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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
.
Abstract
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).
Barlat,
Loss of the G1-S control of cyclin A expression during tumoral progression of Chinese hamster lung fibroblasts.
1993, Pubmed
Barlat,
Loss of the G1-S control of cyclin A expression during tumoral progression of Chinese hamster lung fibroblasts.
1993,
Pubmed
Biggar,
Functional impact of microRNA regulation in models of extreme stress adaptation.
2018,
Pubmed
Biggar,
Identification and expression of microRNA in the brain of hibernating bats, Myotis lucifugus.
2014,
Pubmed
Biggar,
Perspectives in cell cycle regulation: lessons from an anoxic vertebrate.
2009,
Pubmed
Biggar,
Evidence for cell cycle suppression and microRNA regulation of cyclin D1 during anoxia exposure in turtles.
2012,
Pubmed
Biggar,
The emerging roles of microRNAs in the molecular responses of metabolic rate depression.
2011,
Pubmed
Chen,
Understanding mechanism of sea cucumber Apostichopus japonicus aestivation: Insights from TMT-based proteomic study.
2016,
Pubmed
,
Echinobase
Chen,
Large-scale identification and comparative analysis of miRNA expression profile in the respiratory tree of the sea cucumber Apostichopus japonicus during aestivation.
2014,
Pubmed
,
Echinobase
Chen,
Comparative phosphoproteomic analysis of intestinal phosphorylated proteins in active versus aestivating sea cucumbers.
2016,
Pubmed
,
Echinobase
Chen,
The potential contribution of miRNA-200-3p to the fatty acid metabolism by regulating AjEHHADH during aestivation in sea cucumber.
2018,
Pubmed
,
Echinobase
Chen,
High-throughput sequencing reveals differential expression of miRNAs in intestine from sea cucumber during aestivation.
2013,
Pubmed
,
Echinobase
Feng,
MiR-200, a new star miRNA in human cancer.
2014,
Pubmed
Gao,
Phenotypic plasticity of gut structure and function during periods of inactivity in Apostichopus japonicus.
2008,
Pubmed
,
Echinobase
Gramantieri,
Cyclin G1 is a target of miR-122a, a microRNA frequently down-regulated in human hepatocellular carcinoma.
2007,
Pubmed
Hadj-Moussa,
The hibernating South American marsupial, Dromiciops gliroides, displays torpor-sensitive microRNA expression patterns.
2016,
Pubmed
Kanakkanthara,
Cyclin A2 is an RNA binding protein that controls Mre11 mRNA translation.
2016,
Pubmed
Kornfeld,
Differential expression of mature microRNAs involved in muscle maintenance of hibernating little brown bats, Myotis lucifugus: a model of muscle atrophy resistance.
2012,
Pubmed
Lang-Ouellette,
Differential expression of miRNAs with metabolic implications in hibernating thirteen-lined ground squirrels, Ictidomys tridecemlineatus.
2014,
Pubmed
Lee,
The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14.
1993,
Pubmed
Lines,
MiR-15a/miR-16-1 expression inversely correlates with cyclin D1 levels in Men1 pituitary NETs.
2018,
Pubmed
Liu,
Genomic analysis of miRNAs in an extreme mammalian hibernator, the Arctic ground squirrel.
2010,
Pubmed
Luu,
Torpor-responsive expression of novel microRNA regulating metabolism and other cellular pathways in the thirteen-lined ground squirrel, Ictidomys tridecemlineatus.
2016,
Pubmed
Obaya,
Regulation of cyclin-Cdk activity in mammalian cells.
2002,
Pubmed
Pagano,
Cyclin A is required at two points in the human cell cycle.
1992,
Pubmed
Pautke,
Characterization of osteosarcoma cell lines MG-63, Saos-2 and U-2 OS in comparison to human osteoblasts.
2004,
Pubmed
Storey,
Tribute to P. L. Lutz: putting life on 'pause'--molecular regulation of hypometabolism.
2007,
Pubmed
Storey,
Metabolic rate depression in animals: transcriptional and translational controls.
2004,
Pubmed
Swenson,
The clam embryo protein cyclin A induces entry into M phase and the resumption of meiosis in Xenopus oocytes.
1986,
Pubmed
Wang,
MiR-138 induces cell cycle arrest by targeting cyclin D3 in hepatocellular carcinoma.
2012,
Pubmed
Wang,
A potential antiapoptotic regulation: The interaction of heat shock protein 70 and apoptosis-inducing factor mitochondrial 1 during heat stress and aestivation in sea cucumber.
2018,
Pubmed
,
Echinobase
Wu,
Pattern of cellular quiescence over the hibernation cycle in liver of thirteen-lined ground squirrels.
2012,
Pubmed
Xiao,
p38/p53/miR-200a-3p feedback loop promotes oxidative stress-mediated liver cell death.
2015,
Pubmed
Zhang,
MicroRNA-365 inhibits vascular smooth muscle cell proliferation through targeting cyclin D1.
2014,
Pubmed
Zhao,
RNA-seq dependent transcriptional analysis unveils gene expression profile in the intestine of sea cucumber Apostichopus japonicus during aestivation.
2014,
Pubmed
,
Echinobase
Zhu,
Gene structure, expression, and DNA methylation characteristics of sea cucumber cyclin B gene during aestivation.
2016,
Pubmed
,
Echinobase