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.

Sonic vibration ameliorates inflammatory diseases via the up-regulation of IL-10

“…The total RNA was synthesized into first-strand complementary DNA (cDNA) using a random primer (9-mer, Invitrogen, Carlsbad, CA, USA) and Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT, Enzynomics Co., Daejeon, Republic of Korea) (Abdellaoui et al 2023 ). The gene expression was quantified using TOPreal™ qPCR 2X PreMIX (Enzynomics Co.), the Eco Real-Time PCR system (Illumina, San Diego, CA, USA), and the following specific primers: mouse Interleukin ( IL)-1β (Gene Bank ID, NM_008361, 133 bp), 5′-CCC AAG CAA TAC CCA AAG AA-3′ and 5′-GCT TGT GCT CTG CTT GTG AG-3′; mouse IL-6 (NM_031168, 102 bp), 5′-CCG GAG AGG AGA CTT CAC AG-3′ and 5′-TCC ACG ATT TCC CAG AGA AC-3′; mouse IL-10 (NM_010548, 142 bp), 5′-GCC TGG CTC AGC ACT GCT AT-3′ and 5′-GAA GGC AGT CCG CAG CTC TA-3′; mouse glyceraldehyde-3-phosphate dehydrogenase ( Gapdh, NM_001289726, 223 bp), 5′-AAC TTT GGC ATT GTG GAA GG-3′ and 5′-ACA CAT TGG GGG TAG GAA CA-3′; human IL-1β (NM_000576, 180 bp), 5′-CTG TCC TGC GTG TTG AAA GA-3′ and 5′-TTC TGC TTG AGA GGT GCT GA-3′; human IL-6 (NM_000600, 175 bp), 5′-TAC CCC CAG GAG AAG ATT CC-3′ and 5′-TTT TCT GCC AGT GCC TCT TT-3′; human IL-10 (NM_000572, 253 bp), 5′-TGA GAA CAG CTG CAC CCA CT-3′ and 5′-GTT CAC ATG CGC CTT GAT GT-3′; human Gapdh (NM_001256799, 278 bp), 5′-ACT GGC GTC TTC ACC ACC AT-3′ and 5′-GGG CCA TCC ACA GTC TTC TG-3′.…”