Elucidation of activation mechanism of MN/CA9 in human renal cell carcinomas

MN/CA 9基因5 '端低甲基化与肾癌组织中MN/CA 9表达的相关性MN/CA 9基因5'端-74bp的CpG低甲基化与肾癌细胞系和肾癌组织中MN/CA 9的表达密切相关。通过亚硫酸氢盐基因组测序方案检查甲基化状态。用5-氮杂-2 ′-脱氧胞苷（5-aza-2 ′-deoxycytidine）处理MN/CA 9阴性肾癌细胞系，可激活MN/CA 9基因，并鉴定MN/CA 9启动子区。体外DNA甲基化对MN/CA 9启动子活性的影响将MN/CA 9启动子（-158 / +42 bp）/荧光素酶报告质粒分别用CpG甲基化酶（SssI或HhaI）处理，转染MN/CA 9阳性肾癌细胞系。MN/CA 9启动子的所有CpG均被SssI甲基化，仅-74bp处的CpG被HhaI特异性甲基化，SssI和HhaI均能抑制MN/CA 9启动子的活性，提示MN/CA 9启动子-74bp处的CpG低甲基化可能在人肾癌MN/CA 9表达中起重要作用。

Correlation between MN/CA9 expression and hypomethylation of MN/CA9 5'flanking region in human renal cell carcinomasHypomethylation of the CpG at -74 bp of MN/CA9 was strongly correlated with MN/CA9 expression in both human renal cell carcinoma (RCC) cell lines and human RCC tissues. Methylation status was examined by bisulfite genomic sequencing protocol. MN/CA9 expression was examined by RT-PCR.Induction of MN/CA9 expression by treatment with a demethylating agentTreatment with the demethylating agent 5-aza-2'-deoxycytidine resulted in activation of the MN/CA9 gene in MN/CA9 negative RCC cell lines.Identification of MN/CA9 promoter regionDNAs of MN/CA9 5' flanking region (-1246 / +42 bp) were ligated into luciferase reporter plasmids. Deletion analyses revealed that 5' end of minimal MN/CA9 promoter was located between -158 bp and -58 bp.Effect of in vitro DNA methylation on promoter activityMN/CA9 promoter (-158 / +42 bp) / luciferase reporter plasmids were treated with CpG methylases (SssI or HhaI) and transfected into MN/CA9 positive RCC cell lines. All CpGs of MN/CA9 promoter were methylated by SssI, and only CpG at -74 bp was specifically methylated by HhaI.Activity of MN/CA9 promoter was strongly suppressed by treatment with both SssI and HhaI.These data suggest that hypomethylation of the CpG at -74bp in MN/CA9 promoter may have a major role in MN/CA9 expression in human renal cell carcinomas.