炎性肠病易感基因GPR35在肠炎发生发展中的功能研究

doi:10.16288/j.yczz.20-392

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

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 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 出版日期:2021-02-16 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 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 Online:2021-02-16 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/Abstract

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

郑燕森, 卓林刚, 李大力, 刘明耀. 炎性肠病易感基因GPR35在肠炎发生发展中的功能研究[J]. 遗传, 2021, 43(2): 169-181.

Yansen Zheng, Lingang Zhuo, Dali Li, Mingyao Liu. Inflammatory bowel disease susceptible gene GPR35 promotes bowel inflammation in mice[J]. Hereditas(Beijing), 2021, 43(2): 169-181.

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引物名称	引物序列(5?→3?)
GPR35-F	ATGGCACCTACAACACCT
GPR35-R	GGCGAGGGTCACGCACAG
GPR35mut1-R	CGATGGCCATGACCAGGCTG
GPR35mut1-F	CAGCCTGGTCATGGCCATCG
GPR35mut2-R	CTTAGCACGGGGAGCCAC
GPR35mut2-F	GTGGCTCCCCGTGCTAAG

引物名称	引物序列(5′→3′)
m-Gapdh-F	ATGGCACCTACAACACCT
m-Gapdh-R	GGCGAGGGTCACGCACAG
m-IL1β-F	TGCCACCTTTTGACAGTGATG
m- IL1β-R	AAGGTCCACGGGAAAGACAC
m-IL6-F	GGGACTGATGCTGGTGACAA
m-IL6-R	ACAGGTCTGTTGGGAGTGGT
m-TNFα-F	AGGCACTCCCCCAAAAGATG
m-TNFα-R	CCACTTGGTGGTTTGTGAGTG
m-IL10-F	GCTCTTACTGACTGGCATGAG
m-IL10-R	CGCAGCTCTAGGAGCATGTG

Zaprinast	载体	对照		1 μmol/L		10 μmol/L
Zaprinast	载体	活性(归一化)	显著性(P value)	活性(归一化)	显著性(P value)	活性(归一化)	显著性(P value)
钙流检测	GPR35	1±0.24		1.04±0.24		1.1±0.14
	GPR35 ^T108M	0.99±0.02	ns	1.67±0.27	P<0.01	3.8±1.1	P<0.001
	GPR35 ^S294R	1.19±0.26	ns	1.31±0.2	ns	3.0±0.38	P<0.001
β-arrestin 招募检测	GPR35	1±0.1		1.28±0.13		1.35±0.13
	GPR35 ^T108M	1.2±0.38	ns	2.1±0.19	P<0.001	2.03±0.11	P<0.001
	GPR35 ^S294R	1.3±0.1	P<0.05	2.28±0.09	P<0.001	2.56±0.12	P<0.001
CRE转录活性分析	GPR35	1±0.02		0.91±0.11		0.74±0.19
	GPR35 ^T108M	1.03±0.08	ns	0.89±0.22	ns	0.47±0.05	P<0.001
	GPR35 ^S294R	1.04±0.1	ns	817±0.03	ns	0.45±0.05	P<0.001

炎性肠病易感基因GPR35在肠炎发生发展中的功能研究

Inflammatory bowel disease susceptible gene GPR35 promotes bowel inflammation in mice

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