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Transient Delivery of A-C/EBP Protein Perturbs Differentiation of 3T3-L1 Cells and Induces Preadipocyte Marker Genes.

ABSTRACT: Transformation of committed 3T3-L1 preadipocytes to lipid-laden adipocytes involves the timely appearance of numerous transcription factors (TFs); foremost among them, C/EBP? is expressed during the early phases of differentiation. Here, we describe liposome-mediated protein transfection approach to rapidly downregulate C/EBP? by A-C/EBP protein inhibitor. Signals from EGFP-tagged A-C/EBP protein were observed in 3T3-L1 cells within 2 h of transfections, whereas for A-C/EBP gene transfections, equivalent signals appeared in 48 h. Following transient transfections, the expression profiles of 24 marker genes belonging to pro- and anti-adipogenic, cell cycle, and preadipocyte pathways were analyzed. Expectedly, the mRNA and protein expression profiles of adipocyte marker genes showed lower expression in both A-C/EBP protein- and gene-transfected samples. Interestingly, for preadipocytes and cell fate determinant genes, striking differences were observed between A-C/EBP protein- and A-C/EBP gene-transfected samples. Preadipocyte differentiation factors Stat5a and Creb were downregulated in A-C/EBP protein samples. Five preadipocyte markers, namely, Pdgfr?, Pdgfr?, Ly6A, CD34, and Itgb1, showed high expression in A-C/EBP protein samples, whereas only Ly6A and CD34 were expressed in A-C/EBP gene-transfected samples. Pdgfr? and Pdgfr?, two known cell fate markers, were expressed in A-C/EBP protein-transfected samples, suggesting a possible reversal of differentiation. Our study provides evidences for the immediate and efficient knockdown of C/EBP? protein to understand time-dependent preadipocytes differentiation.

SUBMITTER: Sharma N

PROVIDER: S-EPMC7868408 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Transient Delivery of A-C/EBP Protein Perturbs Differentiation of 3T3-L1 Cells and Induces Preadipocyte Marker Genes.

Sharma Nishtha N Kaur Raminder R Yadav Binduma B Shah Koushik K Pandey Harshita H Choudhary Diksha D Jain Prateek P Aggarwal Aanchal A Vinson Charles C Rishi Vikas V

Frontiers in molecular biosciences 20210125

Transformation of committed 3T3-L1 preadipocytes to lipid-laden adipocytes involves the timely appearance of numerous transcription factors (TFs); foremost among them, C/EBPβ is expressed during the early phases of differentiation. Here, we describe liposome-mediated protein transfection approach to rapidly downregulate C/EBPβ by A-C/EBP protein inhibitor. Signals from EGFP-tagged A-C/EBP protein were observed in 3T3-L1 cells within 2 h of transfections, whereas for A-C/EBP gene transfections, e ...[more]

PMID: 33569390

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Project description:Obesity is a risk factor for metabolic diseases, while preadipocyte differentiation or adipogenesis is closely related to obesity occurrence. Long noncoding RNAs (lncRNAs) are a unique class of transcripts in regulation of a variety of biological processes. Using cDNA microarray, we found lncRNA U90926 is negatively correlated with 3T3-L1 preadipocyte differentiation.The aim of this study was to explore the role of lncRNA U90926 (lnc-U90926) in adipogenesis and the underlying mechanisms.Quantitative real-time PCR (qPCR) was performed to determine lnc-U90926 expression in 3T3-L1 preadipocytes, differentiated adipocytes, and in adipose tissues form mice. RNA fluorescent in situ hybridization (FISH) was performed to determine the localization of lnc-U90926 in 3T3-L1 preadipocytes. The effects of lnc-U90926 on 3T3-L1 adipogenesis were analyzed with lentivirus-mediated gain- and loss-of-function experiments. Lipid accumulation was evaluated by oil red O staining; several adipogenesis makers were analyzed by qPCR and western blotting. Dual luciferase assay was applied to explore the transactivation of target genes modulated by lnc-U90926. All measurements were performed at least for three times.Lnc-U90926 expression decreased along the differentiation of 3T3-L1 preadipocytes. In mice, lnc-U90926 is predominantly expressed in adipose tissue. Obese mice have lower lnc-U90926 expression in subcutaneous and visceral adipose tissue than non-obese mice. FISH results showed that lnc-U90926 was mainly located in the cytoplasm. Overexpression lnc-U90926 attenuated 3T3-L1 adipocyte differentiation as evidenced by its ability to inhibit lipid accumulation, to decrease the mRNA levels of peroxisome proliferator-activated receptor gamma 2 (PPAR?2), fatty acid binding protein 4 (FABP4) and adiponectin (AdipoQ) as well as to reduce the protein levels of PPAR? and FABP4 (P<0.05). Knockdown of lnc-U90926 showed opposite effects, which increased mRNA expression of PPAR?2, FABP4, CCAAT/enhancer-binding protein? (C/EBP?) and AdipoQ.Lnc-U90926 attenuates 3T3-L1 adipocyte differentiation via inhibiting the transactivation of PPAR?2 or PPAR?.

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Dataset Information

Transient Delivery of A-C/EBP Protein Perturbs Differentiation of 3T3-L1 Cells and Induces Preadipocyte Marker Genes.

Publications

Transient Delivery of A-C/EBP Protein Perturbs Differentiation of 3T3-L1 Cells and Induces Preadipocyte Marker Genes.

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