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MiR-200-3p Is Potentially Involved in Cell Cycle Arrest by Regulating Cyclin A during Aestivation in Apostichopus japonicus.
Wang S
,
Chen M
,
Yin Y
,
Storey KB
.
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The sea cucumber (Apostichopus japonicus) has become a good model organism for studying environmentally induced aestivation in marine invertebrates. We hypothesized that mechanisms that arrest energy-expensive cell cycle activity would contribute significantly to establishing the hypometabolic state during aestivation. Cyclin A is a core and particularly interesting cell cycle regulator that functions in both the S phase and in mitosis. In the present study, negative relationships between miR-200-3p and AjCA expressions were detected at both the transcriptional and the translational levels during aestivation in A. japonicus. Dual-luciferase reporter assays confirmed the targeted location of the miR-200-3p binding site within the AjCA gene transcript. Furthermore, gain- and loss-of-function experiments were conducted in vivo with sea cucumbers to verify the interaction between miR-200-3p and AjCA in intestine tissue by qRT-PCR and Western blotting. The results show that the overexpression of miR-200-3p mimics suppressed AjCA transcript levels and translated protein production, whereas transfection with a miR-200-3p inhibitor enhanced both AjCA mRNA and AjCA protein in A. japonicus intestine. Our findings suggested a potential mechanism that reversibly arrests cell cycle progression during aestivation, which may center on miR-200-3p inhibitory control over the translation of cyclin A mRNA transcripts.
Figure 1. The complete cDNA sequence and deduced amino acid sequence of AjCA. Coding and noncoding regions are shown by uppercase and lowercase letters, respectively. The asterisk indicates the translational termination codon. 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 AjCA gene. (A) Conservation analysis of the miR-200-3p binding site in the Cyclin A gene from the sea cucumber A. japonicus, hood coral Stylophora pistillata and giant owl limpet Lottia gigantea. (B) Predicted binding structure of miR-200-3p when binding to the 3’UTR of AjCA and the mature miR-200-3p sequence (shown in red), as determined from TargetScan and miRanda programs.
Figure 3. The relative expression of miR-200-3p in the intestine of A. japonicus at non-aestivating (NA), deep-aestivation (DA), and arousal from aestivation (AA) stages. The expression of miR-200-3p was detected by qRT-PCR in the intestine of A. japonicus from NA, DA and AA groups. Data are means ± SE (n = 5 independent trials on tissue from different animals). Different lowercase letters indicate groups that are significantly different from each other (p < 0.05).
Figure 4. The mRNA expression and protein production levels of AjCA in the intestine of A. japonicus at NA, DA and AA stages. (A) Relative mRNA expression levels of AjCA in the intestine of NA, DA and AA groups, determined by qRT-PCR. Values were standardized against β-tubulin and β-actin. Values are means ± SE (n = 5). Different lowercase letters indicate groups that are significantly different from each other (p < 0.05). (B) Relative protein levels of AjCA at the NA, DA and AA stages in intestine as determined by Western blot. Representative bands show blot intensities for NA (lanes 1–3), DA (lanes 4–6) and AA (lanes 7–9) groups. AjCA protein levels were standardized against the corresponding β-tubulin band densities for the same samples. Histograms show the standardized levels for NA, DA and AA. Values are means AjCA ± SE (n = 3). Different lowercase letters indicate groups that are significantly different from each other (p < 0.05).
Figure 5. Validation of the binding sites between miR-200-3p and 3′UTR of AjCA. (A) Schematic representation of the putative miRNA-200-3p targeting sites in AjCA mRNA and the respective mutant sites. (B) HEK-293T cells were co-transfected with the pmiR-RB-REPORT™ vectors, carrying the wild-type (WT) or the mutated (Mut) AjCA 3′-UTR, pRLCMV-Renilla-luciferase, and control miR-200-3p mimics as indicated. ** indicates a significant difference (p < 0.01). NC: negative control without miR-200-3p.
Figure 6. Gain and loss of function analysis of miR-200-3p in the intestine of A. japonicus in vivo. (A) Relative AjCA transcript levels after transfection with miR-200-3p mimics or inhibitor. Values were normalized against β-tubulin and β-actin. Values are means ± SE (n = 5). * indicates a significant difference (p < 0.05); ** (p < 0.01). (B) Relative AjCA protein production after transfection with miRNA mimics or inhibitor. Representative bands show blot intensity. Lanes show the treatments as follows: (1–3) miR-200-3p mimics, negative control; (4–6) miR-200-3p mimics; (7–9) miR-200-3p inhibitor, negative control; (10–12) miR-200-3p inhibitor. Corresponding tubulin bands are also shown. Values were standardized against the corresponding densities for β-tubulin. Values are means ± SE (n = 3). * indicates a significant difference (p < 0.05).
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