系统性硬化症合并肺间质病变患者外周血单个核细胞全基因组DNA甲基化和转录组表达谱

Genome-wide DNA methylation and transcriptome expression profiles of peripheral blood mononuclear cells in patients with systemic sclerosis with interstitial lung disease

目的:分析系统性硬化症(systemic sclerosis,SSc)合并肺间质病变(interstitial lung disease,ILD)患者外周血单个核细胞(peripheral blood mononuclear cells,PBMCs)全基因组DNA甲基化和转录组表达谱,并进一步探讨DNA甲基化对Wnt/β-catenin信号通路和趋化因子信号通路的影响。方法:收集19例SSc患者(SSc组)及18例健康人(对照组)外周血PBMCs。SSc患者中有10例合并ILD(SSc合并ILD亚组)、9例未合并ILD(SSc未合并ILD亚组)。采用Illumina 450K甲基化芯片和Illumina HT-12 v4.0基因表达谱芯片分析全基因组DNA甲基化和基因表达水平,研究DNA甲基化对Wnt/β-catenin和趋化因子信号通路的影响。结果:全基因组DNA甲基化分析发现:与SSc未合并ILD亚组比较,SSc合并ILD亚组存在71个高甲基化位点,98个低甲基化位点。转录组分析发现:与SSc未合并ILD亚组相比,SSc合并ILD亚组有164个基因表达上调,191个基因表达下调。SSc组患者PBMCs中Wnt/β-catenin信号通路中有35个低甲基化基因,其中卷曲同源物1(frizzled-1,FZD1)、丝裂原活化蛋白激酶9(mitogen-activated protein kinase 9,MAPK9)、母亲DPP同源物2(mothers against DPP homolog 2,SMAD2)、转录因子7类似物2(transcription factor 7-like 2,TCF7L2)和无翅型MMTV整合位点家族成员5B(wingless-type MMTV integration site family,member 5B,WNT5B)mRNA在SSc组中的表达显著高于对照组,差异均有统计学意义(均P<0.05)。与SSc未合并ILD亚组比较,SSc合并ILD亚组中Dickkopf相关蛋白2(dickkopf homolog 2,DKK2)、FZD1、MAPK9等多个基因的mRNA表达虽上调,但差异均无统计学意义(均P>0.05)。趋化因子信号通路中有38个低甲基化基因,其中β-抑制蛋白1(β-arrestin 1,ARRB1)、C-X-C基序趋化因子配体10(C-X-C motif chemokine ligand 10,CXCL10)、C-X-C基序趋化因子配体16(C-XC motif chemokine ligand 16,CXCL16)、FGR、中性粒细胞胞浆因子1C(neutrophil cytosolic factor 1C,NCF1C)mRNA在SSc组中的表达显著高于对照组,差异均有统计学意义(均P<0.05)。与SSc未合并ILD亚组比较,SSc合并ILD亚组中ARRB1、CXCL10、CXCL16等多个基因的mRNA表达上调,但差异均无统计学意义(均P>0.05)。结论:SSc合并ILD与SSc无ILD患者存在DNA甲基化和转录组表达谱的差异,且SSc患者PBMCs中存在Wnt/β-catenin和趋化因子信号通路多个基因表达上调,这可能与SSc的发病机制有关。

Objective:This study aims to investigate the genome-wide DNA methylation and transcriptome expression profiles of peripheral blood mononuclear cells(PBMCs)in patients with systemic sclerosis(SSc)with interstitial lung disease(ILD),and to analyze the effects of DNA methylation on Wnt/β-catenin and chemokine signaling pathways.Methods:PBMCs were collected from 19 patients with SSc(SSc group)and 18 healthy persons(control group).Among SSc patients,there were 10 patients with ILD(SSc with ILD subgroup)and 9 patients without ILD(SSc without ILD subgroup).The genome-wide DNA methylation and gene expression level were analyzed by using Illumina 450K methylation chip and Illumina HT-12 v4.0 gene expression profiling chip.The effect of DNA methylation on Wnt/β-catenin and chemokine signal pathways was investigated.Results:Genome-wide DNA methylation analysis identified 71 hypermethylated CpG sites and 98 hypomethylated CpG sites in the SSc with ILD subgroup compared with the SSc without ILD subgroup.Transcriptome analysis distinguished 164 upregulated genes and 191 downregulated genes in the SSc with ILD subgroup as compared with the SSc without ILD subgroup.In PBMCs of the SSc group,35 genes in Wnt/β-catenin signaling pathway were hypomethylated,while frizzled-1(FZD1),mitogen-activated protein kinase 9(MAPK9),mothers against DPP homolog 2(SMAD2),transcription factor 7-like 2(TCF7L2),and wingless-type MMTV integration site family,member 5B(WNT5B)mRNA expressions were upregulated as compared with the control group(all P<0.05).Compared with the SSc without ILD subgroup,the mRNA expressions of dickkopf homolog 2(DKK2),FZD1,MAPK9 were upregulated in the SSc with ILD subgroup,but the differences were not statistically significant(all P>0.05).In PBMCs of the SSc group,38 genes in chemokine signaling pathway were hypomethylated,whileβ-arrestin 1(ARRB1),C-X-C motif chemokine ligand 10(CXCL10),C-X-C motif chemokine ligand 16(CXCL16),FGR,and neutrophil cytosolic factor 1C(NCF1C)mRNA expressions were upregulated as compared with the control group(all P<0.05).Compared with the SSc without ILD subgroup,the mRNA expressions of ARRB1,CXCL10,CXCL16 were upregulated in the SSc with ILD subgroup,but the differences were not statistically significant(all P>0.05).Conclusion:There are differences in DNA methylation and transcriptome profiles between SSc with ILD and SSc without ILD.The expression levels of multiple genes in Wnt/β830 catenin and chemokine signaling pathways are upregulated,which might be associatea with the pathogenesis of SSc.