Characterization of two splice variants of EGFR and their effects on the growth of the razor clam

Knyun Wei,Donghong Niu,b,*,Moxio Peng,Shumei Xie,Shentong Wng,Jile Li,c

aKey Laboratory of Exploration and Utilization of Aquatic Genetic Resources,Ministry of Education,Shanghai Ocean University,Shanghai 201306,China

bNational Demonstration Center for Experimental Fisheries Science Education,Shanghai Ocean University,Shanghai 201306,China

cShanghai Engineering Research Center of Aquaculture,Shanghai 201306,China

1.Introduction

The epidermal growth factor receptor(EGFR)has intrinsic tyrosine kinase activity,and it is present in all Eumetazoa(Kipryushina,Yakovlev&Odintsova,2015).EGFR from the razor clam(Sinonovacula constricta),shares a high level of similarity with other EGFR and contained the 3 typical regions:an extracellular,transmembrane,and intracellular region(Bishayee,2000).In the extracellular region,various ligands,such as the epidermal growth factor(EGF),transforming growth factor-α (TGF-α),amphiregulin(AR),epiregulin(EREG),and betacellulin(BTC),induce the dimerization of EGFR with other family members(e.g.,EGFR,ErbB2,ErbB3,and ErbB4)(Bublil&Yarden,2007).After dimerization,EGFR is phosphorylated and activates multiple intracellular signal pathways,such as the mitogen-activated protein kinase(MAPK)pathway and phosphatidylinositol-3-kinase(PI3K)pathway,that regulate processes,such as proliferation,differentiation,migration,and apoptosis(Bogdan&Kl¨ambt,2001;Magne et al.,2002).

Alternative splicing of immature or precursor mRNAs,can yield different transcript isoforms from a single gene and produce a much larger number of proteins(Matlin,Clark&Smith,2005).To date,at least 4 types of EGFR variants,that have different sensitivities to anti-cancer drugs(Zhu et al.,2008),have been identi fi ed in humans with non-small-cell lung cancers(NSCLCs)(Shigematsu et al.,2005).In addition,mutation in EGFR commonly occur in glioblastoma(GBM)patients,and they upregulate downstream factors like the heterogeneous nuclear ribonucleoprotein A1 splicing factor and delta max to promote cell proliferation in vitro(Babic et al.,2013).In Xiphophorus,two amino acid changes in the extracellular domain of EGFR can result in the generation of a tumorigeniconcoprotein(Gomez,Volff,Hornung,Schartl&Wellbrock,2004).Mutations of EGFR in zebra fish(Danio rerio)leads to a condition similar to the pathology of hypomagnesemia in humans(Arjona,Chen,Flik,Bindels&Hoenderop,2013).In addition,in D.rerio,EGFR and its ligands,such as EGF,heparin-binding EGF-like growth factor(HB-EGF),and BTC(Tse&Ge,2009),have been shown to promote oocyte maturation(Tse&Ge,2010).

Invertebrate species,such as Drosophila,have 3 alternative splice isoforms of EGFR,and these isoforms regulate different cellular signaling pathways(Schejter,Segal,Glazer,&Shilo,1986).In addition,previous studies have shown that alternative splice isoforms are functionally distinct or antagonistic in human diseases(Arinobu et al.,1999).Therefore,it is reasonable to predict that the different EGFR isoforms in S.constricta may have different functions.EGFR transcripts are detectable in the early embryo stages and in Crassostrea angulata,the highest expression levels were observed in the middle-late stages(Coon,Fitt,&Bonar,1990).Schejter performed in situ hybridization and found that EGFR in Drosophila was uniformly distributed in proliferating tissues such as the brain cortex in the early larval stages(Schejter et al.,1986).However,in adult pearl oysters,EGFR is mainly expressed in the mantle and pearl sac(Zhu et al.,2015).In the early stages of wound healing in vivo and in vitro in Crassostrea gigas EGFR was proposed to promote cell proliferation(Sun,Huan,Wang,Liu,&Liu,2014).In Lymnaea stagnalis,EGFR was highly expressed in the synapse,and knockdown of EGFR in vitro resulted in a delay in synapse formation(Kesteren et al.,2008).In addition,EGFR in Gryllus bimaculatus(Dabour et al.,2011)and Macrobrachium rosenbergii(Sharabi,Ventura,Manor,A flalo,&Sagi,2013)appeared to regulate body size.

In this study,we cloned and characterized the full-length cDNA sequence of EGFR from S.constricta and identified 2 transcripts,Sc-EGFR-1a and Sc-EGFR-1b,that were generated by alternative splicing.The involvement of the 2 isoforms in cell proliferation was analyzed by their overexpression in 293T cells and RNAi experiments in vivo revealed their involvement in regulating S.constricta body size.

2.Materials and methods

2.1.Biological material

A total of 20 adult clams used for the gene cloning and qRT-PCR were obtained from Luchaogang Supermarket,Pudong District,China.The adult clams were collected in May,and the gonad tissues were in the rest period(Yan,Li,Liu,Yu&Kong,2010).The shell length of the clams were 6.07±1.92 cm,and 8.93±2.24g of total weight were reared in an aerated artificial seawater tank at 25—27°C for a week.

The embryos and larvae at embryogenesis,trochophore,veliger larvae,umbo larvae,creeping larvae,and juveniles were obtained from Donghang Farm,Sanmen City,Zhejiang Province,China.The specimens were collected using a silk screen and stored in three 1.5 mL centrifuge tubes in RNAstore Reagent(Tiangen,China).

For RNA interference,a total of 300 nine-month-old clams of similar body size(shell length,4.00±0.12cm;body weight,3.90±0.18g)were collected in June from Ninghai Chaoyu Farm,Zhejiang District,China,and reared in an aerated arti fi cial seawater tank at 25—27°C for a week(7.67%clams died).Before the experiment,all the clams were labeled with waterproof tags(Star fish,China)for tracking individual growth traits,such as shell length,shell width,shell height,and body weight.

2.2.Cloning the full-length cDNA of Sc-EGFR

For gene cloning,liver,mantle,gill,foot,gonad,hemolymph,gut,and siphon tissueswere collected from threeadult clams.Expressed sequence tags(ESTs)of EGFR was obtained from a transcriptome library(Niu,Wang,Sun,Liu&Li,2013).The cloned EGFR partial sequence was re-sequenced to con firm the accuracy(Table 1).To obtain full-length cDNA,5′and 3′terminal sequences were generated using the SMARTer RACE cDNA Amplification Kit(Clontech,USA)with gene-specific primers(Table 1).

2.3.Sequence and phylogenetic analyses

The open reading frame(ORF)was predicted using the Open Reading Frame Finder(http://www.ncbi.nlm.nih.gov/gorf/gorf.html).The deduced amino acid sequence of EGFR was analyzed using the ExPASy server(http://www.expasy.org/tools/).Conserved domains were explored using SMART tools(http://smart.emblheidelberg.de/).Aphylogenetic tree was constructed using the neighbor-joining(NJ)method of the Molecular Evolutionary Genetics Analysis(MEGA 5.0)package using the deduced full-length amino acid sequence with 1000 bootstrap replications.The phosphorylation sites and related kinases were predicted using the NetPhos 3.1 Server(http://www.cbs.dtu.dk/services/NetPhos/).The data were analyzed using a Poisson correction approach,and gaps were removed by complete deletion.

2.4.Total RNA extraction and cDNA synthesis

For RNA extraction,tissues and larvae were extracted by TRNzol reagent(Tiangen,China).And after 48 h of transfection,the RNA from HEK293T cells were extracted by TRIzolTMreagent(Thermo Fisher Scientific,USA).RNA quantity,purity,and integrity were verified using a NanoDrop 2000 spectrophotometer(NanoDrop Technologies,USA),and electrophoresis was performed using 1%agarose gels.The RNAs from various tissues were mixed in a tube for gene cloning and preparation of Sc-EGFR dsRNA.

The first-strand cDNA was synthesized using the PrimeScript™1st Strand cDNA Synthesis Kit(TaKaRa,Japan)and stored at-20°C.The generated cDNA was also the template for Sc-EGFR dsRNA in section 2.8.

2.5.qRT-PCR assay of Sc-EGFR mRNAs and human proliferating cell nuclear antigen(PCNA)mRNAs in 293T cells

Total RNA was reverse-transcribed into cDNA by using a PrimeScript™reagentkit with gDNA Eraser(TaKaRa,Japan),according to the manufacturer's instructions.Optimal primer pairs were designed to determine the expression pattern(Table 1)and generated a single PCR product and had an amplification efficiency near 97%and were screened by plotting standard curves.The relative expression of Sc-EGFRs was determined with 18SrRNA as the reference gene(Niu et al.,2014).And GAPDH listed in Table 1 was used as the internal control(Brug`e,Venditti,Tiano,Littarru&Damiani,2011)of PCNA gene.qRT-PCR was performed using a CFX Real Time Detection System(BioRad,USA)with a reaction containing 10μL 2×SYBR Premix Ex Taq™(TaKaRa,Japan),0.8μL of each gene-specific primer(10μmol/L),1.6μL of cDNA,and 6.8μL of nuclease-free water.The qRT-PCR protocol was as follows:1 cycle at 95°C for 30 s,followed by 39 cycles at 95°C for 10s,55°C for 30 s,and 72°C for 30 s and a dissociation curve analysis of 5 s per step from 55 to 95°C.The triplicate fluorescence intensities of each sample were measured by cycle threshold(CT)values,then were compared and converted to fold differences by the 2-ΔΔCTmethod(Livak&Schmittgen,2012)to determine relative expression levels.

Table 1 List of primers used in this study.

2.6.Cell culture,plasmid preparation,and transfection

The HEK293T cell line was obtained from Sangon(China)and cultured in DMEM(Gibco,USA)supplemented with 10%fetal bovine serum and 1%penicillin-streptomycin antibiotics and incubated under a 5%CO2atmosphere at 37°C.

The EGFP-N1 plasmids containing Sc-EGFR-1a cDNA or Sc-EGFR-1b cDNA in Escherichia coli DH5αwere obtained from Youbio Technology(Youbio,China).The empty EGFP-N1 plasmid was used as the negative control.Plasmid mini preparations were performed according to the manufacturer's protocol(Promega,USA).

The HEK293T cell line were seeded into 6-well plates and were transfected when cell confluence reached 60—80%.For transfections 0.4μg of Sc-EGFR-1a or Sc-EGFR-1b plasmid and 3.2μL of Enhancer and 10μL of Effectene transfection reagent(Qiagen,Germany)were added to each well of con fl uent 293T.

2.7.Assessment of viability of 293T cells by using the MTT assay

To study the cell proliferation ability of Sc-EGFR-1a and Sc-EGFR-1b,the MTTassay was conducted.At 24h after transfection with Sc-EGFR-1a,Sc-EGFR-1b,and EGFP-N1,the cells were harvested and reseeded into 96-well plates(Corning,USA)at a density of 5000cells per well.The culture medium alone was used as the negative control.Each group was repeated for 6 times.After 0,12,24,36,48,and 72 h at 37°C in a humidified incubator,the cell density was measured according to the manufacturer's instructions(Roche,USA).The absorbance at 560 nm was then measured using a microplate reader(BioRad,USA).The data are expressed as mean±SD values and assessed with the paired t-test by using SPSS 16.0 software.

2.8.Preparation of Sc-EGFR dsRNA and green fluorescent protein(GFP)dsRNA

To prepare Sc-EGFR dsRNA,2 PCR products,dsRNA-ScEGFR-1 and dsRNA-ScEGFR-2,were amplified using the template mentioned in section 2.4.To prepare GFP dsRNA(gene silencing control),2 PCR products,dsRNA-GFP-1 and dsRNA-GFP-2,were prepared using the EGFP-N1 template mentioned in section 2.7.The primers used to synthesize Sc-EGFR dsRNA and GFP dsRNA are listed in Table 1.The reaction system contained 10μL of 2×Taq PCR Mastermix(Tiangen,China),1μL of each gene-specific primer(10μmol/L),2μL of cDNA,and 6μL of nuclease-free water.The PCR protocol was as follows:94°C for 3min,followed by 35 cycles at 94°C for 30 s,55°C for 30 s,and 72°C for 50 s and 95°C for 5 min.After amplification,the PCR products were excised from 1%agarose gel by using the TGreen Transilluminator(Tiangen,China)and purified with the TIANgel Midi Purification Kit(Tiangen,China).The single-strand RNA(ssRNA)was prepared using a T7 High Efficiency Transcription Kit(Transgene,China)and quantified with a Nano Drop2000spectrophotometer(Nano Drop Technologies,USA).An equal masses of ssRNA-ScEGFR-1 and ssRNA-ScEGFR-2 or ssRNA-GFP-1 and ssRNA-GFP-2 were mixed in a tube.The mixture was annealed by addition of DNase I(Transgene,China)at a ratio of 1 U/μg of the template,followed by incubation at 70°C for 10min and room temperature for 20 min.Immediately,the dsRNA was purified with sodium acetate and isopropyl alcohol at 4°C.Finally,the dsRNAs were diluted to 0.7 μg/μL,monitored with 1%agarose gel,and maintained at-80°C until further use.

2.9.Measurement of growth traits after injection of dsRNA and detection of Sc-EGFR transcripts in the knockdown clams

A total of 240 labeled clams were divided into 3 groups;the dsRNA-injected group(experiment group),GFP-injected group(gene silencing control),and no treatment group(blank control).In the experiment group,30μL of Sc-EGFR dsRNAwas injected into the foot muscle(Lin et al.,2013).Similarly,the GFP group was injected with 30μL of GFP dsRNA.After injection,the clams were returned to the seawater tank.Dead clams were removed and recorded using their labels.After 15 days,the shell length,shell width,shell height,and body weight of the clams were measured,and the relative growth rate(GR)and survival rate(SR)were calculated as follows:

Fig.1.Comparison of the amino acid sequences of Sc-EGFRs,vertebrate EGFRs(Homo sapiens and Danio rerio)and invertebrate EGFRs(Pinctada martensii and Drosophila).The conserved and identical residues are represented by black shading,and the conservative substitutions are represented by gray shading.The regions of the L1-CR1-L2-CR2 domain,tyrosine kinase domain,and C-terminal domain are indicated using lines and arrows.The rectangular frame shows the GDALKKIPSEEILDSGATEIVANHTQE motif in Sc-EGFR-1a.Accession numbers in GenBank:Homo sapiens(NG_007726.3),Danio rerio(NM_194429.1),Pinctada martensii(KJ579134.1),Drosophila melanogaster(NM_057411.4),Sc-EGFR-1a(MF958947),and Sc-EGFR-1b(MF958948).The region marked with the asterisk shows the partial Sc-EGFRs used for RNA interference.

Table 2 Prediction results for the kinase and phosphorylate sites of Sc-EGFR-1a and Sc-EGFR-1b after 1232 amino acids by using the NetPhos 3.1 Server.

where GR:gain rate;Dd15:shell length/shell width/shell height/body weight data on the 15th day;and Dd1:shell length/shell width/shell height/body weight data on the 1st day.

where SR:survival rate;NF: final number of individuals;and NI:initial number of individuals.

To examine Sc-EGFR silencing,dsRNA-injected group was used as the experiment group,while the GFP group was used as the control group.In each group,foot tissues were isolated from three clams for RNA extraction at 2,5,10,and 15 days after injection.The total RNA was extracted,cDNA was synthesized,and qRT-PCR was conducted as described above.The primers used for detecting Sc-EGFR expression are listed in Table 1 and were designed to a region not targeted for Sc-EGFR knock-down to avoid amplification of the dsRNA injected template(Treerattrakool,Panyim&Udomkit,2011).18S rRNA was used as the internal reference gene for qRT-PCR.

3.Results

3.1.cDNA analysis of Sc-EGFR-1a and Sc-EGFR-1b

The full-length Sc-EGFR cDNA sequence was obtained from S.constricta and two splice isoforms named Sc-EGFR-1a(Accession number:MF958947)and Sc-EGFR-1b (Accession number:MF958948)were also isolated.When compared with Sc-EGFR-1a,27 amino acids were absent in the C-terminal region of Sc-EGFR-1b.The 5962 bp full-length cDNAof Sc-EGFR-1a had a 1152 bp 5′-UTR,a 226 bp 3′-UTR with a polyA tail and 4581 bp ORF that encoded a protein of 1527 amino acids.The mature Sc-EGFR-1a had a deduced molecular mass of 176.33 kDa and a theoretical pI of 5.69.Sc-EGFR-1b had a deduced molecular mass of 168.37 kDa and a theoretical pI of 5.70.The functional domains were predicted using the ExPASy online tool(Fig.1).Sc-EGFR-1a and Sc-EGFR-1b were identical apart from 27 amino acids that were deleted from 1232 amino acids onwards. Both isoforms had an extracellular region,transmembrane region,and intracellular region(Fig.1).The extracellular region included 2 leucine rich(L)domains and 2 cysteine rich(CR)domains,forming a leucine rich 1 domaincysteine rich 1 domain-leucine rich 2 domain-cysteine rich 2 domain(L1-CR1-L2-CR2 domain)structure(Fig.1).The L domains were located at 112—288 amino acids and 423—542 amino acids,and the CR domains were positioned at 248—334 amino acids and 561—817 amino acids.

Fig.2.A phylogenetic tree based on the complete EGFR open reading frames of 10 species.The phylogenetic tree was constructed using the neighbor-joining method with a Clustal W-generated multiple sequence alignment of the amino acid sequences.The topological stability was evaluated with 1000 bootstrap replications.Numbers on the branches indicate bootstrap values(%).

The SMART tool predicted a conserved tyrosine kinase domain,spanning 953—1209 amino acid residues in the intracellular region.The Net Phos 3.1 Server was used to predict the phosphorylation sites and corresponding kinases(Table 2).Sc-EGFR-1a had 28 serine phosphorylation sites,10 tyrosine phosphorylation sites,and 10 threonine phosphorylation sites.Sc-EGFR-1b had 23 serine phosphorylation sites,8 tyrosine phosphorylation sites,and 8 threonine phosphorylation sites.These changes may alter at least 15 types of downstream kinases.The phosphorylation sites were classified into the corresponding kinases.In the kinase from GSK3 to CKI,the number and types of phosphorylation sites between the 2 isoforms were identical,while from DNAPK to p38MAPK,the sites and kinases between them were different.The p38MAPK kinase showed the greatest differences between the 2 isoforms:Sc-EGFR-1b has 2 serine phosphorylation sites and 2 tyrosine phosphorylation sites less than Sc-EGFR-1a.

3.2.Phylogenetic analysis

A phylogenetic tree was constructed using the EGFR amino acid sequences from Homo sapiens,Rattus norvegicus,Xiphophorus xiphidium,D.rerio,Drosophila melanogaster,Apis mellifera,Crassostrea gigas,Pinctada martensii,Lingula anatina,and S.constricta(Fig.2).The results revealed that EGFR in both vertebrates and invertebrates could be classi fi ed into their respective clades.The vertebrate group was divided into mammal and fish clades,and the invertebrate cluster was divided into Arthropoda and Mollusca clades.In the Arthropoda clade,D.melanogaster and A.mellifera clustered together.In the Mollusca clade,Sc-EGFR-1a and Sc-EGFR-1b clustered together,C.gigas and P.martensii clustered together,and the isoforms of L.anatina formed a separate clade.

3.3.Expression of Sc-EGFR-1a and Sc-EGFR-1b during early development

In S.constricta,Sc-EGFR-1a and Sc-EGFR-1b mRNAs were detected at different developmental stages(Fig.3A,B).Both Sc-EGFR-1a and Sc-EGFR-1b were detectable in the early embryo stages,and the levels increased up to the middle-late stages.Sc-EGFR-1a transcripts were low abundance in the juvenile stage(Fig.3A),while Sc-EGFR-1b transcripts showed high-level expression at this stage(Fig.3B).

3.4.Tissue distribution of Sc-EGFR-1a and Sc-EGFR-1b transcripts in adult clams

Sc-EGFR-1a and Sc-EGFR-1b had awidespread tissue distribution(Fig.4A,B).Both isoforms were highly expressed in the siphon(Fig.4A,B).High levels of the Sc-EGFR-1a transcripts were also found in the foot,gut,and liver(Fig.4A),while high levels of the Sc-EGFR-1b transcripts were also found in the liver,gill,and hemolymph(Fig.4B).

3.5.Responses of Sc-EGFR-1a and Sc-EGFR-1b to the proliferation of 293T cells

To determine the impact of Sc-EGFR-1a and Sc-EGFR-1b on 293T cell proliferation,the levels of PCNA at 48h post-transfection were tested.The results revealed a significant up-regulation after transfection of Sc-EGFR-1a,while no significant variation was observed after transfection of Sc-EGFR-1b(Fig.5A).Furthermore,cell proliferation was measured using MTT assays to estimate cell viability.Interestingly,cells that overexpressed Sc-EGFR-1a displayed a higher growth rate than those that overexpressed Sc-EGFR-1b,and this difference in the cell population growth rate was apparent after 24h of culture(Fig.5B).

3.6.The silencing effect of Sc-EGFR

The expression of Sc-EGFR in the foot tissue of the treated clams showed a significant reduction from 5 to 15 days post-injection(Fig.6).When compared with the GFP group,the expression showed 24.84%reduction at 2 days,47.54%reduction at 5 days,57.59%reduction at 10 days,and 45.22%reduction at 15 days postinjection(Fig.6).This result indicated that the Sc-EGFR dsRNA was sufficient to silence Sc-EGFR for up to 15 days.

3.7.Effects of Sc-EGFR silencing on the growth traits

At 15 days post-injection,the Sc-EGFR silencing group showed a significant inhibition of shell length when compared with the GFP group.The shell width,shell height,and body weight in the Sc-EGFR dsRNA group showed no significant differences when compared with the control group(Table 3).

The survival curves(Fig.7)showed that the percentage of surviving Sc-EGFR ds RNA-injected clams decreased when compared with the GFP dsRNA-injected and no treatment groups.Therefore,Sc-EGFRs may play a role in survival during the development period.

4.Discussion

In humans,EGFR plays an important role in controlling cell proliferation,migration,survival,motility,and apoptosis(Bishayee,2000).To the best of our knowledge,this is the first report of the full-length cDNA of EGFR from S.constricta(Sc-EGFR),and the 2 isoforms were generated by alternative splicing.The phylogenetic tree showed that the Sc-EGFRs was closer to EGFR in C.gigas and P.martensii.The ExPASy online tool showed that Sc-EGFRs harbored functional domains that were identical to those in species such as D.rerio(Wang&Ge,2004),H.sapiens(Tsiambas et al.,2016),C.gigas(Sun et al.,2014),P.fucata(Zhu et al.,2015),and Apostichopus japonicus(Xia,Wang,Qin,Liu,&Zhou,2012).However,the C-terminal domain of Sc-EGFRs was different:Sc-EGFR-1b has a deletion of 27 amino acids(GDALKKIPSEEILDSGATEIVANHTQE).The C-terminal tail of EGFR has a role in receptor internalization(Chang et al.,1991).Changing tyrosine phosphorylation sites such as SRC,Grb2,Shc,and PI3K may reduce or induce the internalization(Marek,Vacl,Vosatka,Vit&Sixta,1996;Pawson,1995).According to the NetPhos 3.1 Server(Miller&Blom,2005),p38MAPK kinase showed the greatest difference between the Sc-EGFRs.Further studies have shown that the p38MAPK kinase is the major kinase for inducing EGFR internalization after changing a phosphorylate site between 1002 and 1020 residues(Winogradkatz&Levitzki,2006).Therefore,it is reasonable to predict that Sc-EGFRs may have different functions.

Fig.3.Expression patterns of Sc-EGFR-1a and Sc-EGFR-1b transcripts at different developmental stages.18S rRNA was used as the internal reference.(A)qRT-PCR results for Sc-EGFR-1a transcripts at different developmental stages.(B)qRT-PCR results for Sc-EGFR-1b transcripts at different developmental stages.Different letters(a—c)indicate statistical signi fi cance(One-Way ANOVA,P＜0.05).

Fig.4.Expression patterns of Sc-EGFR-1a and Sc-EGFR-1b transcripts in different tissues.18SrRNA was used as the internal reference.(A)qRT-PCR results for Sc-EGFR-1a transcripts in different tissues.(B)qRT-PCR results for Sc-EGFR-1b transcripts in different tissues.Different letters(a—f)indicate statistical signi fi cance(One-Way ANOVA,P＜0.05).

qRT-PCR shows Sc-EGFRs may exist in the early development stages and increased in the middle-late stages when the larvae reorient the mouth,foot and alter their swimming method(Coon et al.,1990).Therefore,we suggest that Sc-EGFRs may participate in the middle-late stages of metamorphosis.Besides,both isoforms were expressed in all the tested tissues,and the highest levels were detected in the siphon.Previous studies have demonstrated that EGFR transcripts are considerably abundant in the pearl sac and mantle of P.fucata(Zhu et al.,2015).In A.japonicas,the EGFR mRNA is highly expressed in the coelomocyte and epidermis(Xia et al.,2012).We collected the samples used in the present study in May,when the gonad is in the resting stage(Yan et al.,2010).Therefore,our results were similar to that for EGFR in D.rerio,in which the expression was low at the beginning of the reproductive cycle(Tse&Ge,2010).Sc-EGFRs expressed in different tissues may have different functions(Livak&Schmittgen,2012).This suggests that Sc-EGFRs may be related to the growth of fibroblasts cells and gonad cells.

Fig.5.Proliferation of 293T cells overexpressing Sc-EGFR-1a and Sc-EGFR-1b.(A)Expression analysis of PCNA mRNA in 293T cells at 48 h post-transfection.Bars represent means± SD(n=3 wells of the 6-well plate).Columns with*are significantly different from each other(One-Way ANOVA,P＜ 0.05).(B)Sc-EGFR-1a and Sc-EGFR-1b overexpression and cell density were determined at the indicated times by using MTT assays.Values are means±SD(n=6 wells of the 96-well plate).

Fig.6.qRT-PCR was used to con firm the silencing effect of Sc-EGFRs in foot tissues at 2,5,10,and 15 days post-injection.18SrRNA was used as the internal reference.The GFP group was injected with partial EGFP-N1 dsRNA,and the dsRNA group was injected with Sc-EGFR dsRNA.Asterisks represent statistically significant differences.(One-Way ANOVA,P＜0.05).

Table 3 Comparison of the relative growth rate(GR)in the dsRNA-injected group,GFP-injected group,and no treatment group.

Fig.7.Survival rate of clams in the Sc-EGFR dsRNA group,GFP dsRNA group,and no treatment group that were monitored daily from day 1 to day 15 post-injection of dsRNA.

To compare the impact of Sc-EGFR-1a and Sc-EGFR-1b on proliferation,we conducted the cell proliferation assay.Since no S.constricta cell line is available,the assay was conducted using the HEK293Tcell line.PCNA has beenproposed tobe a reliable indicator for cell proliferation as it is associated with proteins that control the G1 phase of the cell cycle,con firming DNA replication and cell division(Celis,Madsen,Celis,Nielsen&Gesser,1987).A kinase such as p38MAPK is activated after changes to the serine phosphorylation sites in EGFR(Winogradkatz&Levitzki,2006),which induces EGFR internalization and activates different pathways(Chen et al.,1989).Cancer cells with a mutation in EGFR survive through the activation of the AKT or STAT3 pathway(Akca,Tani,Hishida,Matsumoto&Yokota,2006).In patients with NSCLCs,the survival time and sensitivity to anti-cancer drugs are determined by alternative splicing of EGFR(Chang et al.,1991).In the GBM cell line,mutated EGFR upregulates cell proliferation by downstream factors(Babic et al.,2013).GnRH and EGFR belong to the G protein-coupled receptor family and play key roles in the reproductive cycle and fertilization(Hughes et al.,1992).However,the GnRH gene obtained by alternative splicing may have opposite functions in oyster;the shorter GnRH isoforms probably does not control any functions(Rodet,Lelong,Dubos&Favrel,2008).The mitofusin 2(Mfn2)gene has 2 isoforms that also have opposite functions during cell proliferation(Zhou et al.,2010).

EGFR has a role in organ development and formation of spatial and temporal features(Shilo,2005).In the present study,the RNAi experiments were designed to detect the in vivo effects of Sc-EGFRs during body growth.RNAi is an effective method that has been widely used to silence gene expression in invertebrates(Lin et al.,2014).In body growth experiments,it has been used to detect the in vivo functions of growth-related genes,such as MSTN(Lee et al.,2015)and EGFR(Sharabi et al.,2013).In our study,the expressions of Sc-EGFRs were successfully silenced for at least 15 days postinjection.This result is consistent with that of a study on crickets,and the duration of the effects of dsRNA was longer than 2 weeks(Dabour et al.,2011).In addition,knockdown of Sc-EGFRs resulted in a significant limiton the shell length,which maybe related to the specific time of incremental growth.This study was conducted from June to July,and the greatest amount of shell is added in this period(Beal et al.,2001).This result is supported by the fact that silenced individuals with a small body size have a lesser number of cells or reduced cell size than large-sized individuals(Dabour et al.,2011).Scanning electron microscopy has shown that the eye cells in EGFR-silenced prawns have irregular morphology(Sharabi et al.,2013).EGFR is involved in the regulation of the body size as well as developmental time in the queen honeybee and Drosophila(Kamakura,2011).After silencing Sc-EGFRs,the survival rate declined overthe 15 days,whichshows that Sc-EGFR mayregulatea stage of clams that survive the developmental stage.

To the best of ourknowledge,this is the first report of alternative splicing of EGFR in mollusks.We obtained full-length Sc-EGFR-1a and Sc-EGFR-1b from S.constricta,and performed phylogenetic tree analysis.We also analyzed expression of Sc-EGFR-1a and Sc-EGFR-1b in early developmental stages and in tissues.In vitro,Sc-EGFR-1a and Sc-EGFR-1b may have different functions,and Sc-EGFR-1a caused transfected 293T cells to proliferate more quickly than Sc-EGFR-1b and the EGFP-N1 control.In vivo,the silencing of Sc-EGFR-1a and Sc-EGFR-1b caused a reduction in shell length.This study,as a whole,indicated that EGFR from S.constricta may play a role in body growth and during the early developmental stages.

This work was supported by the National Natural Science Foundation of China(31472278),National High Technology Research and Development Program of China(863 Program)(2012AA10A400),and the Shanghai Universities Knowledge Service Platform(ZF1206).We would like to thank the native English speaking scientists of Elixigen Company(Huntington Beach,California)for editing our manuscript.

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