The MBD4 Gene Plays an Important Role in Porcine Adipocyte Differentiation

Abstract

Background: MBD4 (methyl-CpG binding domain protein 4) is an important G: T glycosylase that can identify T-G mismatches. It plays a role in active demethylation through base excision repair. Overexpression of MBD4 gene can cause the demethylation of numerous genes, and the remethylation of MBD4-associated genes can occur when the MBD4 gene is knocked out. To date, the functions and regulatory mechanisms of the MBD4 gene in the differentiation of porcine preadipocytes have not been clearly established. Methods: Subcutaneous fat cells from 1- to 7-day-old Junmu-1 piglets were cultured in vitro, induced to differentiate, and then identified. A real-time fluorescence-based quantitative polymerase chain reaction (PCR) analysis was conducted to detect MBD4 messenger RNA (mRNA) expression. Cells were treated with MBD4-siRNA (small interfering RNA) and induced to differentiate. Changes in the lipid droplets were observed by oil red O staining. Changes in the mRNA and protein expression levels of MBD4 and the adipose differentiation-associated genes C/EBPα (CCAAT-enhancer-binding protein alpha), PPARγ (peroxisome proliferator-activated receptor gamma), and aP2 (adipocyte protein 2) were detected. In addition, the bisulfite sequencing method was used to detect changes in methylation in the promoters of certain genes associated with adipose differentiation. Results: Levels of MBD4 mRNA and protein expression varied with time over the course of the porcine adipocyte differentiation, with the highest levels of this expression observed on day two of the differentiation process. After silencing MBD4 and inducing differentiation, the production of lipid droplets decreased, the mRNA expression levels of C/EBPα, PPARγ, and aP2 were significantly reduced, and DNA methylation modification levels were significantly elevated in the examined promoter regions. Conclusion: The silencing of the MBD4 gene can influence the DNA methylation levels of preadipocyte differentiation-related genes and subsequently inhibit the differentiation of porcine preadipocytes.

miR‐375 negatively regulates porcine preadipocyte differentiation by targeting BMPR2

“…The cells were then washed twice with PBS, stained with 10% Oil Red O solution (solvent: 70% ethanol) for 15 min, decolorized rapidly in 75% ethanol, and washed twice with PBS. Subsequently, the cells were counterstained with hematoxylin for 15 s and washed three times with PBS . The stained cells were observed under a microscope and photographed.…”

MiR-378 Plays an Important Role in the Differentiation of Bovine Preadipocytes

“…The reaction system was as follows: 0.5 μl (10 pmol/L) of upstream primer, 0.5 μl (10 pmol/L) of downstream primer, 8.0 μl of ddH 2 O, 1 μl of template and 10 μl of SYBR R Premix Ex Taq II (2x), with the following conditions: 95°C for 30 s; and 40 cycles of 95°C for 5 s and 60 °C for 30 s. The U6 small nuclear RNA (snRNA) was used as an internal control for miR-378, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control for other genes. Experimental data were analyzed using a relative quantification method (2 [16,17].…”

The Landscape of DNA Methylation Associated With the Transcriptomic Network of Intramuscular Adipocytes Generates Insight Into Intramuscular Fat Deposition in Chicken

“…Previous studies recommended that DNMT3A inhibited porcine intramuscular preadipocytes differentiation by changing the methylation levels of p21 and PPARγ (Abdalla et al, 2018;Qimuge et al, 2019). Zhang et al (2014) found that MBD4 inhibited porcine preadipocytes differentiation by changing the DNA methylation levels of adipogenic genes. Li et al suggested that DNA methylation regulated chicken PPARG and CEBPA during the development of chicken adipose tissue (Sun et al, 2014;Gao et al, 2015).…”