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

淀粉样变性患者的淀粉样沉积来源于血清淀粉样蛋白A(SAA)。对于日本类风湿关节炎患者，SAA1.3(SAA1γ)单倍型被证明是发生AA淀粉样变性的危险因素。我们对SAA1基因5‘侧翼区进行了分析，在启动子中发现了3个新的SNPs，分别为-61C/G、-13C/T和-2G/A。我们已经证明-13T与AA-淀粉样变性的风险高于SAA1γ，并且与SAA1γ处于连锁不平衡状态。为了研究-13T/C是否与SAA1转录活性的变化有关，我们进行了SAA1基因启动子单倍型的双荧光素酶报告基因分析。本研究扩增了-61C；-13C；-2G、G-C-G、G-C-A和C-T-G四种单倍型基因，将其亚克隆到pGL-3载体中，并将其导入人肝癌细胞系HepG2。在转染后12h，用IL-1β(10 ng/ml)和IL-6(10 ng/ml)刺激细胞，裂解细胞。用发光计检测上清液中的荧光素酶活性。在荧光素酶报告基因检测中，我们证实了含-13T启动子的相对荧光素酶活性高于含-13C的启动子。用含有-13T或-13C的生物素化的SAA1启动子进行的凝胶迁移率改变分析表明，在IL-1β和IL-6刺激的肝癌细胞提取物中，-13T探针结合的核蛋白比-13C探针多得多。这一结果表明，与含有-13C的启动子相比，SAA1启动子上的-13T SNP与淀粉样蛋白的形成有关。

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.