猪SMYD3基因的克隆、序列分析及其对猪成纤维细胞增殖的影响
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摘要
试验旨在克隆猪SMYD3(SET and MYND domain-containing protein 3)基因并对其进行序列分析,研究其对猪成纤维细胞增殖的影响。首先克隆猪SMYD3基因,根据其他物种SMYD3基因siRNA和shRNA序列,经同源性比对分析,获得两条猪SMYD3基因shRNA序列,分别构建pSicoR-GFP-SMYD3 shRNA1/shRNA2表达载体,转染HEK293T细胞,利用实时荧光定量PCR分析干扰效率,筛选出抑制效率较好的shRNA,并构建pLVX-IRES-ZsGreen1-SMYD3及pSicoR-GFP-SMYD3 shRNA真核表达载体,同时分析SMYD3基因对猪成纤维细胞的增殖作用,检测细胞Nanog、DNMT1及DNMT3a基因表达情况。结果显示,试验克隆得到1 404 bp的猪SMYD3基因编码区序列,生物信息学分析发现,德保猪SMYD3基因与野猪、山羊和野耗牛相应氨基酸序列的同源性分别为99.5%、93.8%和92.9%。shRNA1/shRNA2均能显著抑制SMYD3基因表达(P<0.05),抑制效果分别是34%和54%,选择pSicoR-GFP-SMYD3 shRNA2进行后续研究。通过脂质体转染法将构建的pLVX-IRES-ZsGreen1-SMYD3及pSicoR-GFP-SMYD3 shRNA真核表达载体导入HEK293T细胞,均可观察到清晰的绿色荧光。慢病毒感染细胞及实时荧光定量PCR结果显示,与空白对照组及阴性对照组相比,过表达SMYD3基因促进猪成纤维细胞增殖,Nanog和DNMT1基因表达显著升高(P<0.05);抑制SMYD3基因表达,细胞增殖受到抑制,Nanog、DNMT1、DNMT3a基因表达显著降低((P<0.05),说明SMYD3基因的表达与猪成纤维细胞的增殖显著相关。
This experiment was aimed to clone and analyze the sequence of porcine SET and MYND domain-containing protein 3(SMYD3) gene,and study its effect on the proliferation of porcine fibroblasts.Firstly,SMYD3 gene was cloned,according to the siRNA and shRNA sequences of other species SMYD3 gene,two shRNA sequences of SMYD3 gene were obtained by homologous alignment analysis,pSicoR-GFP-SMYD3 shRNA1/shRNA2 expression vector were constructed and transfected into HEK293 T cells.The inhibition efficiency of shRNA was analyzed by Real-time PCR,and screened out shRNA with better inhibition efficiency.Then the pLVX-IRES-ZsGreen1-SMYD3 and pSicoR-GFP-SMYD3 shRNA eukaryotic expression vectors were constructed,the proliferation of porcine fibroblasts was analyzed,and the expression levels of Nanog,DNMT1 and DNMT3a genes were detected by Real-time quantitative PCR.The results showed that the cloned CDS length of porcine SMYD3 gene was 1 404 bp.Bioinformatics analysis results showed that the homology of the SMYD3 gene was 99.5% between pig and Sus scrofa,which shared 93.8% and 92.9% identity with Capra hircus and Bos mutus,respectively.The expression of SMYD3 gene was significantly inhibited by shRNA1/shRNA2(P<0.05),and the inhibitory rates were 34% and 54%,respectively.pSicoR-GFP-SMYD3 shRNA2 was selected for follow-up studies.The pLVX-IRES-ZsGreen1-SMYD3 and pSicoR-GFP-SMYD3 shRNA eukaryotic expression vectors were constructed,and clear green fluorescent signal was observed when the plasmids were transfected into HEK293 T cells by liposome method.The results of lentiviral infection and Real-time quantitative PCR showed that compared with the negative and blank control groups,overexpression of SMYD3 gene promoted the porcine fibroblast proliferation,and the expression of Nanog and DNMT1 genes were significantly increased(P<0.05);And inhibition of SMYD3 gene inhibited porcine fibroblasts proliferation while the expression of Nanog,DNMT1 and DNMT3a genes were significantly decreased(P<0.05).This results indicated that the expression of SMYD3 gene was significantly associated with the proliferation of porcine fibroblasts.
This experiment was aimed to clone and analyze the sequence of porcine SET and MYND domain-containing protein 3(SMYD3) gene,and study its effect on the proliferation of porcine fibroblasts.Firstly,SMYD3 gene was cloned,according to the siRNA and shRNA sequences of other species SMYD3 gene,two shRNA sequences of SMYD3 gene were obtained by homologous alignment analysis,pSicoR-GFP-SMYD3 shRNA1/shRNA2 expression vector were constructed and transfected into HEK293 T cells.The inhibition efficiency of shRNA was analyzed by Real-time PCR,and screened out shRNA with better inhibition efficiency.Then the pLVX-IRES-ZsGreen1-SMYD3 and pSicoR-GFP-SMYD3 shRNA eukaryotic expression vectors were constructed,the proliferation of porcine fibroblasts was analyzed,and the expression levels of Nanog,DNMT1 and DNMT3a genes were detected by Real-time quantitative PCR.The results showed that the cloned CDS length of porcine SMYD3 gene was 1 404 bp.Bioinformatics analysis results showed that the homology of the SMYD3 gene was 99.5% between pig and Sus scrofa,which shared 93.8% and 92.9% identity with Capra hircus and Bos mutus,respectively.The expression of SMYD3 gene was significantly inhibited by shRNA1/shRNA2(P<0.05),and the inhibitory rates were 34% and 54%,respectively.pSicoR-GFP-SMYD3 shRNA2 was selected for follow-up studies.The pLVX-IRES-ZsGreen1-SMYD3 and pSicoR-GFP-SMYD3 shRNA eukaryotic expression vectors were constructed,and clear green fluorescent signal was observed when the plasmids were transfected into HEK293 T cells by liposome method.The results of lentiviral infection and Real-time quantitative PCR showed that compared with the negative and blank control groups,overexpression of SMYD3 gene promoted the porcine fibroblast proliferation,and the expression of Nanog and DNMT1 genes were significantly increased(P<0.05);And inhibition of SMYD3 gene inhibited porcine fibroblasts proliferation while the expression of Nanog,DNMT1 and DNMT3a genes were significantly decreased(P<0.05).This results indicated that the expression of SMYD3 gene was significantly associated with the proliferation of porcine fibroblasts.
引文
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[11] 白海栋.SMYD3在牛着床前胚胎发育及胎儿成纤维细胞生长中作用的研究[D].呼和浩特:内蒙古大学,2015. BAI H D.The function of SMYD3 on bovine preimplantation embryo development and embryonic fibroblast growth[D].Hohhot:Inner Mongolia University,2015.(in Chinese)
[12] BOURC’HIS D,BOURHIS D L,PATIN D,et al.Delayed and incomplete reprogramming of chromosome methylation patterns in bovine clonedembryos[J].Current Biology,2001,11(19):1542-1546.
[13] DEAN W,SANTOS F,STOJKOVIC M,et al.Conservation of methylation reprogramming in mammalian development:Aberrant reprogramming in clonedembryos[J].Proceedings of the National Academy of Sciences of the United States of America,2001,98(24):13734-13738.
[14] MARTIN C,ZHANG Y.The diverse functions of histone lysine methylation[J].Nature Reviews Molecular Cell Biology,2005,6(11):838-849.
[15] SARRIS M E,MOULOS P,HARONITI A,et al.Smyd3is a transcriptional potentiator of multiple cancer-promoting genes and required for liver and colon cancer development[J].Cancer Cell, 2016,29(3):354-366.
[16] BING S,TAN M,MU X,et al.Upregulated SMYD3 promotes bladder cancer progression by targeting BCLAF1 and activating autophagy[J]. Tumor Biology,2015,37(6):7371-7381.
[17] PALADINO D,YUE P,FURUYA H,et al.A novel nuclear Src and p300 signaling axis controls migratory and invasive behavior in pancreatic cancer[J]. Oncotarget,2016,7(6):7253-7267.
[18] LIU Y,LIU H,LUO X,et al.Overexpression of SMYD3 and matrix metalloproteinase-9 are associated with poor prognosis of patients with gastric cancer[J].Tumor Biology,2015,36(6):4377-4386.
[19] WANG S Z,LUO X G,SHEN J,et al.Knockdown of SMYD3 by RNA interference inhibits cervical carcinoma cell growth and invasion in vitro[J].BMB Reports,2008,41(4):294-299.
[20] VIEIRA F Q,COSTA-PINHEIRO P,RAMALHO-CARVALHO J,et al.Deregulated expression of selected histone methylases and demethylases in prostate carcinoma[J].Endocrine-Related Cancer,2013,21(1):51-61.
[21] OLIVEIRA-SANTOS W,RABELLO D A,LUCENA-ARAUJO A R,et al.Residual expression of SMYD2 and SMYD3 is associated with the acquisition of complex karyotype in chronic lymphocytic leuke-mia[J].Tumor Biology,2016,37(7):9473-9481.
[22] HAMAMOTO R,SILVA F P,TSUGE M,et al.Enhanced SMYD3 expression is essential for the growth of breast cancer cells[J]. Cancer Science,2006,97(2):113-118.
[23] REN T N,WANG J S,HE Y M,et al.Effects of SMYD3 over-expression on cell cycle acceleration and cell proliferation in MDA-MB-231 human breast cancercells[J].Medical Oncology,2011,28(1):91-98.
[24] ZOU J N,WANG S Z,YANG J S,et al.Knockdown of SMYD3 by RNA interference down-regulates c-Met expression and inhibits cells migration and invasion induced by HGF[J].Cancer Letters,2010,280(1):78-85.
[25] GUO N,CHEN R,LI Z,et al.Hepatitis C virus core upregulates the methylation status of the RASSF1A promoter through regulation of SMYD3 in hilar cholangiocarcinoma cells[J].Acta Biochim Biophys Sinica,2011,43(5):354-361.
[2] ABU-FARHA M,LAMBERT J P,Al-MADHOUN A S,et al.The tale of two domains:Proteomics and genomics analysis of SMYD2,a new histone methyltransferase[J]. Molecular & Cellular Proteomics,2008,7(3):560-572.
[3] 宋延霞,吴大平,李海艳.猪JHDM2A基因的克隆及表达分析[J].中国畜牧兽医,2017,44(6):1596-1603. SONG Y X,WU D P,LI H Y,et al.Cloning and expression analysis of porcine JHDM2A gene[J].China Animal Husbandry & Veterinary Medicine,2017,44(6):1596-1603.(in Chinese)
[4] SAWADA K,YANG Z,HORTON J R,et al.Structure of the conserved core of the yeast Dot1p,a nucleosomal histone H3 lysine 79 methyltransferase[J].Journal of Biological Chemistry,2004,279(41):43296-43306.
[5] SIRINUPONG N,BRUNZELLE J,DOKO E,et al.Structural insights into the autoinhibition and posttranslational activation of histone methyltransferase SMYD3[J].Journal of Molecular Biology,2011,406(1):149-159.
[6] FOREMAN K W,BROWN M,PARK F,et al.Structural andfunctional profiling of the human histone methyltransferase SMYD3[J]. PLoS One,2011,6(7):e22290.
[7] NAGATA D E D A,TING H A,CAVASSANI K A,et al.Epigenetic control of Foxp3 by SMYD3 H3K4 histonemethyltransferase controls iTreg development and regulates pathogenic T-cell responses during pulmonary viral infection[J].Mucosal Immunology,2015,8(5):1131-1143.
[8] LI B,PAN R R,ZHOUC,et al.SMYD3 promoter hypomethylation is associated with the risk of colorectal cancer[J].Future Oncology,2018,14(18):1825-1834.
[9] DIAO Y F,OQANI R K,LI X X,et al.Changes in histone H3 lysine 36 methylation in porcine oocytes and preimplantation embryos[J]. PLoS One,2014,9(6):e100205.
[10] CAO Z,LI Y,CHEN Z,et al.Genome-wide dynamic profiling of histone methylation during nuclear transfer-mediated porcine somatic cell reprogramming[J].PLoS One,2015,10(12):e144897.
[11] 白海栋.SMYD3在牛着床前胚胎发育及胎儿成纤维细胞生长中作用的研究[D].呼和浩特:内蒙古大学,2015. BAI H D.The function of SMYD3 on bovine preimplantation embryo development and embryonic fibroblast growth[D].Hohhot:Inner Mongolia University,2015.(in Chinese)
[12] BOURC’HIS D,BOURHIS D L,PATIN D,et al.Delayed and incomplete reprogramming of chromosome methylation patterns in bovine clonedembryos[J].Current Biology,2001,11(19):1542-1546.
[13] DEAN W,SANTOS F,STOJKOVIC M,et al.Conservation of methylation reprogramming in mammalian development:Aberrant reprogramming in clonedembryos[J].Proceedings of the National Academy of Sciences of the United States of America,2001,98(24):13734-13738.
[14] MARTIN C,ZHANG Y.The diverse functions of histone lysine methylation[J].Nature Reviews Molecular Cell Biology,2005,6(11):838-849.
[15] SARRIS M E,MOULOS P,HARONITI A,et al.Smyd3is a transcriptional potentiator of multiple cancer-promoting genes and required for liver and colon cancer development[J].Cancer Cell, 2016,29(3):354-366.
[16] BING S,TAN M,MU X,et al.Upregulated SMYD3 promotes bladder cancer progression by targeting BCLAF1 and activating autophagy[J]. Tumor Biology,2015,37(6):7371-7381.
[17] PALADINO D,YUE P,FURUYA H,et al.A novel nuclear Src and p300 signaling axis controls migratory and invasive behavior in pancreatic cancer[J]. Oncotarget,2016,7(6):7253-7267.
[18] LIU Y,LIU H,LUO X,et al.Overexpression of SMYD3 and matrix metalloproteinase-9 are associated with poor prognosis of patients with gastric cancer[J].Tumor Biology,2015,36(6):4377-4386.
[19] WANG S Z,LUO X G,SHEN J,et al.Knockdown of SMYD3 by RNA interference inhibits cervical carcinoma cell growth and invasion in vitro[J].BMB Reports,2008,41(4):294-299.
[20] VIEIRA F Q,COSTA-PINHEIRO P,RAMALHO-CARVALHO J,et al.Deregulated expression of selected histone methylases and demethylases in prostate carcinoma[J].Endocrine-Related Cancer,2013,21(1):51-61.
[21] OLIVEIRA-SANTOS W,RABELLO D A,LUCENA-ARAUJO A R,et al.Residual expression of SMYD2 and SMYD3 is associated with the acquisition of complex karyotype in chronic lymphocytic leuke-mia[J].Tumor Biology,2016,37(7):9473-9481.
[22] HAMAMOTO R,SILVA F P,TSUGE M,et al.Enhanced SMYD3 expression is essential for the growth of breast cancer cells[J]. Cancer Science,2006,97(2):113-118.
[23] REN T N,WANG J S,HE Y M,et al.Effects of SMYD3 over-expression on cell cycle acceleration and cell proliferation in MDA-MB-231 human breast cancercells[J].Medical Oncology,2011,28(1):91-98.
[24] ZOU J N,WANG S Z,YANG J S,et al.Knockdown of SMYD3 by RNA interference down-regulates c-Met expression and inhibits cells migration and invasion induced by HGF[J].Cancer Letters,2010,280(1):78-85.
[25] GUO N,CHEN R,LI Z,et al.Hepatitis C virus core upregulates the methylation status of the RASSF1A promoter through regulation of SMYD3 in hilar cholangiocarcinoma cells[J].Acta Biochim Biophys Sinica,2011,43(5):354-361.
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