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PeerJ
2018 Jan 01;6:e5703. doi: 10.7717/peerj.5703.
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The potential contribution of miRNA-200-3p to the fatty acid metabolism by regulating AjEHHADH during aestivation in sea cucumber.
Chen M
,
Wang S
,
Li X
,
Storey KB
,
Zhang X
.
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The sea cucumber (Apostichopus japonicus) has become a good model organism for studying environmentally-induced aestivation by a marine invertebrate more recently. In the present study, we hypothesized that miRNA-200-3p may contribute to establish rapid biological control to regulate fatty acid metabolism during a estivation. The peroxisomal bi-functional enzyme (EHHADH) is a crucial participant of the classical peroxisomal fatty acid β-oxidation pathway, the relative mRNA transcripts and protein expressions of EHHADH were analyzed in intestine from sea cucumbers experienced long-term aestivation. Both mRNA transcripts and protein expressions of EHHADH in intestine decreased significantly during deep-aestivation as compared with non-aestivation controls. Analysis of the 3'' UTR of AjEHHADH showed the presence of a conserved binding site for miR-200-3p. Level of miR-200-3p showed an inverse correlation with EHHADH mRNA transcripts and protein levels in intestine, implicating miR-200-3p may directly targeted AjEHHADH by inducing the degradation of AjEHHADH mRNA in the aestivating sea cucumber, further dual-luciferase reporter assay validated the predicted role of miRNA-200-3p in regulating AjEHHADH. In order to further understand their regulatory mechanism, we conducted the functional experiment in vivo. The overexpression of miR-200-3p in sea cucumber significantly decreased mRNA and protein expression levels of AjEHHADH. Taken together, these findings suggested the potential contribution of miRNA-200-3p to the fatty acid metabolism by regulating AjEHHADH during aestivation in sea cucumber.
Figure 1. The complete cDNA sequence and deduced amino acid sequence of AjEHHADH from A. japonicus.Amino acids are numbered starting with the N-terminal Met residue. The asterisk indicates the translational termination codon. The open reading frame (ORF) from the initiation codon (ATG) to the termination codon (TAG) is notated by uppercase letters. At the bottom of the page is the schematic diagram of domains and characteristic motifs.
Figure 2. Theoretical binding of miR-200-3p to a conserved region in the 3′ UTR of the AjEHHADH gene.(A) Conservation analysis of the miR-200-3p binding site in the EHHADH gene from sea cucumber A. japonicus and Lingula anatina. The seed region sequence (shaded) shows 100% conservation between these two sequences. (B) Predicted binding structure of miR-200-3p when binding to the 3′ UTR of AjEHHADH, as determined from the TargetScan and miRanda program.
Figure 3. Effect of estivation on the relative expression levels of AjEHHADH.(A) Relative mRNA expression levels of AjEHHADH in the intestine of NA and DA groups respectively. Values were normalized against β-Tubulin. “*” indicates significant statistical differences (P < 0.05). Values are means ± SE (N = 5). (B) Relative protein expression level of AjEHHADH at the NA and DA stages in intestine by Western blot. Representative bands show blot intensity for NA and DA groups. Histograms show normalized expression levels for NA and DA. β-Tubulin was chosen as the internal control. “***” indicates significant differences for NA and DA groups (P < 0.001). Values are means ± SE (N = 4).
Figure 4. Effect of estivation on the relative expression of miRNA-200-3p.Representative bands show RNA transcript levels amplified by RT-PCR. Band intensities from the RT-PCR samples were normalized to either 5.8S rRNA (microRNA) band amplified from the same sample. Data are means ± SE (N = 5 independent trials on tissue from different animals). “*” Indicates a significant difference from the corresponding control (p < 0.05).
Figure 5. Identification and characterization of the miRNA-200-3p binding sites in the 3′ UTR of AjEHHADH and functional effect of miR-200-3p on AjEHHADH.(A) Schematic representation of the putative miRNA-200-3p targeting sites in AjEHHADH mRNA and the respective mutant sites. (B) HEK-293T cells were co-transfected with the pMIRREPORT-BHMT-WT vector, carrying the wild-type and the mutated AjEHHADH 3′-UTR, pRLCMV-Renilla-luciferase, and control miR-200-3p mimics as indicated. “*”indicates significant differences (P < 0.05). WT, Wild-type; MT, Mutant type; 200M, miR-200-3p mimics; NCM, negative control without miR-200-3p.
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