engleski

Methylation status of PTCH promoter in tumors with alterations in HH-GLI signaling pathway

The Hh-Gli or SHH/PTCH/SMO signaling pathway is an important regulator of embrional development and oncogenic transformation. The pathway is normally activated when the ligand Shh binds to Ptch, suspending the inhibition of Smo and turning on the controlled expression of target genes which include GLI1, PTCH itself and other presently unknown genes. PTCH, a known tumor suppressor, has a central role in the pathway. It is a 12-pass transmembrane protein and is involved in signal transduction down to Gli transcriptional factor, along with its coreceptor Smo. It is known that epigenetic changes related to specific methylation patterns contribute to cancer development. Cancer cell genomes are often generally hypomethylated relative to normal tissue. This is due to the loss of methylation from repetitive regions of the genome, and is also linked with overexpression of known oncogenes. On the other hand, it has been shown that many tumor-suppressor genes lose function if their promoter region is hypermathylated, and such CpG sites often show increased mutation rate. Methylation in normal mammalian cells occurs on 3-5% of normal cytosine residues, and all of these cytosines are contained within CpG rich regions. Promoter regions are often composed of these islands. Since functioning of the Hh-Gli pathway is still not fully understood, we wanted to examine DNA methylation of PTCH promoter. Methylation can be determined using bisulfite genomic sequencing method (Frommer et al, PNAS, 1992). 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 specific for both methylated and unmethylated DNA are used to amplify target sequence, which is then sequenced and analyzed. Ptch promoter sequence was acquired from NCBI database (gi 14787441), and locations of the Gli1 binding site and start codon were determined, according to paper from Agren et al. (2004). The part of the sequence encompassing both these locations was screened for CpG islands. A CpG island 1344 bp long, which includes 143 CpG sites, was determined and primers were designed to amplify both methylated and unmethylated DNA. Results confirmed previous observations of epigenetic influence on expression patterns of PTCH and GLI1 in tumors where we detected alterations in Hh-Gli signaling.

methylation ; patched promoter ; ovarian tumors

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