水牛泌乳期与非泌乳期乳腺组织差异miRNAs的表达模式鉴定及分析
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摘要
为了探求水牛泌乳和非泌乳期关键调控差异表达miRNAs及其表达模式,试验结合Solexa高通量测序和生物信息学分析结果筛选出18个水牛乳腺组织在泌乳期和非泌乳期差异表达的miRNAs,设计、筛选可行的miRNAs反转录及实时荧光定量PCR引物,进行实时荧光定量PCR验证差异表达miRNAs的表达模式。结果发现,miRNA的差异表达模式与测序结果基本一致。泌乳期miRNA-103、miRNA-125a、miRNA-30a-5p和miRNA-148a的表达量均显著高于非泌乳期(P<0.05);miRNA-29a在非泌乳期表达量显著高于泌乳期(P<0.05);miRNA-141、miRNA-125b和miRNA-497在泌乳期表达量均高于非泌乳期,但差异不显著(P>0.05);低丰度的miRNA-490和miRNA-592在泌乳期表达量均显著高于非泌乳期(P<0.05)。Novel-123b在泌乳期和非泌乳期差异不显著(P>0.05),Novel-57在泌乳期比非泌乳期高29.79倍(P<0.01)。本试验结果为后续水牛乳腺研究提供了16个miRNA的可用特异性反转录引物和实时荧光定量PCR引物,miRNA-103、miRNA-125a、miRNA-30a-5p、miRNA-148a、miRNA-29a和Novel-57 6个水牛差异表达miRNAs值得进一步研究。
In order to explore the differential expression miRNAs and their expression patterns in the control of buffalo in lactation and non-lactation,the miRNAs of 18 buffalo breast tissues differentially expressed in lactation and non-lactation were screened by Solexa high-throughput sequencing and bioinformatics analysis.Viable miRNAs primers for reverse transcription and Realtime quantitative PCR were used to validate the expression patterns of miRNAs.The Real-time quantitative PCR results were basically consistent with sequencing results.The expression levels of miR-103,miRNA-125 a,miRNA-30 a-5 p and miRNA-148 ain lactation were significantly higher than those in non-lactation(P<0.05).The expression level of miRNA-29 ain non-lactation was higher than that in lactation(P<0.05).The expression levels of miRNA-141,miRNA-125 band miRNA-497 in lactation were higher than those in non-lactation,but the difference was not signif-icant(P>0.05).The low-expression levels of miRNA-490 and miRNA-592 in lactation were significantly higher than those in non-lactation(P<0.05).There was no significant difference in the expression of Novel-123 bbetween lactation and non-lactation(P>0.05).Novel-57 was extremely significantly in lactation(29.79 times)higher than non-lactation(P<0.01).This results provide available specific reverse transcription primers and Real-time quantitative PCR primers of 16 miRNAs for further study on buffalo,and 6 miRNAs such as miRNA-103,miRNA-125 a,miRNA-30 a-5 p,miRNA-148 a,miRNA-29 aand Novel-57 deserve further research.
In order to explore the differential expression miRNAs and their expression patterns in the control of buffalo in lactation and non-lactation,the miRNAs of 18 buffalo breast tissues differentially expressed in lactation and non-lactation were screened by Solexa high-throughput sequencing and bioinformatics analysis.Viable miRNAs primers for reverse transcription and Realtime quantitative PCR were used to validate the expression patterns of miRNAs.The Real-time quantitative PCR results were basically consistent with sequencing results.The expression levels of miR-103,miRNA-125 a,miRNA-30 a-5 p and miRNA-148 ain lactation were significantly higher than those in non-lactation(P<0.05).The expression level of miRNA-29 ain non-lactation was higher than that in lactation(P<0.05).The expression levels of miRNA-141,miRNA-125 band miRNA-497 in lactation were higher than those in non-lactation,but the difference was not signif-icant(P>0.05).The low-expression levels of miRNA-490 and miRNA-592 in lactation were significantly higher than those in non-lactation(P<0.05).There was no significant difference in the expression of Novel-123 bbetween lactation and non-lactation(P>0.05).Novel-57 was extremely significantly in lactation(29.79 times)higher than non-lactation(P<0.01).This results provide available specific reverse transcription primers and Real-time quantitative PCR primers of 16 miRNAs for further study on buffalo,and 6 miRNAs such as miRNA-103,miRNA-125 a,miRNA-30 a-5 p,miRNA-148 a,miRNA-29 aand Novel-57 deserve further research.
引文
[1]林浪,龚云,王梦,等.水牛缝隙连接蛋白43基因克隆及表达[J].中国畜牧兽医,2013,40(9):1-7.
[2]Ahmad S,Gaucher I,Rousseau F,et al.Effects of acidification on physico-chemical characteristics of buffalo milk:A comparison with cow’s milk[J].Food Chemistry,2008,106(1):11-17.
[3]唐善生.广西奶水牛业发展现状与思考[J].广西农学报,2014,29(1):50-52.
[4]简保权,秦学敏,龚芳.世界水牛奶业发展现状和典型模式分析[J].世界农业,2015,3:115-118.
[5]王锦.水牛乳腺基因表达谱与生长激素转基因水牛的初步研究[D].南宁:广西大学,2013.
[6]Bartel D P.microRNAs:Genomics,biogenesis,mechanism,and function[J].Cell,2004,116(2):281-297.
[7]Ambros V.The functions of animal microRNAs[J].Nature,2004,431(7006):350-355.
[8]Kim V N.microRNA biogenesis:Coordinated cropping and dicing[J].Nature Reviews Molecular Cell Biology,2005,6(5):376-385.
[9]赵静雯,陈国荣,吴慧光.miRNA合成途径研究进展[J].中国畜牧兽医,2015,42(12):3186-3191.
[10]Wang K,Long B,Jiao J Q,et al.miRNA-484regulates mitochondrial network through targeting Fis1[J].Nature Communications,2012,3:781.
[11]Wightman B,Ha I,Ruvkun G.Posttranscriptional regulation of the heterochronic gene lin-14by lin-4mediates temporal pattern formation in C.elegans[J].Cell,1993,75(5):855-862.
[12]Weber J A,Baxter D H,Zhang S,et al.The microRNA spectrum in 12 body fluids[J].Clinical Chemistry,2010,56(11):1733-1741.
[13]Kosaka N,Izumi H,Sekine K,et al.microRNA as a new immune-regulatory agent in breast milk[J].Silence,2010,1(1):7.
[14]张抒,张强,张萌,等.miRNA对乳腺发育及泌乳调节作用的研究进展[J].中国畜牧兽医,2015,42(3):663-667.
[15]Grembergen O V.microRNAs regulate KDM5histone demethylases in breast cancer cells[J].Molecular Bio Systems,2016,12(2):404-413.
[16]Phan B,Majid S,Ursu S,et al.Tumor suppressor role of microRNA-1296 in triple-negative breast cancer[J].Oncotarget,2016,7(15):19519.
[17]Cochrane D R,Spoelstra N S,Richer J K.The role of miRNAs in progesterone action[J].Molecular and Cellular Endocrinology,2012,357(1):50-59.
[18]熊显荣,兰道亮,李键,等.牦牛卵巢小RNA高通量测序及生物信息学分析[J].畜牧兽医学报,2016,47(1):55-63.
[19]周华,张新,刘腾云,等.高通量转录组测序的数据分析与基因发掘[J].江西科学,2012,30(5):607-611.
[20]Morin R D,O’Connor M D,Griffith M,et al.Application of massively parallel sequencing to microRNA profiling and discovery in human embryonic stem cells[J].Genome Research,2008,18(4):610-621.
[21]Li Z,Liu H,Jin X,et al.Expression profiles of microRNAs from lactating and non-lactating bovine mammary glands and identification of miRNA related to lactation[J].BMC Genomics,2012,13(1):731.
[22]Huang J,Ju Z,Li Q,et al.Solexa sequencing of novel and differentially expressed microRNAs in testicular and ovarian tissues in Holstein cattle[J].Int J Biol Sci,2011,7(7):1016-1026.
[23]曾献存,贾斌,史洪才,等.利用Solexa测序技术鉴定策勒黑羊microRNA[J].中国畜牧兽医,2016,43(1):204-209.
[24]Elsik C G,Tellam R L,Worley K C.The genome sequence of taurine cattle:A window to ruminant biology and evolution[J].Science,2009,324(5926):522-528.
[25]Ro S,Park C,Jin J,et al.A PCR-based method for detection and quantification of small RNAs[J].Biochemical and Biophysical Research Communications,2006,351(3):756-763.
[26]Larionov A,Krause A,Miller W.A standard curve based method for relative Real-time PCR data processing[J].BMC Bioinformatics,2005,6(1):62.
[27]Livak K J,Schmittgen T D.Analysis of relative gene expression data using Real-time quantitative PCR and the 2-ΔΔCt method[J].Methods,2001,25(4):402-408.
[28]Pfaffl M W.A new mathematical model for relative quantification in Real-time RT-PCR[J].Nucleic Acids Research,2001,29(9):e45.
[29]Ibarra I,Erlich Y,Muthuswamy S K,et al.A role for microRNAs in maintenance of mouse mammary epithelial progenitor cells[J].Genes&Development,2007,21(24):3238-3243.
[30]金晓露,杨建香,李真,等.乳腺发育及泌乳相关miRNA研究进展[J].遗传,2013,35(6):695-702.
[31]陆黎敏,李庆章,王春梅,等.miR-221对小鼠乳腺上皮细胞增殖和泌乳功能的影响[J].中国生物化学与分子生物学报,2009,5:454-458.
[32]Lin X,Luo J,Zhang L,et al.miRNA-103controls milk fat accumulation in goat(Capra hircus)mammary gland during lactation[J].PLoS One,2013,8(11):e79258.
[33]Zhang L,Volinia S,Bonome T,et al.Genomic and epigenetic alterations deregulate microRNA expression in human epithelial ovarian cancer[J].Proceedings of the National Academy of Sciences,2008,105(19):7004-7009.
[34]Guo Y,Chen Z,Zhang L,et al.Distinctive microRNA profiles relating to patient survival in esophageal squamous cell carcinoma[J].Cancer Research,2008,68(1):26-33.
[35]Tanaka T,Haneda S,Imakawa K,et al.A microRNA,miRNA-101a,controls mammary gland development by regulating cyclooxygenase-2expression[J].Differentiation,2009,77(2):181-187.
[36]Liu C H,Chang S H,Narko K,et al.Overexpression of cyclooxygenase-2is sufficient to induce tumorigenesis in transgenic mice[J].Journal of Biological Chemistry,2001,276(21):18563-18569.
[37]Lu S,Yu G,Zhu Y,et al.Cyclooxygenase-2overexpression in MCF-10Fhuman breast epithelial cells inhibits proliferation,apoptosis and differentiation,and causes partial transformation[J].International Journal of Cancer,2005,116(6):847-852.
[38]Wu Z,Huang X,Huang X,et al.The inhibitory role ofmiR-29in growth of breast cancer cells[J].Journal of Experimental&Clinical Cancer Research,2013,32(1):98.
[39]边艳杰.miRNA-29家族对奶牛乳腺上皮细胞泌乳调控机制的研究[D].哈尔滨:东北农业大学,2015.
[40]Watson C J,Burdon T G.Prolactin signal transduction mechanisms in the mammary gland:The role of the Jak/STAT pathway[J].Reviews of Reproduction,1996,1(1):1-5.
[41]Freeman M E,Kanyicska B,Lerant A,et al.Prolactin:Structure,function,and regulation of secretion[J].Physiological Reviews,2000,80(4):1523-1631.
[2]Ahmad S,Gaucher I,Rousseau F,et al.Effects of acidification on physico-chemical characteristics of buffalo milk:A comparison with cow’s milk[J].Food Chemistry,2008,106(1):11-17.
[3]唐善生.广西奶水牛业发展现状与思考[J].广西农学报,2014,29(1):50-52.
[4]简保权,秦学敏,龚芳.世界水牛奶业发展现状和典型模式分析[J].世界农业,2015,3:115-118.
[5]王锦.水牛乳腺基因表达谱与生长激素转基因水牛的初步研究[D].南宁:广西大学,2013.
[6]Bartel D P.microRNAs:Genomics,biogenesis,mechanism,and function[J].Cell,2004,116(2):281-297.
[7]Ambros V.The functions of animal microRNAs[J].Nature,2004,431(7006):350-355.
[8]Kim V N.microRNA biogenesis:Coordinated cropping and dicing[J].Nature Reviews Molecular Cell Biology,2005,6(5):376-385.
[9]赵静雯,陈国荣,吴慧光.miRNA合成途径研究进展[J].中国畜牧兽医,2015,42(12):3186-3191.
[10]Wang K,Long B,Jiao J Q,et al.miRNA-484regulates mitochondrial network through targeting Fis1[J].Nature Communications,2012,3:781.
[11]Wightman B,Ha I,Ruvkun G.Posttranscriptional regulation of the heterochronic gene lin-14by lin-4mediates temporal pattern formation in C.elegans[J].Cell,1993,75(5):855-862.
[12]Weber J A,Baxter D H,Zhang S,et al.The microRNA spectrum in 12 body fluids[J].Clinical Chemistry,2010,56(11):1733-1741.
[13]Kosaka N,Izumi H,Sekine K,et al.microRNA as a new immune-regulatory agent in breast milk[J].Silence,2010,1(1):7.
[14]张抒,张强,张萌,等.miRNA对乳腺发育及泌乳调节作用的研究进展[J].中国畜牧兽医,2015,42(3):663-667.
[15]Grembergen O V.microRNAs regulate KDM5histone demethylases in breast cancer cells[J].Molecular Bio Systems,2016,12(2):404-413.
[16]Phan B,Majid S,Ursu S,et al.Tumor suppressor role of microRNA-1296 in triple-negative breast cancer[J].Oncotarget,2016,7(15):19519.
[17]Cochrane D R,Spoelstra N S,Richer J K.The role of miRNAs in progesterone action[J].Molecular and Cellular Endocrinology,2012,357(1):50-59.
[18]熊显荣,兰道亮,李键,等.牦牛卵巢小RNA高通量测序及生物信息学分析[J].畜牧兽医学报,2016,47(1):55-63.
[19]周华,张新,刘腾云,等.高通量转录组测序的数据分析与基因发掘[J].江西科学,2012,30(5):607-611.
[20]Morin R D,O’Connor M D,Griffith M,et al.Application of massively parallel sequencing to microRNA profiling and discovery in human embryonic stem cells[J].Genome Research,2008,18(4):610-621.
[21]Li Z,Liu H,Jin X,et al.Expression profiles of microRNAs from lactating and non-lactating bovine mammary glands and identification of miRNA related to lactation[J].BMC Genomics,2012,13(1):731.
[22]Huang J,Ju Z,Li Q,et al.Solexa sequencing of novel and differentially expressed microRNAs in testicular and ovarian tissues in Holstein cattle[J].Int J Biol Sci,2011,7(7):1016-1026.
[23]曾献存,贾斌,史洪才,等.利用Solexa测序技术鉴定策勒黑羊microRNA[J].中国畜牧兽医,2016,43(1):204-209.
[24]Elsik C G,Tellam R L,Worley K C.The genome sequence of taurine cattle:A window to ruminant biology and evolution[J].Science,2009,324(5926):522-528.
[25]Ro S,Park C,Jin J,et al.A PCR-based method for detection and quantification of small RNAs[J].Biochemical and Biophysical Research Communications,2006,351(3):756-763.
[26]Larionov A,Krause A,Miller W.A standard curve based method for relative Real-time PCR data processing[J].BMC Bioinformatics,2005,6(1):62.
[27]Livak K J,Schmittgen T D.Analysis of relative gene expression data using Real-time quantitative PCR and the 2-ΔΔCt method[J].Methods,2001,25(4):402-408.
[28]Pfaffl M W.A new mathematical model for relative quantification in Real-time RT-PCR[J].Nucleic Acids Research,2001,29(9):e45.
[29]Ibarra I,Erlich Y,Muthuswamy S K,et al.A role for microRNAs in maintenance of mouse mammary epithelial progenitor cells[J].Genes&Development,2007,21(24):3238-3243.
[30]金晓露,杨建香,李真,等.乳腺发育及泌乳相关miRNA研究进展[J].遗传,2013,35(6):695-702.
[31]陆黎敏,李庆章,王春梅,等.miR-221对小鼠乳腺上皮细胞增殖和泌乳功能的影响[J].中国生物化学与分子生物学报,2009,5:454-458.
[32]Lin X,Luo J,Zhang L,et al.miRNA-103controls milk fat accumulation in goat(Capra hircus)mammary gland during lactation[J].PLoS One,2013,8(11):e79258.
[33]Zhang L,Volinia S,Bonome T,et al.Genomic and epigenetic alterations deregulate microRNA expression in human epithelial ovarian cancer[J].Proceedings of the National Academy of Sciences,2008,105(19):7004-7009.
[34]Guo Y,Chen Z,Zhang L,et al.Distinctive microRNA profiles relating to patient survival in esophageal squamous cell carcinoma[J].Cancer Research,2008,68(1):26-33.
[35]Tanaka T,Haneda S,Imakawa K,et al.A microRNA,miRNA-101a,controls mammary gland development by regulating cyclooxygenase-2expression[J].Differentiation,2009,77(2):181-187.
[36]Liu C H,Chang S H,Narko K,et al.Overexpression of cyclooxygenase-2is sufficient to induce tumorigenesis in transgenic mice[J].Journal of Biological Chemistry,2001,276(21):18563-18569.
[37]Lu S,Yu G,Zhu Y,et al.Cyclooxygenase-2overexpression in MCF-10Fhuman breast epithelial cells inhibits proliferation,apoptosis and differentiation,and causes partial transformation[J].International Journal of Cancer,2005,116(6):847-852.
[38]Wu Z,Huang X,Huang X,et al.The inhibitory role ofmiR-29in growth of breast cancer cells[J].Journal of Experimental&Clinical Cancer Research,2013,32(1):98.
[39]边艳杰.miRNA-29家族对奶牛乳腺上皮细胞泌乳调控机制的研究[D].哈尔滨:东北农业大学,2015.
[40]Watson C J,Burdon T G.Prolactin signal transduction mechanisms in the mammary gland:The role of the Jak/STAT pathway[J].Reviews of Reproduction,1996,1(1):1-5.
[41]Freeman M E,Kanyicska B,Lerant A,et al.Prolactin:Structure,function,and regulation of secretion[J].Physiological Reviews,2000,80(4):1523-1631.