NRBP1在痛风中的作用及机制研究

Gout is a complex disorder caused by the impact of environmental factors on a complement of inherited genetic risk variants. Genome-wide association studies (GWASs) have identified more than 100 loci associated with gout, but for most cases, functionally affected genes and their underlying molecular mechanisms contributing to these associations are unknown. By performing an integrative analysis incorporating multiple “omics” data, we identified a gout risk gene NRBP1 and a potential epigenetic mechanism regulating its expression which were further confirmed in clinical samples. Based on these findings, we hypothesized that the methylation level at the promoter region of NRBP1 affects its expression and downstream urate transporters, which might contribute to the development of hyperuricemia and gout. Through molecular, cellular and genetic approaches, this project aims to investigate (1) whether epigenetic modifications can regulate NRBP1 expression in vitro; (2) the epigenetic regulations of NRBP1 expression in gout patients in vivo; (3) the functional mechanisms of NRBP1 on urate transporters, as well as the development of gout in both cell and mouse model. This project bears substantial significance to reveal the mechanism of gout development, and may provide a potential target for its future prediction and treatment.

痛风是遗传和环境共同作用导致的复杂疾病。全基因组关联分析（GWAS）已经发现了大量和痛风相关的单核苷酸多态性（SNP），但是对它们导致疾病发生的分子机制仍知之甚少。我们前期的工作通过多组学数据分析，筛选出痛风候选风险基因NRBP1及其潜在的表观遗传调控机制，并在临床样本中进行了初步验证。基于此，我们提出科学假设：启动子DNA甲基化异常导致NRBP1过度表达，通过影响尿酸转运体表达，引起高尿酸血症和痛风。本项目拟运用分子、细胞和遗传等手段探索NRBP1在痛风发病中的作用和机制：（1）体外实验研究启动子DNA甲基化和转录因子对NRBP1表达调控的分子机制；（2）体内实验研究临床样品中启动子CpG位点不同甲基化程度对痛风病人NRBP1表达的影响；（3）建立细胞及小鼠模型，研究NRBP1对尿酸转运体及痛风发生的生理机制。通过探索表观调控在痛风发病机制中的作用，为将来的诊治提供新的靶点。

痛风是遗传和环境共同作用导致的复杂疾病，全基因组关联分析（GWAS）已经发现了大量和痛风相关的单核苷酸多态性（SNP），但是对它们导致疾病发生的分子机制仍知之甚少。我们通过多组学数据分析筛选出痛风候选风险基因NRBP1及其启动子区的一段DNA序列（GGCGCAAGG，命名为B1）为其可能的表观遗传学调控位点。首先在痛风患者外周血单个核细胞（PBMC）中发现NRBP1较健康人群明显升高，表明NRBP1是导致痛风产生的风险基因。然后通过体外实验证实转录因子TFAP2A甲基化依赖性地结合NRBP1启动子从而抑制基因表达，我们发现TFAP2A特异性结合NRBP1启动子B1，并且B1甲基化促进其和TFAP2A结合，抑制基因表达，该抑制呈启动子甲基化依赖性。接着通过体内实验证实痛风病人NRBP1启动子低甲基化抑制TFAP2A的结合导致NRBP1表达增加，我们发现在PBMC中NRBP1启动子B1甲基化水平与其表达呈负相关，而痛风病人NRBP1启动子B1甲基化水平较健康人群明显下降，并且B1低甲基化抑制其与TFAP2A的结合。最后通过NRBP1过表达的近端肾小管上皮细胞模型研究证实NRBP1通过促进Wnt/β-catenin信号通路的激活来抑制尿酸转运体ABCG2表达而导致高尿酸血症和痛风，我们发现在细胞模型中NRBP1过表达可以抑制ABCG2表达，此过程受到Wnt/β‑catenin信号通路的调节，阻断该通路后ABCG2则明显恢复。综合以上结果，在痛风中NRBP1启动子低甲基化抑制TFAP2A结合导致NRBP1高表达，通过激活Wnt/β-catenin信号通路，引起ABCG2表达减少，从而导致高尿酸血症和痛风。本研究丰富了痛风的表观遗传学研究，为治疗痛风增加了其作为药物治疗靶点的可能，我们将GWAS研究成果和DNA甲基化进行结合解释了疾病的发病机制，为后GWAS时代的研究提供一些借鉴。