Supplementary Material for: Identification of hub gene and transcription factor related to chronic allograft nephropathy based on WGCNA analysis

Abstract Introduction Kidney transplantation has surpassed dialysis as the optimal therapy for end-stage kidney disease (ESKD). Yet, most patients could suffer from a slow but continuous deterioration of kidney function leading to graft loss mostly due to chronic allograft nephropathy (CAN) after KT. The dysregulated gene expression for CAN is still poorly understood. Methods To explore the pathogenesis of genomics in CAN, we analyzed the differentially expressed genes (DEGs) of kidney transcriptome between CAN and non-rejecting patients by downloading gene expression microarrays from the Gene Expression Omnibus (GEO) database. Then, we used weighted gene co-expression network analysis (WGCNA) to analyze the co-expression of DEGs to explore key modules, hub genes, and transcription factors in CAN. Functional enrichment analysis of key modules was performed to explore pathogenesis. ROC curve analysis was used to validate hub genes. Results As a result, 3 key modules and 15 hub genes were identified by WGCNA analysis. 3 key modules had 21 mutual Gene Ontology (GO) term enrichment functions. Extracellular structure organization, extracellular matrix organization, and extracellular region were identified as significant functions in CAN. Furthermore, transcription factor 12 was identified as the key transcription factor regulating key modules. All 15 hub genes: Yip1 interacting factor homolog B, membrane trafficking protein(YIF1B), toll like receptor 8 (TLR8), neutrophil cytosolic factor 4 (NCF4), glutathione peroxidase 8 (GPX8), mesenteric estrogen dependent adipogenesis (MEDAG), decorin(DCN), serpin family F member 1 (SERPINF1), integrin subunit beta like 1 (ITGBL1), SRY-box transcription factor 15 (SOX15), trophinin associated protein (TROAP), SRY-box transcription factor 1 (SOX1), metallothionein 3 (MT3), lysosomal protein transmembrane (5LAPTM5), FERM domain containing kindlin 3 (FERMT3), cathepsin S (CTSS) had a great diagnostic performance (AUC>0.7). Conclusion This study updates information and provides a new perspective for understanding the pathogenesis of CAN by bioinformatics means. More research is needed to validate and explore the results we have found to reveal the mechanisms underlying CAN.

【摘要】肾移植已超越透析，成为终末期肾病（end-stage kidney disease, ESKD）的最优治疗方案。然而，多数肾移植（kidney transplantation, KT）患者会出现缓慢且持续的肾功能恶化，最终导致移植物失功，这一过程主要由慢性移植物肾病（chronic allograft nephropathy, CAN）引发。目前，人们对CAN相关的基因表达失调机制仍知之甚少。

【方法】为探究CAN的基因组发病机制，我们从基因表达综合数据库（Gene Expression Omnibus, GEO）中下载基因表达微阵列数据，分析了CAN患者与非排斥反应患者的肾脏转录组差异表达基因（differentially expressed genes, DEGs）。随后，我们采用加权基因共表达网络分析（weighted gene co-expression network analysis, WGCNA）对DEGs的共表达模式进行分析，以筛选CAN中的关键模块、枢纽基因及转录因子。通过对关键模块进行功能富集分析，以进一步探究其发病机制；并采用ROC曲线分析对枢纽基因的诊断效能进行验证。

【结果】通过WGCNA分析，我们共筛选出3个关键模块与15个枢纽基因。3个关键模块共富集到21个共有基因本体（Gene Ontology, GO）功能项。其中，细胞外结构组织、细胞外基质组织及细胞外区域被鉴定为CAN的显著富集功能。此外，转录因子12（transcription factor 12）被确定为调控关键模块的核心转录因子。15个枢纽基因分别为：Yip1互作因子同源物B（Yip1 interacting factor homolog B, YIF1B）、膜转运蛋白（membrane trafficking protein, YIF1B）、toll样受体8（toll like receptor 8, TLR8）、中性粒细胞胞质因子4（neutrophil cytosolic factor 4, NCF4）、谷胱甘肽过氧化物酶8（glutathione peroxidase 8, GPX8）、肠系膜雌激素依赖性脂肪生成因子（mesenteric estrogen dependent adipogenesis, MEDAG）、核心蛋白聚糖（decorin, DCN）、丝氨酸蛋白酶抑制剂家族F成员1（serpin family F member 1, SERPINF1）、整合素亚基β样蛋白1（integrin subunit beta like 1, ITGBL1）、SRY框转录因子15（SRY-box transcription factor 15, SOX15）、滋养素相关蛋白（trophinin associated protein, TROAP）、SRY框转录因子1（SRY-box transcription factor 1, SOX1）、金属硫蛋白3（metallothionein 3, MT3）、溶酶体跨膜蛋白5（lysosomal protein transmembrane 5, LAPTM5）、含FERM结构域的kindlin3（FERM domain containing kindlin 3, FERMT3）及组织蛋白酶S（cathepsin S, CTSS），上述基因均具有良好的诊断效能（AUC>0.7）。

【结论】本研究通过生物信息学手段更新了CAN的相关研究数据，为阐明CAN的发病机制提供了新视角。未来仍需开展更多研究以验证本研究发现，并进一步探索CAN的潜在发病机制。