SLCO3A1 單核苷酸多型性與克隆氏症的相關性

Abstract

克隆氏症(Crohn’s disease)和潰瘍性結腸炎(Ulcerative colitis) 為主要慢性腸道發炎疾病。根據基因組關聯研究方法(GWAS),證實 IL23R、 ATG16L1 和 IRGM 為患有克隆氏症西方族群的相關基因。而 TNFSF15 是目前為止 唯一與非西方族群相關的的克隆氏症基因。我們透過兩個階段的分析,成功找 出台灣族群的克隆氏症相關基因。 第一階段,我們利用平均年齡和性別相同的 16 名克隆氏症患者與 16 名 的對照組受試者進行 Illumina 基因晶片分析;第二階段,我們利用 53 名克隆 氏症患者與 41 名的對照組受試者進行 Sequenom MassArray 驗證,從中挑出一 些與克隆氏症相關的單核甘酸多型性(SNPs)。rs207959 為 14 個具有顯著差異 單核苷酸多型性(SNPs)當中的其中一個(其第一階段的 P 值為 2.3 × 10-2,第 二階段的 P 值為 1.0 × 10-3)。透過基因型和表現型的統計分析, 我們發現 rs207959(T)對偶基因是造成克隆氏症患者腸道穿孔的危險指標,而基因型的 不同也造成 SLCO3A1 mRNA 表現量的差異。 接著,我們透過即時定量 PCR 和免疫組織染色(IHC)分析,比較了克隆 氏症患者與對照組受試者 SLCO3A1 的 mRNA 和蛋白質表現量的差別 。我們也透過 Luciferese 冷光酵素活性分析來探討 SLCO3A1 過量表現情況下的 NF-κB 活 性。 透過定量 PCR 和免疫組織染色(IHC),我們也發現 SLCO3A1 主要表現在腸 道上皮細胞,而克隆氏症患者的 SLCO3A1 mRNA 和蛋白質表現量有顯著的增加。 我們接著發現 SLCO3A1 是透過活化 NF-κB 轉錄因子,促進 P65、ERK 和 JNK 的 磷酸化來調控腸道上皮細胞的發炎。在尼古丁誘導的 SLCO3A1 過量表現細胞中 也發現有較多的 NF-κB 被活化,所以我們推測具有 rs207959(T)對偶基因的 克隆氏症抽煙患者,會有更加劇的發炎的現象 。 研究表示 SLCO3A1 為新的台灣族群克隆氏症相關基因,其可透過活化 NF-κB 轉錄因子而引起在腸道上皮細胞的發炎,抽煙可能會加重發炎的程度。 rs207959(T)與克隆氏症患者腸道穿孔的機會相關。

關鍵字:SLCO3A1、克隆氏症、腸道穿孔、NF-κB 、尼古丁、抽煙

Background & Aims: Crohn’s disease (CD) and ulcerative colitis (UC) are two categories of chronic bowel inflammatory disorders. Several CD susceptible genes were identified in Caucasians with genome-wide association studies (GWAS), namely IL23R, ATG16L1 and IRGM, but only gene-TNFSF15 was discovered in non-Caucasian. This two-stage study aims at investigating novel CD-associated genes in Taiwan population. Methods: We analyzed individual’s SNPs using Illumina (first stage: 16 CD patients with 16 age- and sex- matched control patients) and Sequenom MassArray analysis (second stage: 53 CD patients and 41 control patients). We performed quantitative PCR and immunohistochemical staining to demonstrate mRNA and protein expression level of candidate gene SLCO3A1. NF-κB reporter luciferase assay was performed to evaluate NF-κB activity in over-expressed SLCO3A1 cells. Results: SLCO3A1 (rs207959) was a significant finding in the first-stage analysis, with P value of 2.3E-02; and was validated in the second-stage analysis, with P value of 1.0E-03. Genotype and phenotype analysis of rs207959 alleles showed rs207959 (T) allele as a risk allele causing perforation in CD patients, which also altered in SLCO3A1 mRNA expression level. Quantitative PCR and immunohistochemical staining demonstrated SLCO3A1 expression in intestinal epithelium, with significantly higher expression of both mRNA and protein in CD. SLCO3A1 was then proved to mediate inflammation in intestinal epithelial cells via activation of NF-κB transcription factor with consequent stronger activation of P65, ERK and JNK phosphorylation, leading to a more intense and protracted NF-κB activation. Higher NF-κB activation was also discovered in nicotine-induced SLCO3A1 presenting cells, depicting smoking as relevant exacerbating cause in SLCO3A1-susceptible-allele-changed CD patients. Conclusions: SLCO3A1, revealed to be the novel CD-associated gene, was proved to mediate inflammatory process in intestinal epithelial cells through NF-κB transcription activation, associated with higher chance of bowel perforation in CD patients, while nicotine showed add-on impact in inflammation.

Keyword : SLCO3A1, CD, NF-κB, perforated, nicotine