Identification of Potential Methylation Regions of the Smad4 mRNA and Determining Primer Sequences for MS-PCR with the ‘Methprimer’ Program

Abstract

SMAD4 is a member of an intracellular signaling pathway protein family that is widely expressed in human tissues. This protein is responsible for carrying a chemical signal from the cell membrane to the nucleus. Since reduced SMAD4 expression leads to several tumors and neural disease, it is important to elucidate the mechanisms affecting the expression of this protein. Methylation is among the major factors that affect the expression of the SMAD4 gene. While methylation of the promoter and non-coding exons of SMAD4 gene appear to affect expression, there is no information regarding the other regions of this gene in this regard. Furthermore, cytosine methylation in mRNA is also important in gene activity. For this reason, the demonstration of possible cytosine methylation in mRNA of the SMAD4 gene may be important in understanding gene activity. In this study, we aimed to determine the potential methylation regions in the exons corresponding to SMAD4 protein generation which have not been investigated before. In order to do this, we used the MethPrimer program and identified 25 single CpG sequences and a double CpGpCpG across the exons as potential methylation regions. In addition, 5 pairs of methylated/unmethylated primer sequences were designed with the same program. The study results have shown the presence of potential methylation sequences that are candidates to affect SMAD4 gene expression.