Preliminary Study Of The Effect Of SMYD3 Gene In Pig Fibroblast Proliferation And Reprogramming

SET and MYND domain-containing protein 3(SMYD3),a member of the SMYD protein family,was a kind of histone 3 lysine 4(H3K4)methylation transferase.It was reported that SMYD3 gene.could promote the bovine somatic cell reprogramming,and improve the induced efficiency of bovine iPSCs.In the present study,the effect of SMYD 3 gene was explored in pig somatic cell reprogramming,and the method of RNA interference(RNAi)and overexpression gene were used.After the SMYD3 gene of the donor pig fibroblasts was regulated,reprogramming was investigated in the process of pig SCNT pre-implantation embryos.The results were looking forward to lay the foundation for improving the efficiency of pig cell clone.Construction of SMYD3 gene expression vectors and verification of its shRNA inhibition efficiency1.The sequence of Debao pig SMYD3 gene was cloned and analyzed,which were inserted into the lentiviral expression vector pLVX-IRES-Zsgreenl,named as pLVX-IRES-Zsgreenl SMYD3.Bioinformatics analysis showed that the sequence of cloned Debao pig SMYD3 gene was 1404 bp,and the homology of sequence which is SMYD3 gene between Debao pig and sus scrofa was 99%,which shared 93%identify with sheep and bovine.The eukaryotic vector of the SMYD3 gene expression could be expressed in 293T cells by cell transfection test.2.Based on the sequence of siRNA and shRNA reported in other species,two shRNA fragments of pig SMYD3 genes were obtained by sequence comparative analysis,which were synthesized and inserted into the lentiviral expression vector pSicoR-GFP,named as pSicoR-GFP-SMYD3 shRNA 1/2 respectively.The vectors were transfected into 293T cell respectively and investigated the shRNA inhibition efficiency by qRT-PCR.The results revealed that,compared with the control groups,the two shRNA fragments had significant inhibition effect on expression of SMYD3 gene(P<0.05),reached 34%and 54%respectively.We selected pSicoR-GFP-SMYD3 shRNA2 in the following experiments.The effects of SMYD3 genes on cell proliferation of Debao pig fibroblastsTo understand the role of SMYD3 gene on cell proliferation of Debao pig fibroblasts,lentivirus containing SMYD3 gene or its shRNA was obtained by packing three plasmids into 293T.Virus of the SMYD3 gene or its shRNA expression was added to the cell culture medium with different MOI value respectively,and then a suitable MOI value was chosen.Cell growth curve was determined by counting the cell number every 24 hours for 7 days.The cells infected with a suitable MOI value virus were collected and analyzed the expression level of Nanog,DN-MT1 and DNMT3a genes by qRT-PCR.The results revealed that,compared with the negative and blank control groups,overexpressing SMYD3 gene promoted Debao pig fibroblasts proliferation,and inhibition of SMYD3 gene inhibited Debao pig fibroblasts Proliferation.Results of qRT-PCR analysis confirmed that,compared with the negative and blank control groups,the expression of Nanog,DNMT1 and DNMT3a genes were increased significantly in overexpressing SMYD3 gene group(P<0.05).However,the expression of Nanog,DNMT1 and DNMT3a genes were decreased significantly in suppressive SMYD3 gene group(P<0.05).The effects of SMYD3 gene on development potentiality of pig cloned embryos and preliminary mechanism analysisWe firstly analyzed the expression pattern of SMYD3 gene in pig embryos during maturation,parthenogenetic and SCNT embryos.After the SMYD3 gene expression of the donor pig fibroblasts was regulated,its effect on early embryonic development and pluripotent gene expression was studied.The result showed that,SMYD3 gene had lower expression during pig oocyte maturation.The expression of SMYD3 gene firstly increased,then decreased,and had the highest level at 4-and 8-cell stages respectively in early parthenogenetic and SCNT embryos.Compared with the control group,more SCNT embryos developed to blastocyst 'stage in overexpressing SMYD3 gene group(14.63%vs 9.68%,P<0.05),and lesser SCNT embryos developed to blastocyst stage in suppressive SMYD3 gene group(6.74%vs 9.68%,P<0.05).The results showed that,compared with the control group,the expression levels of Nanog and Oct4 genes were significantly increased in overexpressing SMYD3 gene groups(P<0.05).In suppressive SMYD3 gene group,the Nanog gene was not detested.There was no significant difference for Oct4 gene expression between the suppressive SMYD3 gene group and control group.In conclusion,the above results demonstrated that SMYD3 gene was correlated with proliferation and reprogramming in pig fibroblast.overexpressing SMYD3 gene could promote Debao pig fibroblast proliferation,and improve the development ability of pig SCNT embryos in vitro subsequently.