Association of Self-DNA Mediated TLR9-Related Gene, DNA Methyltransferase, and Cytokeratin Protein Expression Alterations in HT29-Cells to DNA Fragment Length and Methylation Status

Abstract EN

To understand the biologic role of self-DNA bound to Toll-like Receptor 9 (TLR9), we assayed its effect on gene and methyltransferase expressions and cell differentiation in HT29 cells.

HT29 cells were incubated separately with type-1 (normally methylated/nonfragmented), type-2 (normally methylated/fragmented), type-3 (hypermethylated/nonfragmented), or type-4 (hypermethylated/fragmented) self-DNAs.

Expression levels of TLR9-signaling and proinflammatory cytokine-related genes were assayed by qRT-PCR.

Methyltransferase activity and cell differentiation were examined by using DNA methyltransferase (DNMT1, -3A, -3B) and cytokeratin (CK) antibodies.

Treatment with type-1 DNA resulted in significant increase in TLR9 expression.

Type-2 treatment resulted in the overexpression of TLR9-related signaling molecules (MYD88A, TRAF6) and the IL8 gene.

In the case of type-3 treatment, significant overexpression of NFkB, IRAK2, and IL8 as well as downregulation of TRAF6 was detected.

Using type-4 DNA, TRAF6 and MYD88A gene expression was upregulated, while MYD88B, IRAK2, IL8, and TNFSF10 were all underexpressed.

CK expression was significantly higher only after type-1 DNA treatment.

DNMT3A expression could also be induced by type-1 DNA treatment.

DNA structure may play a significant role in activation of the TLR9-dependent and even independent proinflammatory pathways.

There may be a molecular link between TLR9 signaling and DNMT3A.

The mode of self-DNA treatment may influence HT29 cell differentiation.