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. 2014 Nov 5;9(11):e111820.
doi: 10.1371/journal.pone.0111820. eCollection 2014.

Characterization and expression analysis of microRNAs in the tube foot of sea cucumber Apostichopus japonicus

Hongdi Wang  1 , Shikai Liu  2 , Jun Cui  1 , Chengze Li  1 , Xuemei Qiu  1 , Yaqing Chang  1 , Zhanjiang Liu  2 , Xiuli Wang  1
Affiliations
Free PMC article

Characterization and expression analysis of microRNAs in the tube foot of sea cucumber Apostichopus japonicus

Hongdi Wang et al. PLoS One. .
Free PMC article

Abstract

MicroRNAs (miRNAs) are a class of endogenous non-coding small RNA with average length of 22 nucleotides, participating in the post-transcriptional regulation of gene expression. In this study, we report the identification and characterization of miRNAs in the tube foot of sea cucumber (Apostichopus japonicus) by next generation sequencing with Illumina HiSeq 2000 platform. Through the bioinformatic analysis, we identified 260 conserved miRNAs and six novel miRNAs from the tube foot small RNA transcriptome. Quantitative realtime PCR (qRT-PCR) was performed to characterize the specific expression in the tube foot. The results indicated that four miRNAs, including miR-29a, miR-29b, miR-2005 and miR-278-3p, were significantly up-regulated in the tube foot. The target genes of the four specifically expressed miRNAs were predicted in silico and validated by performing qRT-PCR. Gene ontology (GO) and KEGG pathway analyses with the target genes of these four miRNAs were conducted to further understand the regulatory function in the tube foot. This is the first study to profile the miRNA transcriptome of the tube foot in sea cucumber. This work will provide valuable genomic resources to understand the mechanisms of gene regulation in the tube foot, and will be useful to assist the molecular breeding in sea cucumber.

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Conflict of interest statement

Competing Interests: The co-author Zhanjiang Liu is a PLOS ONE Editorial Board member. This does not alter the authors' adherence to PLOS ONE Editorial policies and criteria.

Figures

Figure 1
Figure 1. Length distribution of small RNAs identified from the tube foot of sea cucumber (A. japonicus).
Figure 2
Figure 2. Relative abundance of conserved miRNAs identified from the tube foot of sea cucumber (A. japonicus).
Figure 3
Figure 3. Analyses of the nucleotide bias at the first position of miRNAs and each position of miRNAs with length of 22 nucleotides.
A: The nucleotide bias at the first position of miRNAs. B: The nucleotide bias at each position of miRNAs with length of 22 nucleotides.
Figure 4
Figure 4. The secondary structure of predicted novel miRNAs.
Figure 5
Figure 5. GO terms for predicted target genes.
Figure 6
Figure 6. Quantitative realtime PCR analysis of the expression of miR-29a, miR-29b, miR-2005 and miR-278-3p in different tissues of sea cucumber.
The tissues are abbreviated as follows: TF, tube foot; I, intestine; RT, respiratory tree, and C, coelomocytes. A: qRT-PCR using β-actin as the reference gene. B: qRT-PCR using Cytb as the reference gene. Bars are shown as mean ± standard deviation. Bars with different superscripts indicate that they are significantly different from each other (p<0.05).
Figure 7
Figure 7. Quantitative realtime PCR analysis of the expression of 15 selected predicted target genes in different tissues of sea cucumber.
The tissues are abbreviated as follows: TF, tube foot; I, intestine; RT, respiratory tree, and C, coelomocytes. Bars are shown as mean ± standard deviation. Bars with different superscripts indicate that they are significantly different from each other (p<0.05).
Figure 8
Figure 8. GO analysis for predicted target genes of miR-29a at level 2.
Figure 9
Figure 9. Venn diagram of the identified KEGG pathways.
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Grants and funding

This project was supported by the State 863 High-Technology R & D Project of China (2012AA10A412) and the grant of Dalian Ocean University (2012HYDX02). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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