engleski

Methylation status of PTCH promoter in tumors with alterations in Hh-Gli signaling pathway

PTCH, a known tumor suppressor has a central role in the HH-GLI signaling pathway. It is a 12-pass transmembrane glycoprotein, which inhibits Smo in the absence of Shh signal. If Shh is present, the repression of Smo is relieved and signaling can proceed down to Gli trancription factor and cause target gene transcription. It is known that epigenetic changes related to specific methylation patterns contribute to cancer development. Many tumor suppressor gene lose function if their promoter region is hypremethylaed, and such CpG sites often show increased mutation rate. Since functioning of the HH-GLI pathway is still not completely understood, we intend to examine DNA methylation of PTCH promoter. We used bisulfite genomic sequencing method (Frommer et al, PNAS, 1992) for the analysis of the methylation status of the PTCH promoter. The method is based on chemical alteration of DNA, where bisulfite reacts with DNA and, as a result, cytosine is deaminated into uracil, but 5-methylcytosine remains unaltered. Primers specefici for both methylated and unmethylated DNA were used to amplify a target sequence, which was then sequenced and analysed. PTCH promoter sequence was acquired from NCBI database (gi 14787441), and location of the Gli1 binding site and start codon were determined (Agren et al, Gene, 2004). The part of the sequence encompassing both these locations was screened for CpG islands. CpG island 1593 bp long, which includes 163 CpG sites, was determined and primers were designed to amplify both methylated and unmethylated DNA. Comparison of methylation patterns with previous expression analysis showed a significant correltion between PTCH promoter methylation and reduced PTCH expression.

methylation; patched promoter; ovarian tumors

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