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The sea cucumber Apostichopus japonicus, renowned for its remarkable ability to expel and regenerate its internal organs within weeks, serves as a model organism for regeneration research. However, studies on the role of non-coding RNAs, particularly long non-coding RNA (lncRNA), in intestinal regeneration remain limited. In this study, we identified and performed differential expression analysis of lncRNAs in both normal intestines and intestines at 3 days post evisceration (dpe). A total of 2361 lncRNAs were identified, 183 of which were differentially expressed (DE-lncRNAs). The genes targeted by these lncRNAs, either cis- or trans-acting, were involved in oxidative stress, immune response, extracellular matrix remodeling, and energy metabolism during intestinal regeneration. Notably, MSTRG.6200/miR-7847-3p and MSTRG.18440/miR-4220-5p have been confirmed as interacting lncRNA-miRNA pairs. These results suggest that lncRNAs are key regulators of intestinal regeneration in A. japonicus, offering new insights into the underlying mechanisms and potential targets for enhancing regeneration.
Fig. 1. Identification and characterisation of novel lncRNAs expressed in intestines during regeneration. A. Venn diagram showing lncRNAs identified by three tools (CPC2, CNCI, PLEK); B. Classification of the lncRNA according to the source; C. Distribution of novel lncRNAs based on length; D. Distribution of novel lncRNAs based on exon number.
Fig. 2. Differentially expressed lncRNAs at 3dpe. A. Volcano plot analysis of DE-lncRNAs; B. The heatmap for DE-lncRNAs.
Fig. 3. Validation of the six lncRNAs by qRT-PCR. NADH was selected to normalize the gene expression levels. The data are shown as means ( ± SE) of at least three replicates. Red bars: Significantly differentially expressed lncRNAs in RNA-seq (|log2FC= >1, p < 0.05); Blue bars: RT-qPCR-validated lncRNAs (Student’s t-test, ** p < 0.01,*** p < 0.001, ns no significance).
Fig. 4. Enrichment analysis of cis-targeted genes. A. Gene Ontology (GO) enrichment of cis-targeted genes. Categories: Biological Process (BP), Cellular Component (CC), and Molecular Function (MF); B. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment of cis-targeted genes.
Fig. 5. Analysis of trans-targeted genes. A. Gene Ontology (GO) enrichment of trans-targeted genes; B. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment of trans-targeted genes; C. The interaction network of DE-lncRNA and DE-mRNA.
Fig. 6. Secondary structures of MSTRG.1184 (A), MSTRG.6200 (B), MSTRG.14360 (C), MSTRG.14380 (D), MSTRG.17922 (E), MSTRG.18440 (F), and MSTRG.18680 (G).
Fig. 7. The lncRNAs-miRNAs network of 7 candidate lncRNAs.
Fig. 8. Dual-luciferase assay reporter system results of lncRNA-miRNA. A-C. Schematic of target site combination for MSTRG.6200 and miR-7847–3p (A), MSTRG.14380 and miR-4214–5p (B), MSTRG.18440 and miR-4220–5p (C); D-F. The relative luciferase activities were investigated post co-transfection with lncRNA-wt or lncRNA-mut plasmids and modified miRNA mimics or NC mimics. NC mimics means negative control mimics. NC mimics: UUCUCCGAACGUGUCACGUdTdT * ** * p < 0.0001.
