基于生物信息学的结节病诊断标志物的筛选及实验验证

Screening and verification of diagnostic markers for sarcoidosis based on bioinformatics

目的探索结节病的关键基因,并为其分子机制提供理论依据。方法实验性研究。从基因芯片公共数据库(GEO)中下载获得结节病患者外周血的基因芯片数据集GSE34608和GSE18781。使用R软件对芯片数据进行整合并筛选出差异基因。随后进行基因本体论(GO)富集分析和KEGG信号通路分析,利用随机森林和LASSO回归两种算法,筛选出核心基因。为进一步验证基因芯片的结果,选取成都医学院第一附属医院2017年7月至2020年10月收治的肺结节病患者50例设为病例组,并选取于同一时期体检的50例健康志愿者设为健康对照组。采用酶联免疫吸附法对生信分析结果进行验证。结果761个差异基因在免疫反应、炎症反应、GTP酶的活性等生物学过程和功能中均有富集。且富集于T细胞受体,NF-κB,AMPK等信号通路。随机森林和LASSO回归两种算法筛选出核心基因为SPOCK2,ELISA结果显示0~Ⅰ期肺结节病患者(3.24±0.18)μg/L及Ⅱ~Ⅲ期肺结节病患者(5.03±0.12)μg/L,外周血样本中的SPOCK2浓度低于健康对照组(9.31±0.59)μg/L,且差异有统计学意义(F=37.36,P<0.05),且血清SPOCK2对0~Ⅰ期肺结节有较高的诊断效能(AUC=0.836)。结论筛选的核心基因SPOCK2为结节病的发病机制和靶向治疗提供了理论基础,同时通过实验验证血清SPOCK2对于肺结节病的诊断有一定帮助。

Objective To use the bioinformatics for screening differentially expressed genes in peripheral blood of patients with sarcoidosis and healthy subjects,in combination with some relevant experiments for key genes of sarcoidosis in theoretical basis for molecular mechanism.Methods Gene Expression Omnibus(GEO)was used to download the microarray data sets GSE34608 and GSE18781 of peripheral blood of patients with sarcoidosis.R software was used to integrate chip data and screen out the differential genes,in analysis with gene ontology(GO)enrichment analysis and KEGG signal pathway.Two algorithms,random forest and LASSO regression were used to screen out the core genes.To verify the results of the gene chip,50 patients with pulmonary sarcoidosis admitted to the First Affiliated Hospital of Chengdu Medical College from July 2017 to October 2020 were selected as the case group,and 50 healthy volunteers from the physical check during the same period were selected as the healthy control group.The bioinformatics analysis results were verified by enzyme-linked immunosorbent assay(ELISA).Results A total of 761 differential expressed genes were screened out,and GO enrichment analysis showed that the differentially expressed genes were enriched in biological processes and functions such as immune response,inflammatory response and activity of GTPase.KEGG pathway analysis showed that the different genes were enriched in T cell receptor,NF-κB,AMPK and other signaling pathways.SPOCK2 was selected as the core gene by random forest and LASSO regression algorithms.Stage 0-Ⅰsarcoidosis group and stageⅡ-Ⅲsarcoidosis group showed significant decrease in serum SPOCK2 compared with healthy control group(F=37.36,t=-57.37,P<0.05).The serum SPOCK2 provided higher diagnostic power in stage 0-Ⅰsarcoidosis group.Conclusions The selected core gene SPOCK2 provided theoretical basis for the pathogenesis and targeted therapy of sarcoidosis.Meanwhile,the serum SPOCK2 helpful in diagnosis of pulmonary sarcoidosis was verified by experiments.