Effects And Mechanism Of Methylation Status Of Gene Promoter On Secretion Levels Of Tumor Necrosis Factor Alpha In THP-1 Cells

Objective:To detect the expression and secretion of tumor necrosis factor alpha (TNF-a) in THP-1 cells. To investigate the correlation between the expression and secretion of TNF-a and methylation status of the TNF-a promoter. To investigate the mechanism of epigenetic regulation of TNF-a.Methods:Stimulated by 1μg/ml Lipopolysaccharide (LPS) at different times, TNF-a secretion levels of the THP-1 cells were examined with enzyme-linked immunosorbent assay (ELISA). Real-time quantitative PCR were used to investigate the expression of TNF-a. Investigated the peak time of the expression and secretion of TNF-a. The methylation status of the TNF-a promoter was measured with bisulfite sequencing analysis. Investigated the correlation between the expression and secretion of TNF-a and methylation status of the TNF-a promoter. Real-time quantitative PCR were used to investigate the expression of Methyl-CpG binding proteins. Analysed the mechanism of epigenetic regulation of TNF-a.Results:In unstimulated THP-1 cells, secretion levels of TNF-a were very low, and all the eleven CpG dinucleotides in gene promoter ranging from-344 bp to transcription start site (TSS) were completely methylated. In 4 h LPS-stimulated THP-1 cells, secretion levels of TNF-a reached their peak concentrations, and CpG dinucleotides in gene promoter were extensively demethylated, only four in eleven were methylated. In 8 h LPS-stimulated THP-1 cells, secretion levels of TNF-a dropped from the high concentration, and nine CpG dinucleotides in eleven in gene promoter were methylated. The percentages of methylation between unstimulated and 4 h LPS-stimulated THP-1 cells were statistical different (P<0.01). And the percentages of methylation between 4 h and 8 h LPS-stimulated THP-1 cells were statistical different (P<0.05). The expression of Methyl-CpG binding proteins had a relationship with the stimulation of LPS.Conclusion:1. Secretion levels of TNF-a showed strong association with the stimulated time of THP-1 cells. In unstimulated THP-1 cells, secretion levels of TNF-a were very low; In 4 h LPS-stimulated THP-1 cells, secretion levels of TNF-a reached their peak concentrations; In 8 h LPS-stimulated THP-1 cells, secretion levels of TNF-a dropped from the high concentration.2. TNF-a promoter methylation status had an association with the stimulated time of THP-1 cells.3. TNF-a promoter methylation status showed an association with the expression and secretion levels of the TNF-a.4. Methyl-CpG binding protein may play a key role in epigenetic regulation of TNF-a.