1 INTRODUCTION

The insulin-like growth factors (IGFs) play critical roles in vertebrate growth, development, proliferation, and metabolic regulation (Jones and Clemmons, 1995; Wood et al., 2005). The complex system consists of IGF ligands (IGF1 and IGF2), high-affinity binding proteins (IGFBP1 to 6), and high-affinity cell surface receptors consisting of type Ⅰ (IGF-1R) receptor, type Ⅱ (IGF-2R) receptor, and insulin receptor (IR) (Denley et al., 2005). Both mature IGF1 and IGF2 are conserved single-chain polypeptide growth factors (Jones and Clemmons, 1995). They mediate their biological actions via binding on their receptors to activate the PI3K/Akt pathway and MAPK/Erk pathway (Reinecke et al., 2005; Duan et al., 2010). In addition, most circulating IGFs form complexes with IGFBPs that protect them from degradation and modulate their actions (Reinecke et al., 2005; Duan et al., 2010).

Mature IGF2 is a 67 to 70 amino acids (aa) bioactive protein with four domains (B, C, A, and D). It is initially synthesized as a preprohormone containing a signal peptide at the N-terminus and an E domain at the C-terminus that are proteolytically removed (O'Dell and Day, 1998). Three disulfide bonds are formed in IGFs during the processing: two between domains B and A, and a third within the A domain (O'Dell and Day, 1998; Moriyama et al., 2000).

The expression patterns of igf2 are not exactly the same in different species. In mammals, igf2 expression declined precipitously during the early postnatal period in rodents (Soares et al., 1985; Brown et al., 1986; Graham et al., 1986; Lee et al., 1990), while it maintained a certain level through adult life in humans (Rotwein, 1991; Sussenbach et al., 1993). During teleost embryogenesis, igf2 mRNA is maternal deposited in unfertilized eggs, and its expression is detected in all developmental stages (Greene and Chen, 1997; Ayson et al., 2002; Moriyama et al., 2008; Nipkow et al., 2018; Wang et al., 2019a). In adult fish, igf2 mRNA is expressed in a wide range of tissues (Tse et al., 2002; Radaelli et al., 2003; Caelers et al., 2004; Nipkow et al., 2018; Wang et al., 2019a). Therefore, the expression pattern of igf2 in teleost was contrasts with that of mammals. The previous studies in telesost suggests that IGF2 contributes to both early development and adult growth in fish.

Olive flounder (Paralichthys olivaceus), a benthic flatfish, is an important marine economic fish in Asia due to its fast growth rate and high market value. However, the structure and expression of igf2 in olive flounder has remained unknown. In this study, we identified the open reading frame (ORF) of igf2 gene from olive flounder and investigated its expression patterns in different tissues and early developmental stages. In addition, its soluble recombinant protein was produced in human embryonic kidney (HEK293T) cells and its effect on cell proliferation was verified by methyl thiazolyl tetrazolium (MTT) assay in-vitro. This study provides a basis for further physiological function analysis of IGF2 in the flounder and other fishes.

2 MATERIAL AND METHOD 2.1 Ethics statement

All experiments were conducted in accordance with guidelines approved by the Animal Care and Use Committee of the Shandong University of Technology and the Institute of Oceanology, Chinese Academy of Sciences.

2.2 Experimental animal

Olive flounder were cultured at Shenghang Fish Farm (Rongcheng, Weihai, China) under controlled conditions (photoperiod, 14-h light: 10-h dark; temperature, 15±1 ℃; seawater; aeration). The fertilized eggs were prepared by mixing sperm and eggs, which were collected from matured females and males by artificial gently stripping, respectively. The embryos were cultured in nets floating in a 16-m3 water pool under the same controlled conditions as that in adult flounder. Embryos at different stages of development were obtained as previously reported (Jiao et al., 2015). Tissues (eye, brain, gill, heart, liver, head kidney, kidney, spleen, stomach, intestine, gonad, and muscle) of adult flounders (28–34 cm in total length, TL) were respectively dissected from three females and three males and stored immediately in liquid nitrogen for RNA isolation.

2.3 Total RNA isolation and cDNA synthesis

Total RNA was isolated from the embryos (about 50 embryos per group) and tissues (50–100-mg tissue sample per group) using TRIzol reagent (Invitrogen, USA) following the manufacturer's instruction. The quality and quantity of total RNA were checked by gel electrophoresis and NanoDrop 2000 spectrophotometer (Thermo, USA), respectively. After DNase treatment, 1-μg RNA was used for the first-strand cDNA synthesis with the M-MLV reverse transcriptase (Promega, USA). The obtained cDNAs were preserved at -20 ℃.

2.4 Cloning and analysis of the igf2 ORF

For isolation of igf2, PCR with GoldStar Taq MasterMix (CWBIO, Ltd., China) was performed to isolate the predicted ORFs using gene specific primers (Table 1). The primers used for igf2 and β-actin were designed according to GenBank accession nos. XM_020102187.1 and XM_020109620.1, respectively. The template was an equal mixed cDNAs prepared from embryos at 4–6 somites and hatching stage. The target fragment was subcloned into pEASY-T3 (TransGen Biotech, Beijing, China) vector and sequenced. The genomic structure analysis of flounder igf2 was carried out based on sequence file NW_017859687.1 in the GenBank database. The phylogenetic tree was constructed using the Neighbor Joining method with the Mega X (Kumar et al., 2018), and the branch supports were assessed with 1 000 bootstrap replications. The alignments of the amino acid sequences were analyzed with the multi alignment software Bioedit 7.0 (Hall, 1999).

2.5 Different expression analysis by reverse transcription-polymerase chain reaction (RT-PCR)

The expression profile of igf2 during embryonic development and in adult tissues of olive flounder was analyzed by RT-PCR using GoldStar Taq MasterMix. For embryos, each sample was a pool of about 50 embryos in the same developmental stages. For adult tissues, each sample was from an individual fish. The β-actin was used as an internal control. Sequences of primers used are listed in Table 1.

2.6 Whole-mount in-situ hybridization

Single-color in-situ hybridization using digoxigenin-labeled igf2 antisense riboprobes was performed as reported previously (Jiao et al., 2015). Images were captured with a Leica DFC420C camera mounted on a Leica DMLB2 microscope (Leica, Wetzlar, Germany).

2.7 Construction of plasmids

For in-vitro expression of recombinant flounder IGF2 protein in eukaryotic cells, the signal peptide and BCAD domain of flounder IGF2 were amplified by PCR using igf2-F-3.1 and igf2-R-3.1 as the primers (Table 1), and flounder igf2 ORF in pEASY-T3 as the template. The Kozak sequence (GCCACC) was added immediately upstream of the start codon of the forward primers. The amplified sequence was cloned into the EcoR I site of an eukaryotic expression vector pcDNA3.1/myc-his(-)A using Trelief SoSoo Cloning Kit (TsingKe Biotech, Beijing, China).

2.8 Cell culture, transfection, and eukaryotic expression of flounder IGF2

HEK293T cells (ATCC, Manassas, VA) were cultured in DMEM medium (BI, Israel) supplemented with 10% fetal bovine serum (BI, Israel) and antibiotics in a humidified-air atmosphere containing 5% CO2 at 37 ℃. To produce IGF2, 2.5-μg pcDNA3.1-igf2 was transfected into HEK293T cells by using lipofectamine 3000 (Invitrogen, USA) according to the manufacturer's instruction. The pcDNA3.1/myc-his(-)A alone was used as control. Two days after transfection, the cells were washed with serum free media (SFM) and then incubated in fresh SFM for 48–72 h. Conditioned media (CM) with cell excretes were prepared as reported previously (Duan et al., 1999). The CM of pcDNA3.1 or pcDNA3.1-igf2 transfected HEK293T cells were concentrated by centrifugal filters (Amicon Ultra 3K, Millipore) from 1 000 μL to 10 μL per well for 6-well plate, respectively.

2.9 Western blot analysis

To determine the expression of secretory flounder IGF2, CM or cell lysates from transfected cells was subjected to western blotting using anti-his-tag (TransGen Biotech, Beijing, China) or anti-γ-tubulin (Sigma-Aldrich, St. Louis, MO, USA) antibodies, respectively.

2.10 Cell proliferation assay

Cell proliferation was determined by coloric MTT assay (Beyotime Co., Jiangsu, China) according to the manufacture's protocol 24 h after treatment with 3-μL concentrated CM of pcDNA3.1 or pcDNA3.1-igf2. Hela and HEK293T cells were used as the models for the bioactivity assay.

2.11 Statistical Analysis

Data were expressed as mean±standard error of the mean (SEM) and analyzed with GraphPad Prism 8 software package (GraphPad Prism Software Inc, San Diego, CA, USA). Assumptions of normality and homoscedasticity were assessed. The differences of gene expression levels in the tissues were determined by one-way analysis of variance (ANOVA) with Tukey's post hoc test. The differences in cell proliferation were determined by student's t-tests. Significance was accepted at P < 0.05.

3 RESULT 3.1 Isolation and characterization of igf2 homologue from olive flounder

The ORF encoding flounder IGF2 was obtained by RT-PCR method using flounder embryos cDNA as template. It was 648 bp and spanned about 4.7 kb of genomic DNA, containing four exons and three introns (Fig. 1a). The flounder igf2 encoded a protein of 215 aa. This protein was divided into a 47-aa putative signal peptide, a 32-aa B domain, a 11-aa C domain, a 21-aa A domain, a 6-aa D domain, and a 98-aa E domain. It possessed 6 conserved cysteine residues: 2 in the B domain and 4 in the A domain region. It also contained the conserved Arg²⁴ and Phe²⁶-Tyr²⁷-Phe²⁸ motif in B domain, and Phe⁵¹ and Ser⁵³ in A domain, which were in involved in the binding of its receptors or IGFBPs (O'Dell and Day, 1998; Wood et al., 2005). The predicted flounder IGF2 amino acids shared 48.88% with human (Homo sapiens), 56.85% with zebrafish (Danio rerio) IGF2a, and 78.30% with zebrafish IGF2b (Fig. 2). Phylogenetic tree analysis grouped flounder IGF2 into the IGF2 subgroup. The flounder IGF2 and half-smooth tongue sole (Cynoglossus semilaevis) IGF2 clustered together firstly, and then clustered with zebrafish IGF2a and IGF2b in the teleost group (Fig. 3). These results indicated that the cloned gene was igf2.

3.2 Spatiotemporal expression patterns of flounder igf2

The igf2 mRNA was detected in many adult tissues with varying degrees, including the eye, brain, gill, heart, liver, head kidney, kidney, spleen, stomach, intestine, gonad, and muscle (Fig. 4). Compared with female, male liver had higher levels of igf2 mRNA (Fig. 4).

During early development, the igf2 mRNA was easily detected in all stages of embryogenesis, ranging from unfertilized eggs to hatching embryos (Fig. 5). The igf2 mRNA signals were also detected by whole mount in-situ hybridization during early development (Fig. 6). At both 4–6 somites and tail-bud stages, transcripts of igf2 were mainly detected in diencephalon, midbrain, hindbrain, and floor plate (Fig. 6a & b). At heart beating stage, igf2 transcripts were also detected in hypochord (Fig. 6c & f). At hatching stage, additional signals were detected in otic vesicle and pectoral fin (Fig. 6d & e).

3.3 Expression of recombinant flounder IGF2 in eukaryotic cells

Western immunoblotting of CM with HEK293T cells showed the successful expression and secretion of soluble recombinant IGF2 protein from the IGF2-transfected cells (Fig. 7).

3.4 Flounder IGF2 promotes cell proliferation

The effects of flounder IGF2 on Hela and HEK293T cells proliferation were measured by MTT assay. Flounder IGF2 significantly promoted cell proliferation by 1.12-fold (P < 0.01) in Hela cells (Fig. 8a) and 1.17-fold (P < 0.001) in HEK293T cells respectively (Fig. 8b).

4 DISCUSSION

In this study, we cloned and characterized the ORF of igf2 gene from olive flounder, an important commercial flat fish species in East Asia. Its expression pattern during embryogenesis and in adult tissues, and function on cell proliferation were analyzed.

Flounder igf2 mRNA was detected in all tested adult tissues to different extents, and there was obvious sexual dimorphism in liver in favor of male. The wide expression of igf2 in multiple tissues was also found in other teleosts, such as tilapia (Oreochromis niloticus) (Caelers et al., 2004), Japanese eel (Anguilla japonica) (Moriyama et al., 2008), white seabream (Diplodus sargus) (Perez et al., 2016), and yellowtail kingfish (Seriola lalandi) (Wang et al., 2019a). These results indicate that flounder IGF2 may be associated with growth, metabolism, reproduction, osmoregulation, and development as in other teleosts (Wood et al., 2005; Reinecke, 2010). However, the mRNA level of igf2 was higher in the flounder male liver than that of female. The liver-derived endocrine IGF2 may be a somatomedin in teleost fishes (Pierce et al., 2011). Since the plasma levels of IGF1 and IGF2 were unknown, we could not rule out the possibility that the circulating IGF1 and IGF2 levels in female and male flounder may be comparable, and the lower mRNA level of igf2 in female may be a negative feedback on pituitary growth hormone secretion by a transcriptional mechanism.

The expression of igf2 was detected at various embryonic stages including unfertilized eggs indicating its maternal deposition and transcription from embryonic genomes at later stages (Perrot et al., 1999; Ayson et al., 2002; Peterson et al., 2005; Xu et al., 2015; Wang et al., 2019a), though the onset of embryonic transcription is unclear in our particular case. Our in-situ hybridization data indicate that igf2 was ubiquitously expressed in gastrula-stage embryos (data not shown). With development, igf2 transcripts were readily detected in the brain area and floor plate at somitogenesis-stage. The floor plate is a small group cells present in the ventral midline of the neural tube that profoundly influences the development of the vertebrate nervous system by specifying cellular identities and directing axonal trajectories (Placzek and Briscoe, 2005). So, flounder IGF2 may play critical roles in development and maintenance of the central nervous system (CNS) during embryogenesis. Igf2 transcripts were also detected in hypochord at heart beating stage. Hypochord cells are located at the midline of anamniote embryos and are important for aorta development (Latimer and Appel, 2006). Our results further support the involvement of igf2 in the development of the embryo midline (White et al., 2009; Zou et al., 2009). With development, igf2 transcripts were also readily detected in otic vesicle and fin at hatching-stage. Our results are consistent with findings from zebrafish (Li et al., 2014). In zebrafish, igf2a and igf2b were both detected in otic vesicle, and igf2b was also detected in fin (Li et al., 2014). These above evidence suggest the conserved expression of igf2 in teleosts. The expression changes during embryonic stage with the origin of different tissues might suggest the important role of igf2 in the induction of tissue or organ development. It will be interesting to study the role of igf2 during the most critical physiological changes in the transition from embryo, eleutheroembryo, larva, and metamorphosis (eye migration) to the juvenile period.

Since the expression of igf2 can be detected in all embryonic stages and adult tissues, the igf2 expression may be used as an indicator of animal welfare. However, this decision should be made based on understanding the rules of the normal expression patterns of igf2 from unfertilized eggs to adult, such as hatching, specific larva, metamorphosis, specific juvenile, and adult periods. The abnormal expression of igf2 in specific period may indicate that the flounder is under bad culture condition. Once it happens, appropriate aquaculture interventions could be applied to improve the animal welfare.

We obtained the soluble flounder IGF2 protein in HEK293T cell supernatant by eukaryotic expression method and confirmed its biological activity by MTT assay. Consistent with findings in other teleosts and mammals, recombinant flounder IGF2 significantly increased the proliferate activity of HEK293T and Hela cell lines. In fish, IGF2 stimulates DNA synthesis in tilapia (Oreochromis mossambicus) ovary (TO-2) cells in vitro (Chen et al., 1997) and rainbow trout (Oncorhynchus mykiss) myocytes in primary culture (Codina et al., 2008). In mammals, IGF2 can enhance the proliferation of human embryonic stem cells (ESCs) (Bendall et al., 2007), human preadipocytes (Bäck et al., 2011), and rat adipose-derived stromal cells (ADSCs) (Wang et al., 2019b) in vitro. In addition, IGF2 seems to promote proliferation of fish follicular cells in vivo (Reinecke, 2010). Our results further support the physiological importance of IGF2 in promoting cell proliferation in teleost development and growth.

5 CONCLUSION

This study provided the ORF sequence of igf2 gene in olive flounder. Protein comparison and phylogenetic analysis demonstrated that flounder igf2 is the ortholog of teleost igf2 gene. igf2 is expressed in all selected tissues and is widely expressed during embryonic development. In addition, the soluble recombinant IGF2 protein is directly expressed in HEK293T cells, and it is biologically active. These results suggest that IGF2 may play important roles in the growth and development from embryonic to adult stage in olive flounder.

6 DATA AVAILABILITY STATEMENT

All data generated and/or analyzed during the study are available from the corresponding author on reasonable request.