遗传 ›› 2021, Vol. 43 ›› Issue (2): 169-181.doi: 10.16288/j.yczz.20-392

• 研究报告 • 上一篇    

炎性肠病易感基因GPR35在肠炎发生发展中的功能研究

郑燕森1(), 卓林刚1, 李大力1(), 刘明耀1()   

  1. 1 华东师范大学生命科学学院生命医学研究所,上海调控生物学重点实验室,上海 200241
  • 收稿日期:2020-11-18 发布日期:2021-01-27
  • 基金资助:
    国家重点研发计划基金项目(2019YFA0110802);国家自然科学基金面上项目(81670470);国家自然科学基金面上项目(81873685)

Inflammatory bowel disease susceptible gene GPR35 promotes bowel inflammation in mice

Yansen Zheng1(), Lingang Zhuo1, Dali Li1(), Mingyao Liu1()   

  1. 1 Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China
  • Received:2020-11-18 Published:2021-01-27
  • Supported by:
    National Key R&D Program of China(2019YFA0110802);the National Natural Science Foundation of China(81670470);the National Natural Science Foundation of China(81873685)

摘要:

炎性肠病在全球范围内发生极其普遍,具有反复发作、难以治愈的特点,也是诱发结直肠癌的高风险因素之一。肠炎的发生与遗传因素密切相关,有报道发现位于GPR35基因座上的多个单核苷酸多态性(single nucleotide polymorphism, SNP)位点rs4676410、rs3749171和rs3749172与肠炎敏感性高度相关,但是GPR35基因在肠炎的发生发展进程中的功能及相关机制尚没有明确结论。为了研究GPR35在肠炎中的作用,首先通过CRISPR/Cas9技术构建Gpr35敲除小鼠,随后利用DSS诱导的肠炎模型评价Gpr35在肠炎发生中的作用,发现敲除小鼠在体重变化、DAI评分、肠上皮损伤以及炎性细胞浸润等肠炎相关指标显著低于野生型小鼠。为了研究肠炎相关SNP突变对GPR35活性的影响,首先根据rs3749171和rs3749172SNP位点突变信息构建GPR35-T108M和GPR35-S294R两种突变型受体,其次通过多种GPR35下游信号通路活性测试,发现两种突变均能够增强GPR35受体活性。最后通过Western blotting分析发现相较于野生型小鼠,Gpr35敲除小鼠肠上皮Erk1/2磷酸化水平增加,表明Gpr35敲除后可能通过上调Erk1/2信号通路的方式抑制肠炎的发生发展。综上所述,本研究发现人类肠炎易感的rs3749171和rs3749172位点可能通过激活GPR35及下游信号通路的方式促进肠炎的发生发展,为炎性肠病的治疗提供了潜在的药物作用靶点。

关键词: rs3749171, rs3749172, GPR35, 肠炎, 动物模型

Abstract:

Inflammatory bowel disease (IBD) has emerged as a public health challenge with high incidence, recurrence rates and low cure rate. Moreover, sustained inflammation increases the risk of colorectal cancer. The occurrence and progression of IBD are closely related to the genetic mutation. Previous genome-wide association studies (GWAS) analysis demonstrated that the susceptibility loci rs4676410, rs3749171, and rs3749172 in theGPR35 gene locus increase the risk of IBD, but no direct evidence on the function ofGPR35 in IBD progression has been shown. To investigate the role ofGPR35 in IBD, CRISPR/Cas9 technology was employed to construct aGpr35 knockout mouse strain. TheGpr35-/- mice exhibited lower susceptibility to dextran sodium sulfate-induced IBD model than the wildtype group with a significant reduction in body weight loss, DAI score, intestinal epithelial injury, and macrophage cell infiltration. To explore how the IBD susceptibility loci rs3749171 and rs3749172 regulate GPR35 activity, two mutant forms of GPR35 (T108M and S294R) were constructed. By analyzing the activity of GPR35 downstream signaling pathway, the two mutation forms of GPR35 exhibited higher receptor activity to Zaprinast than the wildtype GPR35. Finally, the Western blotting analysis found an elevated phosphorylation level of ERK1/2 inGpr35-/- colon epithelial after DSS treatment, demonstrating that the loss function ofGpr35 alleviates the IBD syndrome by activating the ERK1/2 signaling pathway. In summary, the IBD susceptibility loci rs3749171 and rs3749172 may promote the disease progression by activating GPR35 activity, providing a potential drug target for the treatment of inflammatory bowel disease.

Key words: rs3749171, rs3749172, GPR35, IBD, animal model