signal transducer and activator of transcription 3 (STAT3), interleukin 6 (IL6), angiotensinogen (AGT), FOS, and MYC—were, in fact, significantly upregulated. Further analyses of these regulators by causal networks of the top 14 predicted diseases and functions networks (IPA network score indicated more than 30), suggesting that STAT3 was the most critical upstream regulator. The findings presented herein suggest that STAT3 has a critical role in regulating the unique biological properties of 3D spheroids that are produced from 3T3-L1 preadipocytes.
Highlights
The number and sizes of adipocytes, which are elastic and plastic cells in fat tissue, are altered during adipogenic differentiation or are eliminated through necrosis or apoptosis in response to fluctuations in metabolism states [1,2,3]. Since those characteristic changes in adipocytes occur within the three-dimensional (3D) spaces of the body, a 3D cell culture method would be expected to be more representative than conventional two-dimensional (2D) cell culture methods in adipocyte-related research
We reported on the production of 3D spheroids of 3T3L1 cells and human orbital adipocytes by a 3D drop culture technique [6] and, using those, we were able to establish in vitro models that replicate prostaglandin-induced orbital fat atrophy, a condition called “deepening of the upper eyelid sulcus (DUES)” [7], and thyroidassociated orbitopathy [6]
Included among these differences were that (1) 2.1. Two-Dimensional (2D) 3T3-L1 cells were rapidly dispersed within a few minutes in the presence of 0.2% trypsin, whereas 3D 3T3-L1 preadipocyte spheroids started to disperse only after 3 h, and up to 12 h was needed for this process to reach completion, and 3D 3T3-L1 adipocytes were relatively resistant to 0.2% trypsin for periods of up to 12 h, and (2) adipogenic differentiation was much more efficient in the case of 3D 3T3-L1 spheroids, compared with 2D-cultured 3T3-L1 cells [8,9,10,11,12]
Summary
The number and sizes of adipocytes, which are elastic and plastic cells in fat tissue, are altered during adipogenic differentiation or are eliminated through necrosis or apoptosis in response to fluctuations in metabolism states [1,2,3]. We reported on the production of 3D spheroids of 3T3L1 cells and human orbital adipocytes by a 3D drop culture technique [6] and, using those, we were able to establish in vitro models that replicate prostaglandin-induced orbital fat atrophy, a condition called “deepening of the upper eyelid sulcus (DUES)” [7], and thyroidassociated orbitopathy [6]. During these studies, we identified some rather interesting and unidentified differences, in that the 3D spheroids showed some distinct differences from the conventional 2D cultures. In the present study, to elucidate the above unknown mechanisms for causing such a distinct phenomenon, we carried out an RNA-sequencing analysis, which is a powerful tool for identifying potential target genes for diseases and the underlying pathological mechanisms [13], between 2D- and 3D-cultured 3T3-L1 cells
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