The Identification On Stem Cell Related Factors Of Nanog And Oct4in Megalobrama Amblycephala

Megalobrama amblycephala is one of the major freshwater fish with important economic value and significance in science research in China.At present,there were some researches related on its general biology,genetics and breeding,nutrition and feedstuff,disease and immunity,gene cloning and expression,and genomics.However,still little is known about the stem cells related transcription factors of the M.amblycephala.Among these factors,nanog and oct4 have been proved to play an important role in regulating the early embryonic development in some mammals and teleost,and maintaining the self-renewal and differentiation of the stem cells.But it is still not clear whether there are some differences about these two genes between M.amblycephala and other animals in the gene structures,expression patterns and functions.During this research,a variety of molecular biology and developmental biology experimental techniques including CRISPR/Cas9 were adopted,to explore the gene structures,expression and part of function of nanog and oct4 in M.amblycephala（Mananog,Maoct4）.In this study,firstly,it is demonstrated that Mananog and Maoct4 were maternal inheritance and had some different expression patterns from mammals.Secondly,the antibodies of MaNanog and MaOct4 were obtained through prokaryotic expression and were proved to be effective.Moreover,it was proved that the two factors could promote the proliferation,improve the anchorage-dependent growth ability and migration ability of the HepG2 cells.Finally,it was preliminarily validated that Mananog might rescue the phenotype of nanog-null zebrafish at certain extent.The main results of this paper are as follows.1)nanog gene.Firstly,the gDNA sequence of Mananog is 3326 bp and consisted of four exons and three introns.The full-length cDNA is 1556 bp with 217 bp of 5′UTR,178 bp of 3′UTR and 1161 bp of the CDS.The predicted amino acid sequence of Mananog is 386 residues with deduced molecular weight 42.46 ku,and contains a homodomain（HD）of 63 amino acids at positions 197-259,including a conservative nuclear localization motif YKQVKTWFQN.Two mRNA isoforms of Mananog848 and Mananog683 were found in this study.The protein sequence comparison showed that the similarity of Nanog protein between M.amblycephala and other fish was 33%⁸0%,while only 16%or 15%between the fish and human or mouse.But the similarity of HD was 49%⁹7%in all of the studied species.The phylogenetic analysis further proved that MaNanog was in the same branch with Nanog in other teleost,and was homologous to Nanog in mammalian.Secondly,the semi-quantitative RT-PCR and quantitative real-time RT-PCR analysis showed that Mananog was maternally expressed and mainly existed in the early stage from 1-cell to gastrula stage with the peaking at blastula stage.The subsequent whole mount in situ hybridization（WISH）analyses demonstrated that the Mananog transcripts were present in all blastomeres of these early embryos.In adult tissues,Mananog mRNA was detectable mainly in gonads with weak expression in liver.Moreover,the expression was significantly higher in ovary than in testis.The fluorescence in situ hybridization（FISH）revealed that Mananog transcripts were located in the oogonia,ⅠandⅡstage of oocytes and the vacuole of theⅢstage of oocyte in ovary,while in the spermatogonia,spermatocytes and spermatids in testis,and in certain type of hepatocytes in liver.MaNanog protein is located in the nucleus after transfection HepG2 cells with an EGFP reporter vector pEGFP-N1-Mananog.Thirdly,the recombinant Nanog protein could be highly expressed with 0.5 mmol/L IPTG induction for 4 h at 37℃.And the polyclonal antibody could effectively recognize the induced MaNanog protein in E.coli,the endogenous MaNanog protein in adult organs,and the ectopic expressed MaNanog proteins in HepG2.Fourthly,on the founction of Mananog,it is detected that the expression levels of transcription factors NANOG,OCT4,KLF4 and C-MYC in HepG2 cells were significantly changed after overexpression of MaNanog or other factors.Meanwhile,the transfections with the factors could significantly promote the proliferation of HepG2,enhance the anchorage-dependent growth and migratory ability of the cell.In zebrasfih,knockout of nanog（Drnanog）gene by using CRISPR/Cas9 leaded to a higher rate of mortality and malformation during embryogenesis,as well as the disorder of PGC migration.However,the phenotype of Drnanog mutant in zebrafish could be resuced by microinjection of Mananog mRNA at some extent,while Mananog683 mRNA could not work.These results demonstrated that Mananog was homologous to nanog in teleost and mammals,and was an important factor related to the pluripotency of stem cells.Taken together,this research might provide an important basic data for future study on stem cells of M.amblycephala.2)oct4 gene.Firstly,the gDNA sequence of Maoct4 is 5308 bp and includes five exons and four introns.The full-length cDNA is 3397 bp with 373 bp of 5′UTR,1605 bp of 3′UTR and 1419 bp of the CDS.The deduced amino acid sequence of Maoct4 is 472 residues with molecular weight of 51.92 ku,and possesses a characteristic POU domain that consists of the POU specific domain（POUs）,the POU homeodomain(POU_HD)and a linker region between them.There were five mRNA isoforms of Maoct4-I1（834 bp）,Maoct4-I2（726 bp）,Maoct4-I3（435 bp）,Maoct4-I4（387 bp）and Maoct4-I5（366 bp）.The protein sequence alignment showed that the similarity of Oct4 protein between M.amblycephala and other fish was high to 47%⁹4%,while only 27%³3%between this fish and the tetrapods.But the similarity of POU domain was 57%⁹5%in all of the studied species.The phylogenetic analysis further proved that MaOct4 was in the same branch with Oct4 in other teleost and was homologous to Oct4 in mammalian.Secondly,the semi-quantitative RT-PCR and real-time fluorescence quantitative RT-PCR（qRT-PCR）analysis noted that Maoct4 mRNA was present throughout early development from 1-cell to neurola stage with gradually reduced expression.WISH analyses further demonstrated that the transcripts were present in all blastomeres of the early embryos.In adult tissues,Maoct4 mRNA was detectable mainly in gonads and a weak expression in liver within eight examined tissues.And the Maoct4 transcripts were much more abundance in ovary than in testis.FISH results further revealed that Maoct4 transcripts existed in the oogonia,ⅠandⅡstage of oocytes and the vacuole of theⅢstage of oocyte in ovary,while in the spermatogonia,spermatocytes but no spermatids in testis,and in certain type of hepatocytes.MaOct4 is also located in the nucleus after transfection HepG2 with a RFP reporter vector pCMV-Maoct4-Red.Thirdly,the expression vector pET32a-Maoct4 was transformed into Escherichia coli BL21（DE3）pLysS,and the recombinant Oct4 protein was highly induced by 0.5 mmol/L IPTG for 4 h at 37℃.The polyclonal anti-Oct4 antibody could effectively recognized the purified recombinant MaOct4 antigen,the induced MaOct4 protein in E.coli,the endogenous MaOct4 protein from the fish embryos,and the ectopic expressed MaOct4:DsRed fusion protein in HepG2 cells.Fourthly,on the founction of Maoct4,an overexpression of Maoct4 was carried out in HepG2 cells.It was detected that the expression levels of transcription factors NANOG,OCT4,KLF4 and C-MYC in HepG2 cells were significantly changed,and the HepG2 cells showed an improved ability in cell proliferation,anchorage-dependent growth and migratory after overexpression of this gene.The results showed Maoct4 is homologous to Oct4 gene in teleost and mammals,and plays an important role in early embryonic development and stem cells related processes.More work should be carried out on the mechanism of this crucial transcription factor.In conclusion,the two important genes related to stem cells of nanog and oct4 in M.amblycephala were obtained in this paper.They had a maternal expression pattern,mainly existed in the early embryos and gonadal germ cells,and might participate in the early stage development of embryos and the formation of germ cells.Meanwhile,the antibodies of Nanog and Oct4 were prepared and the HepG2 cell lines stably transfected with Mananog or Maoct4 were gained.Additonally,some of the functions of these two genes were preliminarily verified.These works would lay a foundation for further study on fish stem cells.