基于转录组测序分析转化生长因子-β1诱导肾纤维化机制

Mechanism of transforming growth factor-β1 induce renal fibrosis based on transcriptome sequencing analysis

目的:探究转化生长因子-β1(TGF-β1)诱导肾纤维化的机制。方法:将TGF-β1处理和未处理的肾成纤维细胞NRK-49F进行转录组测序,采用DESeq2分析测序结果,以错误发现率低于0.05且log2FC绝对值大于1为标准筛选差异表达的基因,并对差异表达的基因进行基因本体(GO)、京都基因和基因组百科全书(KEGG)通路富集分析。进一步筛选差异表达基因中编码转录因子的基因,并利用单侧输尿管梗阻诱导的小鼠肾纤维化模型和高通量基因表达数据库GSE104954数据集对这些基因在肾纤维化过程中的表达进行验证。结果:TGF-β1处理6、12和24 h后,分别有552、1209和1028个差异表达基因。GO分析表明,这些差异表达基因在发育、细胞死亡和细胞迁移过程中显著富集。KEGG分析显示,在TGF-β1诱导早期(TGF-β1处理6 h)主要表现为Hippo、TGF-β、Wnt信号通路的变化,而在TGF-β1诱导晚期(TGF-β1处理24 h)主要表现为细胞外基质受体相互作用、局灶黏附和黏附分子连接等相关通路的改变。在TGF-β1处理6 h时291个上调的差异表达基因中,Snai1、Irf8、Bhlhe40、Junb、Arid5a、Vdr、Lef1、Ahr、Foxo1、Myc、Tcf7、Foxc2、Glis1等13个基因编码转录因子。在细胞模型中验证发现,TGF-β1可以诱导其中9个编码转录因子基因(Snai1、Irf8、Bhlhe40、Junb、Arid5a、Vdr、Lef1、Myc、Tcf7)的表达,其余4个基因的表达水平在TGF-β1处理后无显著变化。在动物模型验证中发现,Snai1、Irf8、Bhlhe40、Junb、Arid5a、Myc和Tcf7显著上调,而Vdr显著下调,Lef1无显著变化。在GSE104954数据集中验证发现,IRF8在糖尿病肾病患者和IgA肾病患者的肾小管间质中显著高表达,MYC在糖尿病肾病患者中高表达,而其他7个基因的表达产物与对照组相似。结论:TGF-β1诱导肾成纤维细胞基因差异表达,Irf8和Myc可能是慢性肾脏病和肾纤维化的潜在靶点。

Objective:To explore the mechanism of transforming growth factor-β1(TGF-β1)induce renal fibrosis.Methods:Renal fibroblast NRK-49F cells treated with and without TGF-β1 were subjected to RNA-seq analysis.DESeq2 was used for analysis.Differentially expressed genes were screened with the criteria of false discovery rate<0.05 and|log2FC|>1.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analyses were performed for differentially expressed genes.Genes encoding transcription factors were further screened for differential expression genes.Then,the expression of these genes during renal fibrosis was verified using unilateral ureteral obstruction(UUO)-induced mouse renal fibrosis model and a public gene expression dataset(GSE104954).Results:After TGF-β1 treatment for 6,12 and 24 h,552,1209 and 1028 differentially expressed genes were identified,respectively.GO analysis indicated that these genes were significantly enriched in development,cell death,and cell migration.KEGG pathway analysis showed that in the early stage of TGF-β1 induction(TGF-β1 treatment for 6 h),the changes in Hippo,TGF-βand Wnt signaling pathways were observed,while in the late stage of TGF-β1 induction(TGF-β1 treatment for 24 h),the changes of extracellular matrix-receptor interaction,focal adhesion and adherens junction were mainly enriched.Among the 291 up-regulated differentially expressed genes treated with TGF-β1 for 6 h,13 genes(Snai1,Irf8,Bhlhe40,Junb,Arid5a,Vdr,Lef1,Ahr,Foxo1,Myc,Tcf7,Foxc2,Glis1)encoded transcription factors.Validation in a cell model showed that TGF-β1 induced expression of 9 transcription factors(encoded by Snai1,Irf8,Bhlhe40,Junb,Arid5a,Vdr,Lef1,Myc,Tcf7),while the expression levels of the other 4 genes did not significantly change after TGF-β1 treatment.Validation results in UUO-induced mouse renal fibrosis model showed that Snai1,Irf8,Bhlhe40,Junb,Arid5a,Myc and Tcf7 were up-regulated after UUO,Vdr was down-regulated and there was no significant change in Lef1.Validation based on the GSE104954 dataset showed that IRF8 was significantly overexpressed in the renal tubulointerstitium of patients with diabetic nephropathy or IgA nephropathy,MYC was highly expressed in diabetic nephropathy,and the expressions of the other 7 genes were not significantly different compared with the control group.Conclusion:TGF-β1 induces differentially expressed genes in renal fibroblasts,among which Irf8 and Myc were identified as potential targets of chronic kidney disease and renal fibrosis.