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Front Immunol
2021 Jan 01;12:792040. doi: 10.3389/fimmu.2021.792040.
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Characterization of Host lncRNAs in Response to Vibrio splendidus Infection and Function as Efficient miRNA Sponges in Sea Cucumber.
Zhang S
,
Shao Y
,
Li C
.
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Long non-coding RNAs (lncRNAs) have been reported to play critical roles during pathogen infection and innate immune response in mammals. Such observation inspired us to explore the expression profiles and functions of lncRNAs in invertebrates upon bacterial infection. Here, the lncRNAs of sea cucumber (Apostichopus japonicus) involved in Vibrio splendidus infection were characterized. RNA-seq obtained 2897 differentially expressed lncRNAs from Vibrio splendidus infected coelomocytes of sea cucumbers. The potential functions of the significant differentially expressed lncRNAs were related to immunity and metabolic process based on the gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Moreover, we identify a lncRNA (XLOC_028509), which is downregulated with Vibrio splendidus challenged, further study indicated that XLOC_028509 adsorb miR-2008 and miR-31 as competing endogenous RNAs (ceRNAs) through base complementarity, which in turn decreased the amount of miRNAs (microRNAs) bound to the 3'UTRs (untranslated regions) of mRNAs to reduce their inhibition of target gene translation. These data demonstrated that the lncRNAs of invertebrates might be important regulators in pathogen-host interactions by sponging miRNAs.
Figure 1. Categories and features of the predicted sea cucumber lncRNAs. (A) Categories of sea cucumber lncRNAs divided according to sea cucumber genome annotation. The lncRNAs were classified into six types (intergenic, intron sense-overlapping, antisense, exon sense-overlapping, intronic antisense, bidirectional) in different groups (uninfected, at day 1 post-infection, at day 7 post-infection). (B) Length distribution of sea cucumber lncRNAs in response to Vibrio splendidus infection. The symbols indicated uninfected and at day 1 or day 7 post-infection.
Figure 2. Expression of sea cucumber lncRNAs during Vibrio splendidus infection. (A) Scatter plot of expressed lncRNAs from uninfected (control) sea cucumbers and at day 1 (early) or day 7 (later) after Vibrio splendidus infection of sea cucumbers. X-axis and Y-axis present log2 value of FPKM of different samples, respectively. (B) Heat map analysis of selected differentially expressed lncRNAs based on the high-throughput sequencing data, red and blue represent upregulated and downregulated lncRNAs, respectively. (C) Expression of selected lncRNAs in sea cucumber. At different time post-infection, total RNAs were extracted from the coelomocytes of sea cucumbers, the expression level of lncRNAs were detected using quantitative real-time PCR. The error bars denote the means ± SD of three independent experiments (*p < 0.05; **p < 0.01). β-actin was used as a control.
Figure 3. GO analysis of differentially expressed lncRNAs. (A, B) GO annotations based on the target genes of differentially expressed lncRNAs by co-location analysis, (A) between uninfected and early infected group, (B) between uninfected and later infected group. Different colors were used to distinguish functional category, red columns represented biological process (BP), blue columns represented molecular function (MF), green columns represented cellular component (CC). Only annotations with a significant P-value of < 0.01 were shown.
Figure 4. The enriched KEGG pathways of differentially expressed lncRNAs. (A, B) KEGG analysis of differentially expressed lncRNAs with high enrichment score. (A) between uninfected and early infected group, (B) between uninfected and later infected group. The size of the circle represented the number of genes, red to green indicated that the corrected P-value is gradually becoming smaller. The degree of KEGG enrichment is assessed by the Rich factor, P-value, and Gene number.
Figure 5. Interaction analysis of the differentially expressed lncRNA and miRNAs. (A) Schematic diagram of lncRNA XLOC_028509 binding to miR-2008 and miR-31, the red letters indicated the direct binding sites of miRNAs with XLOC_028509 and the respective mutant sites. (B) Direct interaction between lncRNA XLOC_028509 and miRNAs. HEK-293T cells were co-transfected with target miRNAs and a luciferase reporter fused with XLOC_028509. At 36 h after co-transfection, the luciferase activities were examined. The activity of renilla luciferase was normalized to that of firefly luciferase. As controls, control miRNAs and mutants of XLOC_028509 were included in the co-transfections. Error bars indicate the means ± SD of three independent experiments (**p < 0.01).
Figure 6. The effects of lncRNA XLOC_028509 on target gene expression. (A) Expression level of lncRNA XLOC_028509 after Vibrio splendidus infection of sea cucumbers detected using quantitative real-time PCR. The error bars denote the means ± SD of three independent experiments (*p < 0.05). β-actin was used as a control. (B) Knockdown of XLOC_028509 by sequence-specific siRNA. Sea cucumbers were injected with XLOC_028509-siRNA, as a control, XLOC_028509-siRNA-scrambled was included in the injection. At 36 h after injection, the expression level of XLOC_028509 was examined by quantitative real-time PCR. β-actin was used as a control. Error bars indicate the means ± SD of three independent experiments (**p < 0.01). (C) Influence of lncRNA XLOC_028509 silencing on the target genes expression. Sea cucumbers were respectively injected with XLOC_028509-siRNA and XLOC_028509-siRNA-scrambled, at 36 h after injection, the mRNA levels of BHMT and CTRP9 were analyzed by quantitative real-time PCR (*p < 0.05; **p < 0.01).