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

Abstract

To explore the mechanism of transforming growth factor-β1 (TGF-β1) inducing renal fibrosis. Renal fibroblast NRK-49F cells treated with and without TGF-β1 were subjected to RNA-seq analysis. DESeq2 was used to analyze the sequencing results. Differentially expressed genes were screened with the criteria of false discovery rate<0.05 and l o g 2 F C >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 in differentially expressed genes. Then, the expression of these genes during renal fibrosis were verified using unilateral ureteral obstruction (UUO)-induced mouse renal fibrosis model and data set GSE104954 in high-throughput gene expression database. 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 of differential expression 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 of Hippo, TGF-β and Wnt signaling pathways were mainly manifested, 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 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 cell model showed that TGF-β1 induced 9 transcription factors expressions (encoded by Snai1, Irf8, Bhlhe40, Junb, Arid5a, Vdr, Lef1, Myc, Tcf7), while the expression levels of the other 4 genes did not change significantly 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 in 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. TGF-β1 induces genes differentially expressed in renal fibroblasts, and Irf8 and Myc were identified as potential targets of chronic kidney disease and renal fibrosis.