Effect of TRAF6-knockout on gene expression and lncRNA expression in Epithelioma papulosum cyprini (EPC) cells

Abstract

TRAF6 is a key immune gene that plays a significant role in toll-like receptor signal transduction and activates downstream immune genes involved in antiviral immunity in fish. To explore the role of TRAF6 in Epithelioma papulosum cyprini (EPC) cells, we knocked out the TRAF6 gene using the Clustered Regularly Interspaced Short Palindromic Repeats-Cas9 (CRISPR-Cas9) technique and then analyzed the transcriptomes of the knockout cells. In this study, we identified that 232 transcripts were differentially expressed in naive cells. Using the pipeline, we identified 381 novel lncRNAs in EPC cells, 23 of which were differentially expressed. Gene Ontology enrichment analysis demonstrated that differentially expressed genes (DEG) are implicated in various immune processes, such as neutrophil chemotaxis and mitogen-activated protein kinase binding. In addition, the KEGG pathway analysis revealed enrichment in immune-related pathways (Interleukin-17 signaling pathway, cytokine-cytokine receptor interaction, and TNF signaling pathway). Furthermore, the target genes of the differentially expressed lncRNAs were implicated in the negative regulation of interleukin-6 and tumor necrosis factor production. These results indicate that lncRNAs and protein-coding genes participate in the regulation of immune and metabolic processes in fish.

Keywords: Epithelioma Papulosum Cyprini; RNA-seq; immune process; lncRNAs.

Figure 1.

Workflow showing the procedure used to construct the CRISPR/Cas9 vector targeting Epithelioma papulosum cyprini (EPC) cell's TRAF6 gene (pCRISPR/Cas9 RGR-sgTRAF6).

Figure 2.

Sequence alignment of nucleotides and amino acids of TRAF6 gene. The knockout of the TRAF6 cell clone showed heterozygous insertion/deletion (indel) mutations. The upper black boxes represent exon regions of the TRAF6 gene and the upper numbers of the boxes mean the nucleotides of TRAF6 ORF from 1 to 1629 (A). The asterisk represents the generation of a premature stop codon by out-of-frame indels (B).

Figure 3.

Heatmap of differentially expressed genes in naïve cells (control) and TRAF6-knockout cells(ΔTRAF6). The heatmap was generated using the Complex Heatmap package and the counts of differentially expressed genes. Upregulated genes are indicated in red and downregulated genes are indicated in blue.

Figure 4.

The top 15 enriched gene ontology terms of (A) downregulated genes and (B) upregulated genes. The abscissa represents the proportion to gene ratio of the enriched gene number. The node size represents the count of genes significantly enriched in each gene ontology term.

Figure 5.

KEGG pathway enrichment analysis of (A) downregulated genes and (B) upregulated genes using ClusterProfiler package. The abscissa represents the count of genes significantly enriched in each pathway.

Figure 6.

Gene ontology enrichment analysis (A) and KEGG pathway analysis (B) of cis-target genes of differentially expressed lncRNAs. The gene ontology (A) categories were biological process, cellular component, and molecular function.

Figure 7.

Comparison between transcriptome and qRT-PCR expression levels. The differentially expressed genes were validated by qPCR. The expression level showed a similar pattern to the transcriptome fold change.