The potential contribution of miRNA-200-3p to the fatty acid metabolism by regulating AjEHHADH during aestivation in sea cucumber

Animals

Adult sea cucumbers (males and females mixed) (A. japonicus), 100 ± 8g, were collected from the coast of Qingdao by diving and hand fishing (Jiaozhou Bay of the Yellow Sea, China). Two groups of sea cucumbers were sampled and dissected right after capture. Non-aestivating sea cucumbers (NA) which served as the control group were sampled in May when the seawater temperature was about 15 °C. At this time and temperature, the sea cucumbers have already recovered fully from aestivation. Animals in deep-aestivation (DA) were sampled in mid-August when seawater temperature was above 25 °C. These animals were sampled after about 15 days of continuous aestivation as indicated by cessation of feeding and locomotion, and the degeneration of the intestine into a very thin and small string (about 2 mm). At each stage (NA and DA), 10 individuals were sacrificed and intestine tissues were dissected, cleared of content and flash frozen in liquid nitrogen. All samples were kept at −80 °C for later analysis. Sea cucumbers (A. japonicus) are commercially cultured animals. The study protocol was approved by the Experimental Animal Ethics Committee of the Ocean University of China.

RNA extraction, AjEHHADH cDNA cloning, and sequence analysis

Total RNA was isolated from intestinal tissues of sea cucumbers using Trizol (Cat No. J20921; TransGen Biotech, Beijing, China) following manufacturer’s instructions. RNA concentration and quality were determined using an Agilent 2100 bioanalyzer. Gene-specific primers for cloning the full-length cDNA of AjEHHADH were designed based on the partial sequences of AjEHHADH genes obtained from our previously constructed transcriptome library of A. japonicus (Data S11). The full-length cDNA sequences were cloned using the SMART RACE cDNA Amplification kit (Cat No. ST0258; Clontech, Mountain View, USA) in accordance with the manufacturer’s instructions. The primer sets for AjEHHADH 5′/3′ RACE are listed in Table 1. Polymerase chain reaction (PCR) amplification was carried out using Advantage 2 polymerase mix (Cat No. S1798; Clontech, Mountain View, CA, USA) in a volume of 50 µL. PCR was performed under the following conditions: 95 °C for 1 min; five cycles of 94 °C for 30 s, 66 °C for 30 s, and 72 °C for 3 min; five cycles of 94 °C for 30 s, 64 °C for 30 s, and 72 °C for 3 min; five cycles of 94 °C for 30 s, 62 °C for 30 s, and 72 °C for 3 min; 20 cycles of 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 3 min, followed by a final cycle of 72 °C for 10 min. The expected PCR products were eluted from a 1% agarose gel using a gel extraction kit (Cat No. D2500-02; Omega, Norcross, GA, USA) following the manufacturer’s instructions, then cloned into the pMD19-T vector (Cat No. 6013; Takara, Kusatsu, Japan) and transformed into JM109 competent cells (Cat No. 9057; Takara, Kusatsu, Japan) following the manufacturer’s instructions. Transformed cells were cultured overnight on Luria-Bertani (LB) agar plates containing 100 µg/mL ampicillin. White clones were chosen and cultured in SOC medium (Cat No. HBDC002; Hopebio, Qingdao, China) containing 100 µg/mL ampicillin for 10 h at 37 °C. Positive recombinant clones were sequenced by Sangon Biotech (Shanghai, China). The sequences were analyzed and assembled using DNAStar software (DNAStar Inc., Madison, WI, USA) to identify the open reading frame (ORF). The deduced amino acid sequence of sea cucumber AjEHHADH was analyzed using an on-site program http://www.bio-soft.net/sms/index.html. The functional sites or domains in the amino acid sequence were predicted using the simple modular architecture research tool (SMART) version 4.0 (http://smart.embl-heidelberg.de/) and Interpro (http://www.ebi.ac.uk/interpro/).

Prediction of the miR-200-3p target

Based on our previous transcriptome (Data S11) and proteome (Chen et al., 2016) data for A. japonicus, potential miR-200-3p targets were predicted using TargetScan and miRanda software. The target genes for different miRNAs were predicted by the TargetScan algorithm complying with the criteria in the seed region: no mismatch between two to eight nt on the end of miRNA (7mer-m8) The potential targets of miRNA-200-3p were also predicted using the Miranda toolbox based on the complementary region between the miRNA and the 3′-UTR of mRNA, as well as the thermodynamic stability of the miRNA-mRNA duplex. The candidate targets with S >90 (single-residue pair scores) and a minimum free energy lower than −20 kcal/mol were selected for further analysis. The TargetScan program searches for microRNA binding sites (seed matches) conserved between several organisms.

Dual-luciferase reporter assays

Both wildtype (WT) and mutant (MT) segments of the AjEHHADH 3′-UTR (about 500 nt before and after the binding sites) were cloned into pmiR-RB-REPORT luciferase reporter vectors (Ribobio, Guangzhou, China). The primers selected are listed in Table 1. For the transfection experiment, the 293T cells were plated into 96-well white plates 24 h before transfection. Plasmids constructed with pMIR-REPORT vectors were cotransfected with a control Renilla luciferase plasmid (pRL-CMV) (Cat No. E2261; Promega, Madison, WI, USA). The ratio of experimental plasmid to control plasmid was 5:1. Then vectors with 3′ UTR of AjEHHADH (WT and MT) were cotransfected with miRNA-200-3p mimics or a negative control. Luciferase assays were performed using the Dual-Luciferase Reporter Assay System (Cat No.E1910; Promega, Madison, WI, USA). In brief, 48 h after transfection, cell lysates were prepared by incubating with 1 × passive lysis buffer for 15 min at room temperature. Cell lysates were transferred to 96-well plates and analyzed using the luciferase dual reporter assay kit (Cat No.E1910; Promega, Madison, WI, USA). The firefly luciferase values were normalized to Renilla.

qRT-PCR analysis of miRNA-200-3p and AjEHHADH, Western blotting analysis of AjEHHADH

Total RNA was isolated as described above from sea cucumber intestinal tissues of the control and estivated groups. RNA concentration and quality were determined using an Agilent 2100 bioanalyzer and then stored at −80 °C for further analysis. The Mir-X miRNA First-Strand Synthesis Kit (Cat No. 638313; Takara, Kusatsu, Japan) was used to synthesize first-strand cDNA as the qRT-PCR template. The entire mature miR-200-3p sequence was synthesized as the specific forward primer (miR-200-3p-F in Table 1) for qRT-PCR and the mRQ 3′ primer supplied with the kit was used as the reverse primer. The 5.8s rRNA was selected as the internal control (see Table 1 for primer sets). SYBR^® Premix Ex TaqTM (Cat No. RR820; Takara, Kusatsu, Japan) master mix was used to determine the expression levels of miR-200-3p in both control and estivated groups using the StepOnePlus system (ABI Inc., Foster City, CA, USA). Each sample was run in triplicate along with the internal control gene. Melting-curve analysis of the amplification products was performed at the end of the PCR reaction to determine the purity of the PCR product. The 2^−△△CT method was used to analyze the comparative expression levels.

Total RNA was isolated as described above from the intestine of five individuals for both control and estivated groups. First-strand cDNA was synthesized using the M-MLV reverse transcriptase enzyme (Cat No. RR047A; Takara, Kusatsu, Japan) by qRT-PCR. The AjEHHADH-specific primer pairs for qRT-PCR were designed based on the obtained open reading frame (ORF) using an online version of Primer 3.0 (Table 1). β-tubulin was previously shown to remain unchanged during aestivation in A. japonicus and therefore we used it as the internal control (Zhao et al., 2014). Gene expression levels of AjEHHADH were analyzed using SYBR^® Premix Ex Taq™ master mix (Cat No. RR420; Takara, Kusatsu, Japan) and amplification was measured by the StepOnePlus system (ABI Inc., Foster, CA, USA). Each sample was run in triplicate along with the internal control. Melting-curve analysis of the amplified products was performed at the end of each PCR reaction to ensure that only one PCR product was produced. The 2^−△△CT method was used to analyze the comparative expression levels.

Protein extraction and Western blot analysis were performed as previously described (Chen et al., 2016). In brief, total protein was extracted from intestinal tissues using Cell Lysis buffer (Cat No. P0013; Beyotime, Shanghai, China) following the manufacturer’s instructions. The protein concentration was measured using an enhanced BCA protein assay kit (Cat No. P0010S; Beyotime, Shanghai, China) and detected by the SpectraMax Plus system (Molecular Devices, San Jose, CA, USA). The positive control (PC) and the negative control were all set. For 10% SDS-PAGE analysis, 13 µg of protein was loaded and electrophoresed until an appropriate separation was reached. The separated proteins were then transferred to polyvinylidene fluoride membranes (PVDF membrane) (Cat No. IPVH00010; Millipore, Bedford, MA, USA) using wet transfer (BIO-RAD, Singapore). After blocking with 5% non-fat milk in TBS-Tween 20 buffer (TBST) for 2 h at room temperature on a shaker, the membranes were incubated with rabbit polyclonal AjEHHADH antibody (1:600, prepared by Abmart, Shanghai, China) or β-tubulin antibody (1:1,000, CST, Cat No. 2146S) overnight at 4 °C. The brief protocol for AjEHHADH antibody development was as follows. Two conserved fragments with antigenic determinant using MacVector version 9 (MacVector Inc., Cambridge, UK) were designed based on the ORF. Constructed plasmids containing the specific sequences were extracted and purified as previously described ( Moore et al., 2015), then injected into two healthy rabbits in vivo per antigen. Rabbits were euthanized after seven days and whole blood was withdrawn. Purified antibodies were retrieved using Protein A/G-Plus Beads (Shang Hai Yue-ke Biotechnology CO., Shanghai, China) followed by Elisa verification. The membranes were washed with TBST buffer five times for 5 min each and then incubated with goat anti-rabbit IgG labeled with HRP (1:10,000; Cat No. 7074S; CST, Danvers, MA, USA) for 1 h at room temperature. After washing with TBST for five times (5 min each), the membranes were incubated in ECL Western blot detection reagents (Cat No. P0018; Beyotime, Shanghai, China) and detected using the photo processing machine. Bands were quantified using the Image-Pro Plus 6.0 software (Media Cybernetics Inc., Rockville, MD, USA).

Functional analysis of miRNA-200-3p in vivo

Mimics and negative controls for miR-200-3p were synthesized by GenePharma (Shanghai, China) as shown in Table 1. MiR-200-3p mimics were dissolved in RNase-free water to obtain a working solution of 20 mM. Aliquots of 10 µL of mimics or negative control were mixed with an equal volume of transfection reagent (Beyotime, Shanghai, China) and 80 µL of PBS to serve as the working solution (Lu et al., 2015). Eighteen sea cucumbers (100 ± 8 g) were injected with 100 µl of miR-200-3p mimics or the negative control and a booster injection was given after 24 h. After a further 24 h, treated and control animals were sacrificed and intestine tissues were collected and stored in liquid nitrogen for expression analysis using the qRT-PCR and Western blot protocols described above. The assays were performed with three biological and three technical replicates.

Statistics

qRT-PCR data are given as mean ± S.E. (N = 5) and were analyzed using one-way analysis of variance (ANOVA) followed by a Tukey’s post hoc test (SPSS 17.0 software, Chicago, IL, USA). The level of statistical significance was set at P < 0.05 unless otherwise stated. Relative protein abundance was analyzed using a t-test (SPSS 17.0) (Chicago, IL, USA) and all the results are given as mean ± S.E. (N = 4) .

Dual-luciferase reporter data are given as mean ± S.E. (N = 6), qRT-PCR data are given as mean ± S.E. (N = 5) and relative protein abundance is given as mean ± S.E. (N = 3). These data were used to demonstrate the interaction between miRNA-200-3p and the AjEHHADH mRNA transcript. All data were analyzed using an independent sample t-test by SPSS 17.0 software. The level of statistical significance was set at P <  0.05. P < 0.001 indicated an extremely significant difference.

Sequence characterization of AjEHHADH and target identification of miR-200-3p

The full-length cDNA of AjEHHADH spanned 2,352 bp, including a 179 bp 5′-UTR and a 1,048 bp 3′ UTR. The open reading frame (ORF) contained 1,125 bp and encoded a 375 long amino acid polypeptide with a calculated molecular weight of 67 kDa (Fig. 1). SMART analysis indicated that the sea cucumber AjEHHADH protein contained two enoyl-CoA hydratase/isomerase (ECH) domains and a 3-hydroxyacyl-CoA dehydrogenase NAD binding domain (Fig. 1) (Gene accession No. KY328747KY). Analysis of the putative miR-200-3p binding sites was then performed using TargetScan 5.2 and Miranda 3.3a programs. Results showed that the 3′ UTR of sea cucumber AjEHHADH contained binding sequences for miR-200-3p starting from residue 2,139 within the 3′ UTR sequence. Importantly, the binding site was also identified and found to be conserved in Lingula anatine, a member of another marine invertebrate group, the brachiopods (Fig. 2). The theoretical minimum free energy of binding (kcal/mol) was also calculated using the program Miranda. Theoretical prediction of thermodynamic binding parameters for miRNA-200-3p binding to AjEHHADH was calculated to be −22.34 kcal/mol (below the given theoretical parameters −20 kcal/mol), suggesting that AjEHHADH is a likely target of miRNA-200-3P in sea cucumbers (Fig. 2).

The expression of miR-200-3p and AjEHHADH during aestivation

The mRNA transcript levels of AjEHHADH decreased significantly in the intestine by 0.34 ± 0.11-fold at the DA stage compared to the NA stage (P < 0.05) (Fig. 3A). Moreover, the protein expression levels of AjEHHADH also showed a significant decrease by 0.72 ± 0.08-fold at the DA compared with NA stage (P < 0.05) (Fig. 3B). The expression of miR-200-3p was assessed in the intestine of A. japonicus from non-aestivation and deep-aestivation stages. MiR-200-3p expression levels showed an inverse correlation with AjEHHADH transcript and protein levels in the intestine. Expression levels of miR-200-3p increased significantly in the intestine by 2.53 ± 0.3-fold in the deep-estivated group compared to controls (P < 0.05) (Fig. 4).

Validation of the interaction between 3′ UTR of AjEHHADH and miR-200-3p by dual-luciferase reporter assays

To further validate the predicted role of miR-200-3P in regulating AjEHHADH, plasmids containing the putative binding site for miRNA-200-3p and the mutation site (Fig. 2) of the 3′ UTR of AjEHHADH were constructed and used in the classical reporter assay. Information concerning the binding and mutation sites of miRNA-200-3p in the 3′ UTR of AjEHHADH is shown in Fig. 5A. A significant reduction (11.9%, P = 0.044) was observed in luciferase activity transfected with WT UTR compared with control (Fig. 5B).

MiR-200-3p modulates AjEHHADH at the post-transcriptional level in vivo

To fully understand the potential roles of miR-200-3p in regulating AjEHHADH, gain-of-function assays for miR-200-3p were performed in vivo. As shown in Fig. 6A, the qRT-PCR results indicated that overexpression of miR-200-3p decreased the transcript levels of AjEHHADH, although it was not significant in statistical analysis (P > 0.05). Western blot analysis revealed that AjEHHADH protein levels were also reduced significantly upon miR-200-3p overexpression (Fig. 6B), consistent with the AjEHHADH transcript expression levels (Fig. 3). These results indicate that miR-200-3p may directly target AjEHHADH gene expression by inducing the degradation of the AjEHHADH mRNA transcripts.