Hypomethylation of the MN/CA9 promoter and upregulated MN/CA9 expression in human renal cell carcinoma.
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.
Cho
M
M
Department of Urology, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8522, Japan.
Uemura
H
H
Kim
S C
SC
Kawada
Y
Y
Yoshida
K
K
Hirao
Y
Y
Konishi
N
N
Saga
S
S
Yoshikawa
K
K
D002292:Carcinoma, Renal Cell / Q000235:genetics* / Q000473:pathology; D019175:DNA Methylation; D017931:DNA Primers; D004273:DNA, Neoplasm; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007680:Kidney Neoplasms / Q000235:genetics* / Q000473:pathology; D011401:Promoter Regions, Genetic / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015533:Transcriptional Activation; D014407:Tumor Cells, Cultured; D015854:Up-Regulation
D017931:DNA Primers; D004273:DNA, Neoplasm