Scriptaid联合5-Aza-CdR对水牛成纤维细胞DNA甲基化和组蛋白乙酰化修饰的影响
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摘要
旨在探讨Scriptaid和5-Aza-CdR共同处理水牛耳部成纤维细胞(buffalo adult fibroblasts,AFs)对细胞生长特性、DNA甲基化和组蛋白乙酰化的影响,为提高水牛体细胞重编程效率提供一定的理论基础。首先使用不同浓度的Scriptaid(0,0.05,0.10μmol/L)和5-Aza-CdR(0,0.02,0.10μmol/L)分别处理AFs,筛选出最佳处理浓度后联合处理AFs 24h。随后采用免疫荧光染色技术分析联合处理后细胞的DNA甲基化和组蛋白H3K18乙酰化变化情况,并使用实时荧光定量PCR(QPCR)技术对其甲基化和乙酰化相关基因的表达进行检测。结果发现,适宜浓度的Scriptaid(0.05μmol/L)和5-Aza-CdR(0.02μmol/L)对细胞正常的形态、生长无显著影响。与对照组相比,联合处理后AFs中DNA甲基化水平显著降低,而组蛋白H3K18位点的乙酰化水平显著上升(P<0.05)。QPCR分析结果显示,联合处理后AFs中的DNA甲基化相关基因Dnmt1、TET1和TET2的表达水平均出现下调,其中Dnmt1的表达水平显著低于对照组(P<0.05);组蛋白去乙酰化酶HDAC1和HDAC2的表达水平也出现下调,其中HDAC1下调显著(P<0.05);而组蛋白乙酰化转移酶CBP,P300和HAT1的表达水平出现上升,其中P300和HAT1的表达水平显著高于对照组(P<0.05)。结果表明,适宜浓度的Scriptaid(0.05μmol/L)和5-Aza-CdR(0.02μmol/L)联合处理AFs,对细胞形态和生长无显著影响,但可以有效降低细胞甲基化水平并提高组蛋白H3K18位点的乙酰化水平。
The aim of this study was to investigate the effects of scriptaid and 5-Aza-CdR treatment on cell morphology and growth,DNA methylation and histone acetylation of buffalo adult fibroblasts(AFs).Firstly different concentrations of scriptaid(0,0.05,0.10μmol/L)and 5-Aza-CdR(0,0.02,0.10μmol/L)were used to select an optimal concentration for the treatment.Next AFs were jointly treated with optimal concentration of scriptaid and 5-Aza-CdR for 24 h,then the level of DNA methylation and histone H3K18 acetylation were detected by immunofluorescence technique,and the expression level of DNA methylation and histone acetylation related genes was also detected using quantitative real-time PCR(QPCR).The results showed that morphology and growth of AFs were not affected with the treatment of proper concentrations of scriptaid(0.05μmol/L)and 5-Aza-CdR(0.02μmol/L).Compared to the control,the level of DNA methylation was decreased while the level of histone H3K18 acetylation was increased significantly after treated with 0.05μmol/L scriptaid and 0.02μmol/L 5-Aza-CdR.QPCR results showed the expression level of Dnmt1TET1,TET2 was downregulated and Dnmt1 was downregulated significantly(P<0.05).Meanwhile,the expression level HDAC1 and HDAC2was also decreased and HDAC1 was decreased significantly(P<0.05).At the same time CBP,P300 and HAT1were up-regulated,among of them,P300 and HAT1were upregulated significantly(P<0.05).In a word,proper concentration of scriptaid(0.05μmol/L)and 5-Aza-CdR(0.02μmol/L)treatment has no significant effects on cell morphology and growth,while it can decrease the methylation level and increase H3K18 acetylation level efficiently.
The aim of this study was to investigate the effects of scriptaid and 5-Aza-CdR treatment on cell morphology and growth,DNA methylation and histone acetylation of buffalo adult fibroblasts(AFs).Firstly different concentrations of scriptaid(0,0.05,0.10μmol/L)and 5-Aza-CdR(0,0.02,0.10μmol/L)were used to select an optimal concentration for the treatment.Next AFs were jointly treated with optimal concentration of scriptaid and 5-Aza-CdR for 24 h,then the level of DNA methylation and histone H3K18 acetylation were detected by immunofluorescence technique,and the expression level of DNA methylation and histone acetylation related genes was also detected using quantitative real-time PCR(QPCR).The results showed that morphology and growth of AFs were not affected with the treatment of proper concentrations of scriptaid(0.05μmol/L)and 5-Aza-CdR(0.02μmol/L).Compared to the control,the level of DNA methylation was decreased while the level of histone H3K18 acetylation was increased significantly after treated with 0.05μmol/L scriptaid and 0.02μmol/L 5-Aza-CdR.QPCR results showed the expression level of Dnmt1TET1,TET2 was downregulated and Dnmt1 was downregulated significantly(P<0.05).Meanwhile,the expression level HDAC1 and HDAC2was also decreased and HDAC1 was decreased significantly(P<0.05).At the same time CBP,P300 and HAT1were up-regulated,among of them,P300 and HAT1were upregulated significantly(P<0.05).In a word,proper concentration of scriptaid(0.05μmol/L)and 5-Aza-CdR(0.02μmol/L)treatment has no significant effects on cell morphology and growth,while it can decrease the methylation level and increase H3K18 acetylation level efficiently.
引文
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[13]MIYOSHI K,MORI H,MIZOBE Y,et al.Valproic acid enhances invitrodevelopment and Oct-3/4expression of miniature pig somatic cell nuclear transfer embryos[J].Cell Reprogram,2010,12(1):67-74.
[14] WEI Y C,ZHU J A,YANJUN H,et al.Aberrant expression and methylation status of putatively imprinted genes in placenta of cloned piglets[J].Cell Reprogram,2010,12(2):213-222.
[15] VAN T N,BUI H T,KIM J H,et al.The histone deacetylase inhibitor scriptaid enhances nascent mRNA production and rescues full-term development in cloned inbred mice[J].Reproduction,2009,138(2):309-317.
[16] AKAGI S,MATSUKAWA K,MIZUTANI E,et al.Treatment with a histone deacetylase inhibitor after nuclear transfer improves the preimplantation development of cloned bovine embryos[J].J Reprod Dev,2011,57(1):120-126.
[17]高川,熊显荣,符梅,等.Scriptaid处理牦牛成纤维细胞对组蛋白乙酰化及重编程的影响[J].农业生物技术学报,2013,21(1):62-68.
[18] ZHAO J,ROSS J W,HAO Y,et al.Significant improvement in cloning efficiency of an inbred miniature pig by histone deacetylase inhibitor treatment after somatic cell nuclear transfer[J].Biol Reprod,2009,81(3):525-530.
[19]曹慧,李俊杰,苏文龙,等.Scriptaid和TSA对牛体细胞核移植重构胚发育效果的影响[J].中国畜牧杂志,2015,51(15):20-23.
[20] LYKO F,BROWN R.DNA Methyltransferase inhibitors and the development of epigenetic cancer therapies[J].J Natl Cancer Inst,2005,97(20):1498-1506.
[21] WANG Q,YIN S,AI J S,et al.Histone deacetylation is required for orderly meiosis[J].Cell Cycle,2006,5(7):766-774.
[22] BOURC′HIS D,BOURHIS D L,PATIN D,et al.Delayed and incomplete reprogramming of chromosome methylation patterns in bovine cloned embryos[J].Curr Biol,2001,11(19):1542-1546.
[23] IAGER A E,RAGINA N P,ROSS P J,et al.Trichostatin A improves histone acetylation in bovine somatic cell nuclear transfer early embryos[J].Cloning Stem Cells,2008,10(3):371-379.
[24] KANG Y K,PARK J S,KOO D B,et al.Limited demethylation leaves mosaic-type methylation states in cloned bovine pre-implantation embryos[J].EMBO J,2002,21(5):1092-1100.
[25] RODRIGUEZ-OSORIO N,URREGO R,CIBELLI J B,et al.Reprogramming mammalian somatic cells[J].Theriogenology,2012,78(9):1869-1886.
[26] ZLATANOVA J,CAIAFA P,VAN H K.Linker histone binding and displacement:versatile mechanism for transcriptional regulation[J].FASEB J,2000,14(12):1697-1704.
[27]胡广卫,苏建民,曹泽磊,等.5-Aza-CdR联合TSA对牛核移植胚胎体外发育及表观遗传状态的影响[J].西北农林科技大学学报(自然科学版),2009(12):1-5.
[28] OKANO M,BELL D W,HABER D A,et al.DNA methyltransferases Dnmt3aand Dnmt3bare essential for de novo methylation and mammalian development[J].Cell,1999,99(3):247-257.
[29]刘谋泽,何发忠,张伟.抗肿瘤药物的表观遗传学研究进展[J].药学学报,2013(11):1629-1636.
[2]BAGUISI A,BEHBOODI E,MELICAN D T,et al.Production of goats by somatic cell nuclear transfer[J].Nat Biotechnol,1999,17(2):456-461.
[3]KATO Y,TANI T,SOTOMARU Y,et al.Eight calves cloned from somatic cells of a single adult[J].Science,1998,282(5396):2095-2098.
[4]REIK W.Stability and flexibility of epigenetic gene regulation in mammalian development[J].Nature,2007,447(7143):425-432.
[5]LI E.Chromatin modification and epigenetic reprogramming in mammalian development[J].Nat Rev Gen,2002,3(9):662-673.
[6]CEDAR H,BERGMAN Y.Linking DNA methylation and histone modification:patterns and paradigms[J].Nat Rev Genet,2009,10(5):295-304.
[7]GEIMAN T M,ROBERTSON K D.Chromatin remodeling,histone modifications,and DNA methylation-how does it all fit together[J].J Cell Biochem,2002,87(2):117-125.
[8]CERVONI N,SZYF M.Demethylase activity is directed by histone acetylation[J].J Biol Chem,2001,276(44):40778-40787.
[9]JULTTERMANN R,LI E,JAENIS R.Toxicity of 5-aza-2′-deoxycytidine to mammalian cells is mediated primarily by covalent trapping of DNA methyltransferase rather than DNA demethylation[J].PNAS,1994(91):11797-11801.
[10]刘宇,张日欣,李钟淑.小鼠卵母细胞及体细胞核移植重构早期端粒酶活性的测定[J].中国兽医学报,2018,38(3):586-590.
[11] YUN F D,NARUSE K,XIAO X L,et al.Effects of 5-aza-2’-deoxyctidine on the development of porcine parthenogenetic and nuclear transfer embryos[J].Nat Sci,2013,5(7A):31-37.
[12]陆杏蓉,李志鹏,孙俊铭,等.DNA甲基化酶抑制剂5-Aza-CdR对多能基因及甲基化相关基因在徒手克隆胚胎中表达模式的影响[J].中国畜牧兽医,2016,43(9):2240-2248.
[13]MIYOSHI K,MORI H,MIZOBE Y,et al.Valproic acid enhances invitrodevelopment and Oct-3/4expression of miniature pig somatic cell nuclear transfer embryos[J].Cell Reprogram,2010,12(1):67-74.
[14] WEI Y C,ZHU J A,YANJUN H,et al.Aberrant expression and methylation status of putatively imprinted genes in placenta of cloned piglets[J].Cell Reprogram,2010,12(2):213-222.
[15] VAN T N,BUI H T,KIM J H,et al.The histone deacetylase inhibitor scriptaid enhances nascent mRNA production and rescues full-term development in cloned inbred mice[J].Reproduction,2009,138(2):309-317.
[16] AKAGI S,MATSUKAWA K,MIZUTANI E,et al.Treatment with a histone deacetylase inhibitor after nuclear transfer improves the preimplantation development of cloned bovine embryos[J].J Reprod Dev,2011,57(1):120-126.
[17]高川,熊显荣,符梅,等.Scriptaid处理牦牛成纤维细胞对组蛋白乙酰化及重编程的影响[J].农业生物技术学报,2013,21(1):62-68.
[18] ZHAO J,ROSS J W,HAO Y,et al.Significant improvement in cloning efficiency of an inbred miniature pig by histone deacetylase inhibitor treatment after somatic cell nuclear transfer[J].Biol Reprod,2009,81(3):525-530.
[19]曹慧,李俊杰,苏文龙,等.Scriptaid和TSA对牛体细胞核移植重构胚发育效果的影响[J].中国畜牧杂志,2015,51(15):20-23.
[20] LYKO F,BROWN R.DNA Methyltransferase inhibitors and the development of epigenetic cancer therapies[J].J Natl Cancer Inst,2005,97(20):1498-1506.
[21] WANG Q,YIN S,AI J S,et al.Histone deacetylation is required for orderly meiosis[J].Cell Cycle,2006,5(7):766-774.
[22] BOURC′HIS D,BOURHIS D L,PATIN D,et al.Delayed and incomplete reprogramming of chromosome methylation patterns in bovine cloned embryos[J].Curr Biol,2001,11(19):1542-1546.
[23] IAGER A E,RAGINA N P,ROSS P J,et al.Trichostatin A improves histone acetylation in bovine somatic cell nuclear transfer early embryos[J].Cloning Stem Cells,2008,10(3):371-379.
[24] KANG Y K,PARK J S,KOO D B,et al.Limited demethylation leaves mosaic-type methylation states in cloned bovine pre-implantation embryos[J].EMBO J,2002,21(5):1092-1100.
[25] RODRIGUEZ-OSORIO N,URREGO R,CIBELLI J B,et al.Reprogramming mammalian somatic cells[J].Theriogenology,2012,78(9):1869-1886.
[26] ZLATANOVA J,CAIAFA P,VAN H K.Linker histone binding and displacement:versatile mechanism for transcriptional regulation[J].FASEB J,2000,14(12):1697-1704.
[27]胡广卫,苏建民,曹泽磊,等.5-Aza-CdR联合TSA对牛核移植胚胎体外发育及表观遗传状态的影响[J].西北农林科技大学学报(自然科学版),2009(12):1-5.
[28] OKANO M,BELL D W,HABER D A,et al.DNA methyltransferases Dnmt3aand Dnmt3bare essential for de novo methylation and mammalian development[J].Cell,1999,99(3):247-257.
[29]刘谋泽,何发忠,张伟.抗肿瘤药物的表观遗传学研究进展[J].药学学报,2013(11):1629-1636.
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