Influence of the polymorphism of 5'-flanking region of SAA1 gene on SAA1 transcriptional activity

SAA1基因5侧翼区多态性对SAA1转录活性的影响

• 批准号: 13670479
• 负责人: TERAI Chihiro
• 金额: $ 1.98万
• 依托单位: Tokyo Women's 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: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13670479/
• 关键词: Rheumatoid Arthritis; AA-amyloidosis; SAA; SAA1; gene; promotor; haplotype; amyloid; RAGE; 慢性関節リウマチ; CRP; HepG2

Amyloid deposit in patients with AA-amyloidosis is derived from serum amyloid A (SAA). For Japanese patients with rheumatoid arthritis, SAA1.3 (SAA1γ) haplotype was shown to be a risk factor for developing AA-amyloidosis. We have analyzed 5'-flanking region of SAA1 gene and found 3 new SNPs, -61 C/G, -13 C/T, and -2 G/A, in the promotor. We have demonstrated that the -13T is associated with a higher risk of AA-amyloidosis than SAA1γ and is in linkage disequilibrium with SAA1γ.To address whether -13T/C is related to change in transcriptional activity of SAA1, dual luciferase reporter gene assay with promotor haplotypes of SAA1 gene has performed. In this study, four haplotypes consisted of -61C ;-13C ;-2G, G-C-G, G-C-A, and C-T-G, were amplified, subcloned into pGL-3 vector, and transfected into HepG2 cells. At 12 hours after transfection, the cells were stimulated with IL-1β (10ng/ml) and IL-6 (10ng/ml) and harvested in the lysis. The supernatants were assayed for luciferase activity using a Luminometer. In the luciferase reporter gene assay, we confirmed that the relative luciferase activity of the -13T-containing promoter was higher than that of the -13C-containing promoters. Electrophoretic mobility shift assay using biotinylated SAA1 promoters containing the -13T or -13C, showed that a much greater amount of nuclear proteins were bound to the -13T probe than the -13C probe in extracts from HepG2 cells stimulated with IL-1β and IL-6.These results indicated that -13T SNP at the SAA1 promoter correlates to the amyloidogenicity as a result of increased transcriptional activity compared to the -13C-containing promoter.

AA-淀粉样变性患者的淀粉样存款来源于血清淀粉样蛋白A（SAA）。对于患有类风湿性关节炎的日本患者，SAA 1.3（SAA 1 γ）单倍型被证明是发生AA-淀粉样变性的风险因素。我们对SAA 1基因5 '侧翼区进行了分析，发现了3个新的SNPs，分别为-61 C/G、-13 C/T和-2 G/A。我们已经证明-13 T与AA-淀粉样变性的危险性比SAA 1 γ高，并且与SAA 1 γ处于连锁不平衡状态，为了研究-13 T/C是否与SAA 1基因转录活性的改变有关，我们用SAA 1基因启动子单倍型进行了双荧光素酶报告基因检测。本研究扩增了-61C、-13C、-2G、G-C-G、G-C-A和C-T-G四种单倍型，并将其亚克隆到pGL-3载体中，转染HepG 2细胞。转染后12小时，用IL-1β（10 ng/ml）和IL-6（10 ng/ml）刺激细胞，并在裂解液中收获细胞。使用光度计测定上清液的荧光素酶活性。在荧光素酶报告基因测定中，我们证实了含-13 T的启动子的相对荧光素酶活性高于含-13 C的启动子。使用含有-13 T或-13 C的生物素化SAA 1启动子的电泳迁移率变动测定，显示在用IL-1β和IL-6刺激的HepG 2细胞提取物中，与-13 T探针结合的核蛋白量远多于与-13 C探针结合的核蛋白量。这些结果表明-SAA 1启动子处的13 T SNP与淀粉样蛋白生成相关，这是与含-13C启动子相比转录活性增加的结果。

项目成果

Chihiro Terai, Hirotaka Kaneko, Masato Moriguchi, Yumi Koseki, Hiroshi Kajiyama, CPJ Maury, S Tiitinen, K Kaarela, M Hurme, H Direskeneli, P Atagunduz, E Akoglu, Serhan Tuglular, Naoyuki Kamatani.: "SAA1 gene analysis in the patients with AA-amyloidosis f

Chihiro Terai、Hirotaka Kaneko、Masato Moriguchi、Yumi Koseki、Hiroshi Kajiyama、CPJ Maury、S Tiitinen、K Kaarela、M Hurme、H Direskeneli、P Atagunduz、E Akoglu、Serhan Tuglular、Naoyuki Kamatani。：“患者中的 SAA1 基因分析

H Kaneko, C Terai, M Moriguchi, Y Koseki, H Kajiyama, Kamatani N: "Up-regulation of transcriptional activity by a single nucleotide polymorphism at the 5' flanking region of SAA1 gene that was associated with risk for AA-amyloidosis secondary to RA"Arthri

H Kaneko、C Terai、M Moriguchi、Y Koseki、H Kajiyama、Kamatani N：“SAA1 基因 5 侧翼区域的单核苷酸多态性对转录活性的上调与继发性 AA 淀粉样变性风险相关

Moriguchi M, Terai C, Koseki Y, Uesato M, Nishikawa T, Kamatani N.: "Influence of serum amyloid A (SAA) on lipid metabolismin patients with rheumatoid arthritis (RA)."Amyloid. 8(2). 115-120 (2001)

Moriguchi M、Terai C、Koseki Y、Uesato M、Nishikawa T、Kamatani N.：“血清淀粉样蛋白 A (SAA) 对类风湿性关节炎 (RA) 患者脂质代谢的影响。”淀粉样蛋白。

Moriguchi M, Terai C, Kaneko H, Koseki Y, Kajiyama H, Uesato M, Inada S, Kamatani N: "A novel single-nucleotide polymorphism at the 5'-flanking region of SAA1 associated with risk of type AA amyloidosis secondary to rheumatoid arthritis"Arthritis Rheumati

Moriguchi M、Terai C、Kaneko H、Koseki Y、Kajiyama H、Uesato M、Inada S、Kamatani N：“SAA1 5侧翼区域的一种新型单核苷酸多态性与继发于类风湿的 AA 型淀粉样变性风险相关

Masato Moriguchi, Chihiro Terai, Yumi Koseki, Masashi Uesato, Toshio Nishikawa, Naoyuki Kamatani.: "Genotypes at SAA1 locus correlate with the clinical severity of AA-amyloidosis."Amyloid. 8. 115-120 (2001)

Masato Moriguchi、Chihiro Terai、Yumi Koseki、Masashi Uesato、Toshio Nishikawa、Naoyuki Kamatani.：“SAA1 位点的基因型与 AA 淀粉样变性的临床严重程度相关。”淀粉样蛋白。