Cho M, Uemura H, Kim S C, Kawada Y, Yoshida K, Hirao Y, Konishi N, Saga S, Yoshikawa K
Department of Urology, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8522, Japan.
Br J Cancer. 2001 Aug 17;85(4):563-7. doi: 10.1054/bjoc.2001.1951.
MN/CA9 is a cancer-related gene, frequently activated in human renal cell carcinomas (RCCs). To reveal the activation mechanism, we investigated the relationship between methylation status of the MN/CA9 promoter region and gene expression using 13 human RCCs, and examined the effect of in vitro CpG methylation on the MN/CA9 promoter activity using a human RCC cell line (SK-RC-44), expressing MN/CA9. MN/CA9 expression was evaluated by RT-PCR and observed in 10 of 13 RCCs (77%). A total of 9 out of 10 MN/CA9 -positive RCCs (90%) contained clear cell components. Methylation status of 6 CpGs in the MN/CA9 promoter region was decided by using the bisulfite genomic sequencing protocol. Out of 13 RCCs 9 (69%) showed partial hypomethylation of the CpG at -74 bp, while the other 4 RCCs and 3 normal kidney tissue samples showed complete methylation. Hypomethylation of the CpG at -74 bp was strongly correlated with MN/CA9 expression. Luciferase assay revealed that the MN/CA9 promoter activity was strongly suppressed by methylation of the CpG at -74 bp. These findings suggest that hypomethylation of the CpG at -74 bp in the MN/CA9 promoter region might play an important role in this gene activation of human RCC.
MN/CA9是一种与癌症相关的基因，在人类肾细胞癌（RCC）中经常被激活。为了揭示其激活机制，我们使用13例人类RCC研究了MN/CA9启动子区域的甲基化状态与基因表达之间的关系，并使用表达MN/CA9的人类RCC细胞系（SK-RC-44）检测了体外CpG甲基化对MN/CA9启动子活性的影响。通过RT-PCR评估MN/CA9的表达，在13例RCC中有10例（77%）观察到该基因表达。在10例MN/CA9阳性的RCC中，共有9例（90%）含有透明细胞成分。采用亚硫酸氢盐基因组测序方法确定MN/CA9启动子区域6个CpG的甲基化状态。在13例RCC中，9例（69%）在-74 bp处的CpG显示部分低甲基化，而其他4例RCC和3例正常肾组织样本显示完全甲基化。-74 bp处的CpG低甲基化与MN/CA9表达密切相关。荧光素酶检测显示，-74 bp处的CpG甲基化强烈抑制MN/CA9启动子活性。这些发现表明，MN/CA9启动子区域-74 bp处的CpG低甲基化可能在人类RCC的该基因激活中起重要作用。
