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A. Sultana, H.-S. Lee, N. Nakaya, S. Tomarev; Up-Regulation of WIF1 Gene Expression by Over-Expression of Olfactomedin 1. Invest. Ophthalmol. Vis. Sci. 2009;50(13):2912.
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The olfactomedin 1 (Olfm1) gene is expressed in the developing and adult retina and induces outgrowth of retinal ganglion cell axons. Its over-expression in stably transfected rat pheochromocytoma PC12 cells changes the expression pattern of several genes including the antagonist of Wnt signaling, Wif1. Since WIF1 expression is down-regulated in several cancers, the aim of our present study is to determine whether over-expression of Olfm1 may induce WIF1 expression in other cancer cell lines and whether up-regulation of the Wif1 gene in PC12 cells affects the methylation status of its promoter.
Lung and breast cancer cell lines were stably transfected with empty retroviral vector, retroviral vector containing EGFP or with vector containing rat Olfm1 cDNA. Quantitative RT-PCR was used to analyze the expression levels of WIF1 in different cell lines and control PC12 cells treated with 5-azadeoxycytidine and TSA Methylation of the Wif1 proximal promoter and 5’ untranslated region in stably transfected PC12 and control cells was analyzed by combined bisulfite sequencing and pyrosequencing.
Over-expression of Olfm1 in PC12 cells increased the levels of Wif1 mRNA by more than three hundred fold compared to control PC12 cells. However no significant changes in the levels of WIF1 mRNA were observed in cancer cell lines over-expressing rat Olfm1 cDNA. Treatment of control PC12 cells with 5-azadeoxycytidine and TSA increased levels of Wif1 mRNA by four and two hundred fold respectively. The majority of the CpG analyzed were unmethylated in PC12 cells stably transfected with Olfm1 or vector. A single CpG site in the 5’ untranslated region showed 32.3 % methylation in the vector as compared to 17.5% in Olfm1 transfected cells by pyrosequencing as well as bisulfite sequencing.
Our results indicate that up-regulation of Wif1 expression by Olfm1 might be a tissue specific phenomenon restricted to the cells with neural origin. Methylation of CpG islands in the Wif1 proximal promoter may not be a significant cause of up-regulation of Wif1 gene expression in Olfm1 stably transfected PC12 cells. Mechanisms such as histone modification or other transactivating factors might play an important role.
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