TNF-α-Induced NF-κB Alter the Methylation Status of Some Stemness Genes in HT-29 Human Colon Cancer Cell

Background: Acquisition of stem-like properties requires overcoming the epigenetic barrier of differentiation and re-expression of several genes involved in stemness and the cell cycle. DNA methylation is the classic epigenetic mechanism for de/differentiation. The writers and erasers of DNA methylation are not site-specific enzymes for altering specific gene methylation. Thus, the aim of the present study is investigation of the in vitro interaction of ten eleven translocations (TETs) with nuclear factor kappa B (NF-κB) in hypomethylation of stemness genes. Materials and Methods: This experimental study was performed on HT-29 cells as human colorectal cancer cell lines. The interaction between TETs and DNA-methyltransferases 3 beta (DNMT3s) with p65 was achieved by coimmunoprecipitation. TETs were knocked down using siRNA, and the efficacy was analyzed by reverse-transcriptase polymerase chain reaction. The promoter methylation status of the target genes (NANOG, MYC) was determined by the methylation-sensitive high-resolution melting method. Results: TET3 and DNMT3b functionally interacted with p65 in samples through 25 ng/ml TNF-α treatment for 48 h in HT-29 cells. Transfection with siRNA significantly decreased the expression of TET enzymes after 72 h. Interestingly, treatment with TET siRNAs enhanced methylation of MYC and NANOG genes in samples with 25 ng/ml TNF-α treatment for 72 h in HT-29 cells. Moreover, methylation effects of TET3 were stronger than those of TET1 and TET2. Conclusions: These results suggest that inflammation may alter the methylation status of genes required for stemness and predispose the cells to neoplastic alterations.