| Project/Area Number |
11670224
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Experimental pathology
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| Research Institution | NARA MEDICAL UNIVERSITY |
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Principal Investigator |
CHO Masaki Dept.Urol.Nara Med.Univ. Research Associate, 医学部, 助手 (90285362)
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| Co-Investigator(Kenkyū-buntansha) |
KONISHI Noboru 2nd Dept.Pathol.Nara Med.Univ. Professor, 医学部, 教授 (20145832)
HIRAO Yoshihiko Dept.Urol.Nara Med.Univ. Professor, 医学部, 教授 (00133207)
UEMURA Hirotsugu Dept.Urol.Nara Med.Univ. Assistant Professor, 医学部, 講師 (90213397)
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| Project Period (FY) |
1999 – 2000
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| Project Status |
Completed (Fiscal Year 2000)
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| Budget Amount *help |
¥3,100,000 (Direct Cost: ¥3,100,000)
Fiscal Year 2000: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1999: ¥2,000,000 (Direct Cost: ¥2,000,000)
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| Keywords | Renal cell carcinoma / MN / CA9 / methylation / DNAメチル化 / DNA メチル化 / プロモータ |
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| Research Abstract |
Correlation between MN/CA9 expression and hypomethylation of MN/CA9 5'flanking region in human renal cell carcinomas
Hypomethylation 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 agent
Treatment 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 region
DNAs 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 activity
MN/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.
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