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

阐明 MN/CA9 在人肾细胞癌中的激活机制

• 批准号: 11670224
• 负责人: CHO Masaki
• 金额: $ 1.98万
• 依托单位: NARA MEDICAL UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11670224/
• 关键词: Renal cell carcinoma; MN; CA9; methylation; DNAメチル化; DNA メチル化; プロモータ

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.

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。

项目成果

Uemura, H., Nakagawa, Y., Hirao, Y., Okajima, E., Yoshikawa, K., Oosterwijk, E.: "Expression of MN antigen in urologic cancer : potential tool as a therapeutic target."Aktuel Urology. 31. 52-54 (2000)

Uemura, H.、Nakakawa, Y.、Hirao, Y.、Okajima, E.、Yoshikawa, K.、Oosterwijk, E.：“泌尿系统癌症中 MN 抗原的表达：作为治疗靶点的潜在工具。”Aktuel Urology。

Uemura, H., Yoshikawa, M., Shimizu, K., Cho, M., Nakagawa, Y., Ozono, S., Hirao, Y., Yoshikawa, K.: "MN/CA IX as a potential target for active specific immunotherapy in renal cell carcinomas."Biotherapy. 14. 487-489 (2000)

Uemura, H.、Yoshikawa, M.、Shimizu, K.、Cho, M.、Nakakawa, Y.、Ozono, S.、Hirao, Y.、Yoshikawa, K.：“MN/CA IX 作为潜在目标

Cho M: "Activation of the MN/CA9 gene is associated with hyponethylation in human venal cell carcinomas cell lines"Molecular Carcinogenesis. 27(3). 184-189 (2000)

Cho M：“MN/CA9 基因的激活与人静脉细胞癌细胞系中的低甲基化有关”分子癌发生。

Uemura H: "MN/CA IX/G250 as a potential target for immuno-therapy of renal cell carcinoma"British Journal of Cancer. 81. 741-746 (1999)

Uemura H：“MN/CA IX/G250 作为肾细胞癌免疫治疗的潜在靶点”英国癌症杂志。

Uemura, H.: "MN/CA9 Novel biomarker for renal cell carcinoma"Rinsho-Hinyoukika. 55 (in press). (2001)

Uemura, H.：“MN/CA9 肾细胞癌新型生物标志物”Rinsho-Hinyoukika。