Reduced Expression And Aberrant Promoter Methylation Of Wnt Inhibitory Factor-1 In Astrocytomas

Background:Wnt/β-catenin pathway may play a major role in the tumorigenesis and tumor progression in many tumor types including astrocytomas.Wnt inhibitory factor-1(WIF-1) acts as a Wnt-antagonists and tumor suppressor, but hypermethylation of WIF-1 gene promoter and low expression activate Wnt signaling aberrantly and induce the development of various human tumors. With this work we intended to investigate the relation between expression and promoter methylation status of WIF-1 gene in human astrocytomas, and illuminate the impact of WIF-1 gene in astrocytomas with WIF-1 silencing.Methods:Firstly,we analysed expression and methylation status of WIF-1 in 35 astrocytomas and 4 normal brain tissues by immunohistochemistry, semiquantitative RT-PCR and methylation specific PCR. The relationship between methylation and expression of the genes was analyzed. Secondly, U251 cell lines and primary-culture cells derived from 3 patients with glioblastoma were treated with demethylating agent,5-aza-2'-deoxycytidine (5-aza-CdR), to further examine the role of methylation in silencing of WIF-1. Finally, we restored the expression of WIF-1 by transfer with a pReceiver-M03-WIF1 vector into U251 cell lines lacking WIF-1 expression and analysed the effects of WIF-1 on U251 cell lines by cell proliferation, colony formation, and Flowcytometry(FCM).Results:Significant downregulation of WIF-1 mRNA and protein expression was observed in astrocytoma tissues compared with normal brain tissues. WIF-1 gene aberrant methylation was observed in 20(57.14%) of 35 tumor samples. Hypermethylation in the WIF-1 promoter region correlated with downregulation of WIF-1 expression in astrocytoma tissues. Moreover, treatment with demethylating 5-aza-2'-deoxycytidine restored WIF-1 expression in U251 cell lines and primary-culture cells derived from 3 patients with glioblastoma. Transfection of the WIF-1 gene into U251 cell lines resulted in a significant inhibition on cell proliferation and colony formation and an increased fraction of cells in the G0/G1-phase and subsequent decrease in S-phase and G2/M-phase.Conclusion:Our results demonstrate that the WIF-1 gene is frequently down-regulated or silenced in astrocytomas by aberrant promoter methylation and that this epigenetic alteration is involved in glioma cell proliferation and cell cycle. This may be an important mechanism in astrocytoma carcinogenesis.