Transcriptome Studies for Gut and Liver in Turbot (Scophthalmus maximus L.) During Vibrio anguillarum Challenge and Exploration of Occludin Gene Function

Xin CAI Cai

China contributes to more than 60 % of global aquaculture production, but recent disease outbreaks have harmed the aquaculture industry and caused immense economic losses. Turbot (Scophthalmus maximus) is a flatfish with increasing commercial value, while Vibrio anguillarum, a Gram-negative pathogenic bacterium, is becoming a major constraint on the development of the turbot aquaculture industry because of its characteristics of worldwide distribution, broad host range and potentially devastating impacts. The immune response of turbot to bacterial infection has not been well studied and a deeper understanding of this response is essential for the effective development of control strategies, such as vaccination programs, probiotics and immunostimulants, genetic selection and breeding, and broad-spectrum antibacterial drugs. In this study, I used, for the first time, whole transcriptome sequencing to screen the key long non-coding RNAs (lncRNAs), circular RNAs (circRNAs), micro RNAs (miRNAs) and messenger RNAs (mRNAs) involved in the immune response to bacterial infection in turbot liver, and to predict and verify the role of competitive endogenous RNA networks in this response. The results identified 184 differentially expressed (DE) lncRNAs, 31 DEcircRNAs, 53 DEmiRNA, and 948 DEmRNAs, in 12 immune-related KEGG pathways involved in the early stages of innate immunity, including cell adhesion molecules, calcium signaling pathway, alanine, aspartate and glutamate metabolism, and ABC transporters. Finally, 65 circRNA-miRNA-mRNA pathways were constructed, based on the hypothesis of ceRNA regulatory networks. Next, in order to demonstrate immunological cooperation in the turbot gut-liver axis, I constructed libraries of immune genes and performed functional annotation and enrichment analyses on immune genes that were expressed in both the turbot intestine and liver. Additionally, bioinformatic analyses were conducted on transcriptomic data to investigate the regulatory relationships between non-coding RNAs (ncRNAs) and their target immune genes, based on the ceRNA theory. This led to the construction of a hypothesis about the anti-inflammatory regulatory mechanism in turbot. This hypothesis proposes that the Toll-like receptor signaling pathway (TLR) activated the recognition and binding abilities of bacteria. Consequently, the TLR/MyD88 signaling pathway activated NF-κB, leading to the up-regulation of its target genes in the intestine and associated tissues of the digestive tract. Finally, inflammatory cytokines, including IL1β, IL6, IL12, TNFα and were generated and transferred out of the nucleus to participate in the inflammatory response. Moreover, a long non-coding RNA (LNC_002468) can also activate the TLR signaling pathway by upregulating the TLR9 gene through the sequestration of a miRNA (novel_709). These hypothesized regulatory relationships were validated through RT-qPCR analyses and dual-luciferase reporter assays. Histological examination of the intestines and livers of infected fish revealed consistent pathological changes indicative of inflammation. This joint analysis suggests that ncRNAs have regulatory functions in the antibacterial immune response of teleost fishes. Finally, based on the sequencing data of turbot gut and liver during bacterial infection, tight junction genes were identified as early-stage participants in the antibacterial process in the turbot intestine, especially the occludin (OCLD) gene which is highly expressed in turbot intestine. The functions of OCLD, which has been seldom studied in teleost, were examined in a gene knock-down turbot intestine cell line, using CCK- 8 kit, wound healing assay, annexin V/PE apoptosis detection kit and flow cytometry, and intracellular bacterial infection. This showed that OCLD has functions in proliferation, migration, apoptosis and prevention of bacterial infection. Thus, based on the vital roles of the OCLD gene, maintaining the integrity of OCLD protein may be a strategy for preventing and treating intestinal diseases.

Transcriptome Studies for Gut and Liver in Turbot (Scophthalmus maximus L.) During Vibrio anguillarum Challenge and Exploration of Occludin Gene Function

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