中国荷斯坦牛金黄色葡萄球菌诱导型乳房炎CDH13基因甲基化分析
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摘要
为探索钙黏蛋白13基因(cadherin 13,CDH13)甲基化在奶牛金黄色葡萄球菌乳房炎中的调控机制,本试验在已建立的金黄色葡萄球菌诱导型荷斯坦牛乳房炎模型的基础上利用重亚硫酸盐测序技术(bisulfite sequencing PCR,BSP)检测正常乳腺组织和乳房炎乳腺组织候选基因CDH13的甲基化程度,并分析其甲基化程度与基因表达水平的关系。结果显示,除金黄色葡萄球菌致病组(试验组)第12个CpG岛外,其余所有CpG岛均呈现不同程度的甲基化:-259处甲基化水平最高(50%~60%),-144、-135和-126处甲基化均较低,为5%左右。试验组和对照组总体甲基化率分别为10.13%±1.81%和14.43%±0.55%,不同位点和总体甲基化率无显著差异(P>0.05)。与正常乳腺组织相比,乳房炎感染组乳腺组织CDH13基因表达上调,CDH13基因-162和-93两个CpG岛甲基化水平与其基因表达呈显著负相关(P<0.05)。本试验结果表明,CDH13基因甲基化影响其基因表达,从而影响乳房炎的发生,为进一步探索其在金黄色葡萄球菌致病型乳房炎中的发病机制提供了参考依据。
In order to explore the regulatory mechanism of cadherin 13(CDH13)gene methylation in mastitis caused by Staphylococcus aureus in dairy cows,the CDH13 gene methylation levels of healthy and mastitis infected mammary tissue were tested by bisulfite sequencing PCR(BSP),and the relationship between the level of CDH13 gene methylation and expression was analyzed.The results showed that,all of the CpG islands except the twelfth CpG of experimental group showed different levels of methylation.The methylation level was the highest in-259(50% to 60%),and those at-144,-135 and-126 were low,which were all about 5%.The overall methylation level of experimen and control groups ware 10.13%±1.81% and 14.43%±0.55%,respectively,and there was no significant difference between different loci and overall methylation rate of this two groups(P>0.05).Compared with the healthy tissue,the expression of CDH13 gene in mammary tissue which were inflected by mastitis was up-regulation,the methylation level of -162 and -93 CpG islands were significantly negatively correlated with gene expression(P<0.05).Themethylation of CDH13 gene affected its expression,thus affecting the occurrence of mastitis.These provided a reference for further exploring the pathogenesis of Staphylococcus aureus pathogenic mastitis.
In order to explore the regulatory mechanism of cadherin 13(CDH13)gene methylation in mastitis caused by Staphylococcus aureus in dairy cows,the CDH13 gene methylation levels of healthy and mastitis infected mammary tissue were tested by bisulfite sequencing PCR(BSP),and the relationship between the level of CDH13 gene methylation and expression was analyzed.The results showed that,all of the CpG islands except the twelfth CpG of experimental group showed different levels of methylation.The methylation level was the highest in-259(50% to 60%),and those at-144,-135 and-126 were low,which were all about 5%.The overall methylation level of experimen and control groups ware 10.13%±1.81% and 14.43%±0.55%,respectively,and there was no significant difference between different loci and overall methylation rate of this two groups(P>0.05).Compared with the healthy tissue,the expression of CDH13 gene in mammary tissue which were inflected by mastitis was up-regulation,the methylation level of -162 and -93 CpG islands were significantly negatively correlated with gene expression(P<0.05).Themethylation of CDH13 gene affected its expression,thus affecting the occurrence of mastitis.These provided a reference for further exploring the pathogenesis of Staphylococcus aureus pathogenic mastitis.
引文
[1]SCHBELER D.Function and information content of DNA methylation[J].Nature,2015,517(7534):321-326.
[2]ROTHBART S B,STRAHL B D.Interpreting the language of histone and DNA modifications[J].Biochim Biophys Acta,2014,1839(8):627-643.
[3]王志刚,吴建新.DNA甲基转移酶分类、功能及其研究进展[J].遗传,2009,31(9):903-912.WANG Z G,WU J X.DNA methyltransferases:Classification,functions and research progress[J].Hereditas,2009,31(9):903-912.(in Chinese)
[4]DAHL C,GULDBERG P.DNA methylation analysis techniques[J].Biogerontology,2003,4(4):233-250.
[5]徐玉薇,罗小青,魏学良,等.不同地区3个奶牛场临床型乳房炎细菌病原种类分析[J].中国畜牧兽医,2016,43(2):548-552.XU Y W,LUO X Q,WEI X L,et al.Analysis of clinical mastitis pathogenic bacterial species in three dairy farms of different areas[J].China Animal Husbandry&Veterinary Medicine,2016,43(2):548-552.(in Chinese)
[6]姚伟,于学武,曹东,等.辽宁地区奶牛乳房炎细菌性病原分离鉴定及大肠杆菌药敏试验[J].中国畜牧兽医,2016,43(11):3080-3084.YAO W,YU X W,CAO D,et al.Isolation and identification of pathogenic bacteria causing dairy cow mastitis in Liaoning and drug sensibility test of pathogenic E.coli[J].China Animal Husbandry&Veterinary Medicine,2016,43(11):3080-3084.(in Chinese)
[7]王丹,杨峰,李新圃,等.奶牛乳房炎病原菌诊断技术研究进展[J].中国畜牧兽医,2017,44(6):1811-1817.WANG D,YANG F,LI X P,et al.Research progress on diagnosis technology of dairy cow mastitis pathogens[J].China Animal Husbandry&Veterinary Medicine,2017,44(6):1811-1817.(in Chinese)
[8]VANSELOW J,YAND W,HERRMANN J,et al.DNA-remethylation around a STAT5-binding enhancer in the alphaS1-casein promoter is associated with abrupt shutdown of alphaS1-casein synthesis during acute mastitis[J].Journal of Molecular Endocrinology,2006,37(3):463-477.
[9]刘利,高雪,高会江,等.DNA甲基化在奶牛金黄色葡萄球菌性乳房炎中的调控[J].畜牧兽医学报,2012,43(10):1554-1558.LUI L,GAO X,GAO H J,et al.Regulation of DNA methylation during Staphylococcus aureus mastitis in dairy cattle[J].Acta Veterinaria Et Zootechnica Sinica,2012,43(10):1554-1558.(in Chinese)
[10]WANG X S,ZHANG Y,HE Y H,et al.Aberrant promoter methylation of the CD4gene in peripheral blood cells of mastitic dairy cows[J].Genetics&Molecular Research,2013,12(4):6228-6239.
[11]孙昀,耿小平,朱立新.CDH13基因的研究进展[J].医学分子生物学杂志,2005,2(3):224-226.SUN Y,GENG X P,ZHU L X.Advances in the study of CDH13gene[J].Journal of Medical Molecular Biology,2005,2(3):224-226.(in Chinese)
[12]PUTKU M,KALS M,INNO R,et al.CDH13promoter SNPs with pleiotropic effect on cardiometabolic parameters represent methylation QTLs[J].Human Genetics,2015,134(3):291-303.
[13]CHOTCHAEVA F R,BALATSKIY A V,SAMOKHODSKAYA L M,et al.Association between T-cadherin gene(CDH13)variants and severity of coronary heart disease manifestation[J].International Journal of Clinical&Experimental Medicine,2016,9(2):4059-4064.
[14]洪超,李盈甫,马千里,等.CDH13基因变异与非小细胞肺癌的相关性[J].昆明医科大学学报,2016,37(6):48-51.HONG C,LI Y F,MA Q L,et al.The correlation of CDH13 gene variation with non-small cell lung cancer[J].Journal of Kunming Medical University,2016,37(6):48-51.(in Chinese)
[15]刘检波,毛平,应逸,等.急性髓系白血病CDH13基因启动子甲基化的定量研究[J].临床血液学杂志,2013,26(9):606-610.LIU J P,MAO P,YING Y,et al.Quantitive analysis of promoter methylation of CDH13gene in patients with acute myeloid leukemia[J].Journal of Clinical Hematology,2013,26(9):606-610.(in Chinese)
[16]吴燕飞,张乐鸣.CDH13基因在乳腺癌细胞株MCF-7中表达及甲基化调控[J].现代实用医学,2010,22(8):855-858.WU Y F,ZHANG L M.Expression and methylation regulation of CDH13in MCF-7cell line[J].Modern Practical Medicine,2010,22(8):855-858.(in Chinese)
[17]李蕊,张成龙,耿晓晗,等.金黄色葡萄球菌诱导型乳腺炎对中国荷斯坦奶牛乳中脂肪酸组成的影响[J].动物营养学报,2016,28(2):426-435.LI R,ZHANG C L,GENG X H,et al.Effects of Staphylococcus aureus-induced mastitis on milk fatty acid composition of Chinese Holstein cows[J].Chinese Journal of Animal Nutrition,2016,28(2):426-435.(in Chinese)
[18]BOCK C,REITHER S,MIKESKA T,et al.BiQ Analyzer:Visualization and quality control for DNA methylation data from bisulfite sequencing[J].Bioinformatics,2005,21(21):4067-4068.
[19]CARTHARIUS K,FRECH K,GROTE K,et al.MatInspector and beyond:Promoter analysis based on transcription factor binding sites[J].Bioinformatics,2005,21(13):2933-2942.
[20]汪艳杰,龙鸿,姚家玲.亚硫酸氢钠测序法检测水稻FIE基因CpG岛甲基化状态[J].植物科学学报,2011,29(1):134-139.WANG Y J,LONG H,YAO J L.Analysis of methylation at CpG island of PIE in rice by bisulfite sequencing[J].Plant Science Journal,2011,29(1):134-139.(in Chinese)
[21]郑宜文.奶牛泌乳性状相关基因启动子甲基化分析[D].哈尔滨:东北农业大学,2013.ZHENG Y W.Analysis of gene promoter methylation related to milk production traits in dairy cow[D].Harbin:Northeast Agricultural University,2013.(in Chinese)
[22]汤华阳,杜卫东,张学军.焦磷酸测序技术及应用[J].医学分子生物学杂志,2007,4(3):272-275.TANG H Y,DU W D,ZHANG X J.Pyrosequencing and its application[J].Journal of Medical Molecular Biology,2007,4(3):272-275.(in Chinese)
[23]MOYES K M,DRACKLEY J K,MORIN D E,et al.Gene network and pathway analysis of bovine mammary tissue challenged with Streptococcus uberis reveals induction of cell proliferation and inhibition of PPARgamma signaling as potential mechanism for the negative relationships between immune response and lipid metabolism[J].Bmc Genomics,2009,10(1):542.
[24]TAO W,MALLARD B.Differentially expressed genes associated with Staphylococcus aureus mastitis of Canadian Holstein cows[J].Veterinary Immunology Immunopathology,2007,120(3-4):201-211.
[25]黄晖,孔勇,冯旭,等.非小细胞肺癌CDH13及DAPK基因CpG岛甲基化的研究[J].广西医学,2015,4:449-451.HUANG H,KONG Y,FENG X,et al.Research on CpG-island methylation of CDH13and DAPK genes in patients with non-small cell lung carcinoma[J].Guangxi Medical Journal,2007,4:449-451.(in Chinese)
[26]ZUCCHINI C,BIANCHINI M,VALVASSORI L,et al.Identification of candidate genes involved in the reversal of malignant phenotype of osteosarcoma cells transfected with the liver/bone/kidney alkaline phosphatase gene[J].Bone,2004,34(4):672-679.
[27]ZHONG Y H,PENG H,CHENG H Z,et al.Quantitative assessment of the diagnostic role of CDH13promoter methylation in lung cancer[J].Asian Pacific Journal of Cancer Prevention,2015,16(3):1139-1143.
[28]WIDSCHWENDTER A,IVARSSON L,BLASSNIG A,et al.CDH1and CDH13 methylation in serum is an independent prognostic marker in cervical cancer patients[J].International Journal of Cancer,2004,109(2):163-166.
[29]陈丹.奶牛诱导性链球菌型乳腺炎表达谱及部分候选基因甲基化特征分析[D].扬州:扬州大学,2014.CHEN D.Gene expression profiles and methylation analysis on the mammary tissue challenged with Streptococcus in Chinese Holstein[D].Yangzhou:Yangzhou University,2014.(in Chinese)
[2]ROTHBART S B,STRAHL B D.Interpreting the language of histone and DNA modifications[J].Biochim Biophys Acta,2014,1839(8):627-643.
[3]王志刚,吴建新.DNA甲基转移酶分类、功能及其研究进展[J].遗传,2009,31(9):903-912.WANG Z G,WU J X.DNA methyltransferases:Classification,functions and research progress[J].Hereditas,2009,31(9):903-912.(in Chinese)
[4]DAHL C,GULDBERG P.DNA methylation analysis techniques[J].Biogerontology,2003,4(4):233-250.
[5]徐玉薇,罗小青,魏学良,等.不同地区3个奶牛场临床型乳房炎细菌病原种类分析[J].中国畜牧兽医,2016,43(2):548-552.XU Y W,LUO X Q,WEI X L,et al.Analysis of clinical mastitis pathogenic bacterial species in three dairy farms of different areas[J].China Animal Husbandry&Veterinary Medicine,2016,43(2):548-552.(in Chinese)
[6]姚伟,于学武,曹东,等.辽宁地区奶牛乳房炎细菌性病原分离鉴定及大肠杆菌药敏试验[J].中国畜牧兽医,2016,43(11):3080-3084.YAO W,YU X W,CAO D,et al.Isolation and identification of pathogenic bacteria causing dairy cow mastitis in Liaoning and drug sensibility test of pathogenic E.coli[J].China Animal Husbandry&Veterinary Medicine,2016,43(11):3080-3084.(in Chinese)
[7]王丹,杨峰,李新圃,等.奶牛乳房炎病原菌诊断技术研究进展[J].中国畜牧兽医,2017,44(6):1811-1817.WANG D,YANG F,LI X P,et al.Research progress on diagnosis technology of dairy cow mastitis pathogens[J].China Animal Husbandry&Veterinary Medicine,2017,44(6):1811-1817.(in Chinese)
[8]VANSELOW J,YAND W,HERRMANN J,et al.DNA-remethylation around a STAT5-binding enhancer in the alphaS1-casein promoter is associated with abrupt shutdown of alphaS1-casein synthesis during acute mastitis[J].Journal of Molecular Endocrinology,2006,37(3):463-477.
[9]刘利,高雪,高会江,等.DNA甲基化在奶牛金黄色葡萄球菌性乳房炎中的调控[J].畜牧兽医学报,2012,43(10):1554-1558.LUI L,GAO X,GAO H J,et al.Regulation of DNA methylation during Staphylococcus aureus mastitis in dairy cattle[J].Acta Veterinaria Et Zootechnica Sinica,2012,43(10):1554-1558.(in Chinese)
[10]WANG X S,ZHANG Y,HE Y H,et al.Aberrant promoter methylation of the CD4gene in peripheral blood cells of mastitic dairy cows[J].Genetics&Molecular Research,2013,12(4):6228-6239.
[11]孙昀,耿小平,朱立新.CDH13基因的研究进展[J].医学分子生物学杂志,2005,2(3):224-226.SUN Y,GENG X P,ZHU L X.Advances in the study of CDH13gene[J].Journal of Medical Molecular Biology,2005,2(3):224-226.(in Chinese)
[12]PUTKU M,KALS M,INNO R,et al.CDH13promoter SNPs with pleiotropic effect on cardiometabolic parameters represent methylation QTLs[J].Human Genetics,2015,134(3):291-303.
[13]CHOTCHAEVA F R,BALATSKIY A V,SAMOKHODSKAYA L M,et al.Association between T-cadherin gene(CDH13)variants and severity of coronary heart disease manifestation[J].International Journal of Clinical&Experimental Medicine,2016,9(2):4059-4064.
[14]洪超,李盈甫,马千里,等.CDH13基因变异与非小细胞肺癌的相关性[J].昆明医科大学学报,2016,37(6):48-51.HONG C,LI Y F,MA Q L,et al.The correlation of CDH13 gene variation with non-small cell lung cancer[J].Journal of Kunming Medical University,2016,37(6):48-51.(in Chinese)
[15]刘检波,毛平,应逸,等.急性髓系白血病CDH13基因启动子甲基化的定量研究[J].临床血液学杂志,2013,26(9):606-610.LIU J P,MAO P,YING Y,et al.Quantitive analysis of promoter methylation of CDH13gene in patients with acute myeloid leukemia[J].Journal of Clinical Hematology,2013,26(9):606-610.(in Chinese)
[16]吴燕飞,张乐鸣.CDH13基因在乳腺癌细胞株MCF-7中表达及甲基化调控[J].现代实用医学,2010,22(8):855-858.WU Y F,ZHANG L M.Expression and methylation regulation of CDH13in MCF-7cell line[J].Modern Practical Medicine,2010,22(8):855-858.(in Chinese)
[17]李蕊,张成龙,耿晓晗,等.金黄色葡萄球菌诱导型乳腺炎对中国荷斯坦奶牛乳中脂肪酸组成的影响[J].动物营养学报,2016,28(2):426-435.LI R,ZHANG C L,GENG X H,et al.Effects of Staphylococcus aureus-induced mastitis on milk fatty acid composition of Chinese Holstein cows[J].Chinese Journal of Animal Nutrition,2016,28(2):426-435.(in Chinese)
[18]BOCK C,REITHER S,MIKESKA T,et al.BiQ Analyzer:Visualization and quality control for DNA methylation data from bisulfite sequencing[J].Bioinformatics,2005,21(21):4067-4068.
[19]CARTHARIUS K,FRECH K,GROTE K,et al.MatInspector and beyond:Promoter analysis based on transcription factor binding sites[J].Bioinformatics,2005,21(13):2933-2942.
[20]汪艳杰,龙鸿,姚家玲.亚硫酸氢钠测序法检测水稻FIE基因CpG岛甲基化状态[J].植物科学学报,2011,29(1):134-139.WANG Y J,LONG H,YAO J L.Analysis of methylation at CpG island of PIE in rice by bisulfite sequencing[J].Plant Science Journal,2011,29(1):134-139.(in Chinese)
[21]郑宜文.奶牛泌乳性状相关基因启动子甲基化分析[D].哈尔滨:东北农业大学,2013.ZHENG Y W.Analysis of gene promoter methylation related to milk production traits in dairy cow[D].Harbin:Northeast Agricultural University,2013.(in Chinese)
[22]汤华阳,杜卫东,张学军.焦磷酸测序技术及应用[J].医学分子生物学杂志,2007,4(3):272-275.TANG H Y,DU W D,ZHANG X J.Pyrosequencing and its application[J].Journal of Medical Molecular Biology,2007,4(3):272-275.(in Chinese)
[23]MOYES K M,DRACKLEY J K,MORIN D E,et al.Gene network and pathway analysis of bovine mammary tissue challenged with Streptococcus uberis reveals induction of cell proliferation and inhibition of PPARgamma signaling as potential mechanism for the negative relationships between immune response and lipid metabolism[J].Bmc Genomics,2009,10(1):542.
[24]TAO W,MALLARD B.Differentially expressed genes associated with Staphylococcus aureus mastitis of Canadian Holstein cows[J].Veterinary Immunology Immunopathology,2007,120(3-4):201-211.
[25]黄晖,孔勇,冯旭,等.非小细胞肺癌CDH13及DAPK基因CpG岛甲基化的研究[J].广西医学,2015,4:449-451.HUANG H,KONG Y,FENG X,et al.Research on CpG-island methylation of CDH13and DAPK genes in patients with non-small cell lung carcinoma[J].Guangxi Medical Journal,2007,4:449-451.(in Chinese)
[26]ZUCCHINI C,BIANCHINI M,VALVASSORI L,et al.Identification of candidate genes involved in the reversal of malignant phenotype of osteosarcoma cells transfected with the liver/bone/kidney alkaline phosphatase gene[J].Bone,2004,34(4):672-679.
[27]ZHONG Y H,PENG H,CHENG H Z,et al.Quantitative assessment of the diagnostic role of CDH13promoter methylation in lung cancer[J].Asian Pacific Journal of Cancer Prevention,2015,16(3):1139-1143.
[28]WIDSCHWENDTER A,IVARSSON L,BLASSNIG A,et al.CDH1and CDH13 methylation in serum is an independent prognostic marker in cervical cancer patients[J].International Journal of Cancer,2004,109(2):163-166.
[29]陈丹.奶牛诱导性链球菌型乳腺炎表达谱及部分候选基因甲基化特征分析[D].扬州:扬州大学,2014.CHEN D.Gene expression profiles and methylation analysis on the mammary tissue challenged with Streptococcus in Chinese Holstein[D].Yangzhou:Yangzhou University,2014.(in Chinese)