Role of DNA methylation in WNT5A Promoter B expression in Osteosarcoma (SaOS-2) cells.

UNCG Author/Contributor (non-UNCG co-authors, if there are any, appear on document)
Himani Vaidya (Creator)
The University of North Carolina at Greensboro (UNCG )
Web Site:
Karen Katula

Abstract: WNT5A, a member of the WNT family of secreted proteins, activates the non-canonical WNT pathway, regulates developmental events and is involved in tissue homeostasis. Misregulation of WNT5A has been associated with various types of cancer including pancreatic, colorectal, breast, lung and osteosarcoma. Recent studies have shown that WNT5A expression in cancer cells involves non-genetic (epigenetic) changes. The WNT5A gene has two similar transcription start sites (termed promoter A and promoter B in this study) and transcription from these sites give rise to two different messages that encode different protein isoforms. Epigenetic studies to date have focused on the WNT5A promoter A, however our preliminary analysis found that promoter B transcripts are nearly absent in osteosarcoma cells, but present in normal osteoblasts. The goal of this study was to determine if the decrease in promoter B transcripts in osteosarcoma was due to DNA methylation. We identified 6 CpG islands in the WNT5A intron 1 region which contains promoter B and exon 1B, and compared them with data available in NCBI epigenomics website. The NCBI database was also screened for data on the methylation status of these islands in various other cell lines and cancer types. The database analysis shows that there is little or no methylation of Regions 1 - 6 in cells, that normally express WNT5A (fibroblast, chondrocytes, and mesenchymal stem cells). However, in some colorectal tumor tissue there is extensive methylation within intron 1. In adjacent normal colon mucosa tissue, Regions 3 and 4 showed some degree of methylation. Methylation status of the CpG islands in normal osteoblasts and osteosarcoma (SaOS-2) was determined using Sodium bisulfite sequencing. The sequencing data showed that CpG Regions 1 and 2 are unmethylated. Regions 3, 4 and 5 are completely methylated and Region 6 is partially methylated. In normal osteoblasts all 6 regions were unmethylated. A demethylation experiment using 5-Aza-cytidine was performed to determine if decrease in methylation would increase promoter B transcripts. Treatment of SaOS-2 cells with 1µM 5-Aza-cytidine resulted in a 120 fold increase of promoter B transcripts. The methylation data of SaOS-2 cells treated with 1µM 5-Aza-cytidine shows that CpG Region 6 is more prone to demethylation when compared to CpG islands 3, 4, 5. Two clones of Region 6 showed that 4 out of 5 CpG's analyzed were demethylated. 5 clones of Region 3 and 4 showed that only 1-2 CpG's were demethylated out of 24 CpG's in Region 3 and 27 CpG's in Region 4. Overall these results suggest that promoter B transcription is down regulated by DNA methylation of CpG Region 6 and Region 6 is more prone to demethylation compared to Regions 3, 4 and 5.

Additional Information

Language: English
Date: 2013
Epigenetics, Osteosarcoma, WNT5A
Wnt proteins
DNA $x Methylation
Cancer $x Molecular aspects

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