基于自噬筛选英夫利昔单抗治疗类风湿性关节炎不应答的标志物及其鉴定

Identification and validation of the potential early diagnostic biomarkers of infliximab non-response to treatment of rheumatoid arthritis based on autophagy

目的从自噬角度对英夫利昔单抗(infliximab, IFX)治疗类风湿性关节炎(rheumatoid arthritis, RA)不应答基因数据芯片进行生物信息学分析,筛选IFX不应答相关特征基因,并初步研究特征基因对细胞自噬的影响,探讨IFX治疗RA不应答的可能机制。方法利用R语言对筛选出的GEO (gene expression omnibus database)数据集进行自噬相关基因的差异表达分析,使用单因素logistic回归、Lasso (least absolute shrinkage and selection operator)回归、多因素logistic回归对差异自噬相关基因进行筛选,得到IFX治疗RA不应答的特征自噬基因,利用受试者工作特征曲线(receiver operating characteristic curve, ROC)对特征自噬基因进行诊断价值分析。Real-time PCR检测特征基因在RA临床样本中的表达,Western blotting检测特征基因对RA滑膜细胞系MH7A细胞自噬的影响。结果部分自噬相关基因在IFX治疗RA不应答者与应答者间差异表达(P<0.05),自噬相关基因集主要高表达于不应答组。筛选出HGS(hepatocyte growth factor-regulated tyrosine kinase substrate)和ATG4B(autophagy related 4 homolog B)作为IFX治疗RA不应答的特征基因。ROC曲线显示,使用该特征基因构建的预测模型在内部验证数据集GSE78068(AUC=0.708, P<0.05)和外部验证数据集GSE58795(AUC=0.719, P<0.05)中具有较好的区分能力。Real-time PCR结果显示,HGS在不应答组外周血白细胞中表达升高;MH7A细胞过表达HGS后,自噬相关蛋白LC3BⅡ/LC3BⅠ增加,P62表达降低,细胞自噬增强。结论 IFX治疗RA不应答者与应答者外周血中自噬相关基因表达谱不同,提示IFX治疗RA不应答可能与细胞自噬有关;以HGS和ATG4B构建的预测模型对IFX治疗RA不应答具有一定的预测能力,且HGS可促进MH7A自噬增强,为进一步研究IFX治疗RA不应答的分子机制提供新思路。

Objective The purpose of this study was to perform bioinformatics analysis of infliximab(IFX)treatment of rheumatoid arthritis(RA)non-responders and responders gene data chip based on autophagy, and to screen out the characteristic autophagy-related genes, carry out a preliminary experiment on the role of characteristic genes, and explore the possible mechanism of IFX treatment of RA non-response. Methods R language was used to analyze the differential expressions of autophagy-related genes on the selected gene expression omnibus database(GEO) data set.Univariate logistic regression, least absolute shrinkage and selection operator(Lasso) regression, and multiple logistic regression analyses were made to screen out the characteristic autophagy-related genes;receiver operating characteristic curve(ROC)was used to assess the diagnostic value of the characteristic autophagy-related genes. Real-time PCR was used to detect the expressions of characteristic autophagy-related genes in RA clinical samples,and Western blot was used to detect the role of characteristic autophagy-related genes in autophagy in RA synovial cell line MH7A cells. Results Part of autophagy-related genes were differentially expressed between non-responders and responders to IFX treatment of RA(P<0. 05), and autophagy-related gene sets were mainly expressed in non-responders. Hepatocyte growth factorregulated tyrosine kinase substrate(HGS) and autophagy related 4 homolog B(ATG4B) were screened out as characteristic genes of non-response to IFX treatment of RA. The ROC showed that the predictive model constructed using the selected markers had good discrimination ability in the internal validation data set GSE78068(AUC=0. 708,P<0. 05)and the external validation data set GSE58795(AUC=0. 719, P<0. 05). The Real-time PCR results showed that the expression of HGS was increased in peripheral blood leukocytes in the non-responders. After overexpression of HGS in MH7A cells, the ratio of LC3B Ⅱ/LC3B Ⅰ was increased while the expression of P62 was decreased, indicating that autophagy increased. Conclusion The autophagy-related gene expression profiles were different between IFX-treated RA non-responders and responders,suggesting that the non-response of IFX treatment of RA may be related to autophagy. The prediction model constructed with HGS and ATG4B had a certain ability to predict IFX-treated RA non-response,and HGS promoted autophagy in MH7A cells,which provides a new idea for future research on the molecular mechanism of IFX in treating RA non-response.