牦牛Linc24063的克隆鉴定及其与miRNAs表达水平的相关性分析
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摘要
【目的】通过对牦牛长链非编码RNA Linc24063进行克隆鉴定,分析其在乳腺组织中与miRNAs表达量的相关性,为Linc24063通过调控miRNA发挥功能的调控机制研究提供试验依据。【方法】选取4.5岁处于第一泌乳期的健康母牦牛12头,清晨空腹屠宰后,迅速采集大脑、乳腺、肾脏、心脏、肝脏和卵巢组织,提取组织总RNA,利用5′RACE和3′RACE方法克隆牦牛Linc24063序列;利用生物信息学对其序列保守性、染色体定位及编码潜能进行分析,然后利用原核表达试验对其编码能力进行验证;利用RT-qPCR检测其在大脑、乳腺、肾脏、心脏、肝脏和卵巢的表达水平。利用普通牛和绵羊的miRNA数据库,结合miRanda和mireap软件预测与Linc24063具有相互作用的物种间保守miRNAs,与前人研究表明在牛不同泌乳时期乳腺组织中具有差异表达的miRNA取交集,然后对其靶基因进行GO富集和KEGG信号通路分析;最后使用皮尔逊相关系数在乳腺组织中进行Linc24063与miRNAs表达量的相关性分析。【结果】Linc24063 5′RACE和3′RACE片段大小分别为476 bp和356 bp,测序分析表明Linc24063大小为758 bp,位于牦牛21号染色体的Dlk1-Dio3印记域,与普通牛的序列保守性最高。生物信息学预测其编码潜能较低,原核表达试验进一步验证Linc24063不能有效的翻译蛋白,表明Linc24063是一个真正的长链非编码lncRNA。组织表达谱分析表明,Linc24063在牦牛乳腺组织中表达量最高,在肝脏和卵巢中表达量较低。生物信息学分析后共筛选到与Linc24063具有相互作用的物种间保守miRNAs 21个,其中有研究报道在乳腺中有差异表达的13个;13个miRNAs靶基因富集到TGF-β、PI3K-Akt、胰岛素等信号通路中,表明Linc24063可能通过这些信号通路参与牦牛乳脂和乳蛋白的生物合成过程。在乳腺组织中,Linc24063表达水平与miR-200a(P=0.001)、miR-141(P=0.02)显著负相关,与miR-27a(P=0.023)显著正相关,与miR-24(P=0.601)不具备相关性。【结论】Linc24063位于Dlk1-Dio3印记域内,在乳腺组织中具有较高表达量,且可能通过与miR-200a、miR-141和miR-27a相互作用从而参与牦牛乳脂和乳蛋白的生物合成过程。为深入探讨牦牛Linc24063在乳脂和乳蛋白合成中的生物学功能提供了基础数据。
【Objective】The aim of this study was to clone and to identify the protein coding potential of the long-chain non-coding RNA Linc24063 in yak,and then to analyze its correlation with the expression of miRNAs in mammary gland,which might provide basis for studying the function of Linc24063 in yak.【Method】Twelve healthy female yaks at 4.5-year old in the first lactation period were selected as experiment animal.After fasting slaughter,the tissue samples of cerebrum,mammary gland,kidney,heart,liver and ovary were collected for total RNA extraction.Firstly,the sequence of Linc24063 was cloned by 5'RACE and 3RACE,and the sequence conservation,chromosomal location and coding potential were analyzed by bioinformatics,then the prokaryotic expression assays were used to analyze its protein coding ability.Its expression levels in cerebrum,mammary gland,kidney,heart,liver and ovary tissue were determined by using RT-qPCR.Secondly,the conserved miRNAs interacting with Linc24063 were predicted by using the miRNA database of cattle and sheep,combined with miRanda and mireap software.The same miRNAs were obtained from the published differentially expressed miRNAs during different stages of lactation in cattle and the conserved miRNAs,and then GO enrichment and KEGG pathway analysis were performed on their target genes.Finally,Pearson correlation coefficient was used for analyzing the correlation between the expression of Linc24063 and miRNAs in the mammary gland of yak.【Result】The length of Linc24063 5'RACE and 3'RACE fragments were 476 bp and 356 bp,respectively.Sequencing analysis showed that Linc24063 was 758 bp in length and located in the Dlk1-Dio3 imprinting domain of 21 chromosomes in yak.Bioinformatics predicts results showed that its coding potential was lower,and prokaryotic expression experiments further demonstrated that Linc24063had no protein coding ability,suggesting that Linc24063 was a real lncRNA.Tissue expression profiling showed that Linc24063 had highest expression in mammary gland and lowest expression in liver and ovary.After bioinformatics analysis,Twenty-one conserved miRNAs interacting with Linc24063 were screened,among which 13 miRNA were differentially expressed in the mammary gland reported previously.The 13 miRNAs targets were enriched in TGF-β,PI3K-Akt,insulin and other signaling pathways,suggesting that Linc24063 might participate in the biosynthesis of milk fat and protein via these signaling pathways.In mammary gland tissue the expression of Linc24063 was significantly negatively correlated with miR-200a(P=0.001)and miR-141(P=0.02),and significantly positively correlated with miR-27a(P=0.023),but no correlation with miR-24(P=0.601).【Conclusion】The results showed that Linc24063 was located in the Dlk1-Dio3 imprinting domain and expressed higher in mammary gland,and might play an important roles in the biosynthesis of milk fat and protein via miR-200a,miR-141 and miR-27a,which would be beneficial for revealing the regulatory mechanism of Linc24063 in milk fat and protein synthesis.
【Objective】The aim of this study was to clone and to identify the protein coding potential of the long-chain non-coding RNA Linc24063 in yak,and then to analyze its correlation with the expression of miRNAs in mammary gland,which might provide basis for studying the function of Linc24063 in yak.【Method】Twelve healthy female yaks at 4.5-year old in the first lactation period were selected as experiment animal.After fasting slaughter,the tissue samples of cerebrum,mammary gland,kidney,heart,liver and ovary were collected for total RNA extraction.Firstly,the sequence of Linc24063 was cloned by 5'RACE and 3RACE,and the sequence conservation,chromosomal location and coding potential were analyzed by bioinformatics,then the prokaryotic expression assays were used to analyze its protein coding ability.Its expression levels in cerebrum,mammary gland,kidney,heart,liver and ovary tissue were determined by using RT-qPCR.Secondly,the conserved miRNAs interacting with Linc24063 were predicted by using the miRNA database of cattle and sheep,combined with miRanda and mireap software.The same miRNAs were obtained from the published differentially expressed miRNAs during different stages of lactation in cattle and the conserved miRNAs,and then GO enrichment and KEGG pathway analysis were performed on their target genes.Finally,Pearson correlation coefficient was used for analyzing the correlation between the expression of Linc24063 and miRNAs in the mammary gland of yak.【Result】The length of Linc24063 5'RACE and 3'RACE fragments were 476 bp and 356 bp,respectively.Sequencing analysis showed that Linc24063 was 758 bp in length and located in the Dlk1-Dio3 imprinting domain of 21 chromosomes in yak.Bioinformatics predicts results showed that its coding potential was lower,and prokaryotic expression experiments further demonstrated that Linc24063had no protein coding ability,suggesting that Linc24063 was a real lncRNA.Tissue expression profiling showed that Linc24063 had highest expression in mammary gland and lowest expression in liver and ovary.After bioinformatics analysis,Twenty-one conserved miRNAs interacting with Linc24063 were screened,among which 13 miRNA were differentially expressed in the mammary gland reported previously.The 13 miRNAs targets were enriched in TGF-β,PI3K-Akt,insulin and other signaling pathways,suggesting that Linc24063 might participate in the biosynthesis of milk fat and protein via these signaling pathways.In mammary gland tissue the expression of Linc24063 was significantly negatively correlated with miR-200a(P=0.001)and miR-141(P=0.02),and significantly positively correlated with miR-27a(P=0.023),but no correlation with miR-24(P=0.601).【Conclusion】The results showed that Linc24063 was located in the Dlk1-Dio3 imprinting domain and expressed higher in mammary gland,and might play an important roles in the biosynthesis of milk fat and protein via miR-200a,miR-141 and miR-27a,which would be beneficial for revealing the regulatory mechanism of Linc24063 in milk fat and protein synthesis.
引文
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[17]HAN Z,LIU Q,HUANG Z,CUI W,TIAN Y,YAN W,WU Q.Expression and imprinting analysis of AK044800,a transcript from the Dlk1-Dio3 imprinted gene cluster during mouse embryogenesis.Molecules and Cells,2013,35(4):285-290.
[18]杨文志,张明月,王冠楠,赵宇鹏,张巍巍,李世杰.2个印记的基因间lncRNAs位于牛Dlk1-Dio3印记区域.畜牧兽医学报,2016,47(9):1848-1852.YANG W Z,ZHANG M Y,WANG G N,ZHAO Y P,ZHANG W W,LI S J.Two imprinted long non-coding RNAs located in cattle dlk1-dio3 domain.Acta Veterinaria Et Zootechnica Sinica,2016,47(9):1848-1852.(in Chinese)
[19]ZHANG M,ZHAO Y,WANG G,LI D,CHEN W,ZHANG C,L S.An imprinted long noncoding RNA located between genes Meg8 and Meg9 in the cattle Dlk1-Dio3 domain.Genetica,2017,145(1):1-7.
[20]LI M,SUN X,CAI H,SUN Y,PLATH M,LI C,LAN X,LEI C,LINF,BAI Y,CHEN H.Long non-coding RNA ADNCR suppresses adipogenic differentiation by targeting miR-204.Biochimcia et Biophysica Acta(BBA)-Gene Regulatory Mechanisms,2016,1859(7):871-882.
[21]WANG H,SHI H,LUO J,YI Y,YAO D,ZHANG X,MA G,LOOR JJ.Mir-145 regulates lipogenesis in goat mammary cells via targeting insig1 and epigenetic regulation of lipid-related genes.Journal of Cellular Physiology,2017,232(5):1030-1040.
[22]CHEN C,RIDZON D A,BROOMER A J,ZHOU Z,LEE D,NGUYEN J,BARBISIN M,XU N,MAHUVAKAR V,ANDERSENM,LAO K,LIVAK K,GUEGLER K.Real-time quantification of microRNAs by stem-loop RT-PCR.Nucleic Acids Research,2005,33(20):e179.
[23]LI Z,LIU H,JIN X,LO L,LIU J.Expression profiles of microRNAs from lactating and non-lactating bovine mammary glands and identification of miRNA related to lactation.BMC Genomics,2012,13(1):731.
[24]CAI X,LIU Q,ZHANG X,REN Y,LEI X,LI S,CHEN Q,DENG K,WANG P,ZHANG H,SHI D.Identification and analysis of the expression of microRNA from lactating and nonlactating mammary glands of the Chinese swamp buffalo.Journal of Dairy Science,2017,100(3):1971-1986.
[25]ROCHA S T D,EDWARDS C A,ITO M,OGATA T,FERGUSON-SMITH A C.Genomic imprinting at the mammalian Dlk1-Dio3domain.Trends in Genetics,2008,24(6):306-316.
[26]WAKEFIELD L M,PIEK E,B?TTINGER E P.TGF-βSignaling in Mammary Gland Development and Tumorigenesis.Journal of Mammary Gland Biology and Neoplasia,2001,6(1):67-82.
[27]R?DLER P D,WEHDE B L,WAGNER K U.Crosstalk between STAT5 activation and PI3K/AKT functions in normal and transformed mammary epithelial cells.Molecular and Cellular Endocrinology,2017,451:31-39.
[28]ZHANG T,HUANG J,YI Y,ZHANG X,JUAN L L,CAO Y,SHI H,LUO J.Akt Serine/Threonine Kinase 1 Regulates De Novo Fatty Acid Synthesis through m TOR/SREBP1 Axis in Dairy Goat Mammary Epithelial Cells.Journal of Agricultural and Food Chemistry,2018,66(5):1197-1205.
[29]NAGAOKA K,ZHANG H,WATANABE G,TAYA K.Epithelial Cell Differentiation Regulated by MicroRNA-200a in Mammary Glands.Plos One,2013,8(6):e65127.
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[2]WOOD A J,OAKEY R J.Genomic Imprinting in Mammals:Emerging Themes and Established Theories.PloS Genetics,2006,2(11):e147.
[3]SLEUTELS F,DENISE P B.The origins of genomic imprinting in mammals.Advances in genetics,2002,46:119-164.
[4]SAITO T,HARA S,KATO T,TAMANO M,MURAMATSU A,ASAHARA H,TAKADA S.A tandem repeat array in IG-DMR is essential for imprinting of paternal allele at the Dlk1-Dio3 domain during embryonic development.Human Molecular Genetics,2018,27(18):3283-3292.
[5]ENTERINA J R,KSS E,ANDERSON C,MARSHALL E A,NG K W,LAM W L.DLK1-DIO3 imprinted locus deregulation in development,respiratory disease,and cancer.Expert Review of Respiratory Medicine,2017,11(9):749-761.
[6]BENETATOS L,VARTHOLOMATOS G,HATZIMICHAEL E.DLK1-DIO3 imprinted cluster in induced pluripotency:landscape in the mist.Cellular and Molecular Life Science,2014,71(22):4421-4430.
[7]WüST S,DR?SE S,HEIDLER J,WITTIG I,KLOCKNER I,FRANKO A,BONKE E,GüNTHER S,G?RTNER U,BOETTGERT,BRAUN T.Metabolic Maturation during Muscle Stem Cell Differentiation Is Achieved by miR-1/133a-Mediated Inhibition of the Dlk1-Dio3 Mega Gene Cluster.Cell Metabolism,2018,27(5):1026-1039.
[8]CHARLIER C,SEGERS K,WAGENAAR D,KARIM L,BERGHMANS S,JAILLON O,SHAY T,WEISSENBACH J,COCKETT N,GYAPAY G,GEORGES M.Human-Ovine Comparative Sequencing of a 250-kb Imprinted Domain Encompassing the Callipyge(clpg)Locus and Identification of Six Imprinted Transcripts:DLK1,DAT,GTL2,PEG11,antiPEG11,and MEG8.Genome Research,2001,11(5):850-862.
[9]LIU L,LUO G Z,YANG W,ZHAO X Y,ZHENG Q Y,LV Z,LI W,WU H J,WANG L,WANG X J,ZHOU Q.Activation of the Imprinted Dlk1-Dio3 region correlates with pluripotency levels of mouse stem cells.Journal of Biological Chemistry,2010,285(25):19483-19890.
[10]STADTFELD M,APOSTOLOU E,FERRARI F,CHOI J,WALSH RM,CHEN T,OOI S,KIM S Y,BESTOR T H,SHIODA T,PARK P J,HOCHEDLINGER K.Ascorbic acid prevents loss of Dlk1-Dio3imprinting and facilitates generation of all-iPS cell mice from terminally differentiated B cells.Nature Genetics,2012,44(4):398-405.
[11]ZHONG Z,YE Y,GUO W,HE Y,HU W.Relationship between DLK1gene promoter region DNA methylation and non-small cell lung cancer biological behavior.Oncology Letters,2017,13(6):4123-4126.
[12]吴昊,张运海,凌英会.非编码RNA在胚胎发育过程中的作用.畜牧兽医学报,2017,48(1):1-4.WU H,ZHANG Y H,LING H Y.The progress of recent advances in the ncrna-mediated regulation during embryogenesis.Acta Veterinaria Et Zootechnica Sinica,2017,48(1):1-4.(in Chinese)
[13]李燕,陈明明,张俊星,张林林,李新,郭宏,丁向彬,刘新峰.牛LncRNA-133a对骨骼肌卫星细胞增殖分化的影响.中国农业科学,2019,52(1):143-153.LI Y,CHEN M M,ZHANG J X,ZHANG L L,LI X,GUO H,DINGX B,LIU X F.Effects of bovine LncRNA-133a on the proliferation and differentiation of skeletal muscle satellite cells.Scientia Agricultura Sinica,2019,52(1):143-153.(in Chinese)
[14]陈瑞,于帅,陈晓旭,杜健,朱振东,潘传英,曾文先.非编码RNA对哺乳动物精子发生过程的调控.中国农业科学,2017,50(2):380-390.CHEN R,YU S,CHEN X X,DU J,ZHU Z D,PAN C Y,ZENG W X.Regulatory role of noncoding RNAs during spermatogenesis.Scientia Agricultura Sinica,2017,50(2):380-390.(in Chinese)
[15]ZHU X,WU Y B,ZHOU J,KANG D M.Upregulation of lncRNAMEG3 promotes hepatic insulin resistance via increasing FoxO1expression.Biochemical and Biophysical Research Communications,2016,469(2):319-325.
[16]ZHANG F W,ZENG T B,HAN Z B,HE H J,CHEN Y,GU N,JIANG H J,WU Q.Imprinting and expression analysis of a non-coding RNA gene in the mouse Dlk1-Dio3 domain.Journal of Molecular Histology,2011,42(4):333-339.
[17]HAN Z,LIU Q,HUANG Z,CUI W,TIAN Y,YAN W,WU Q.Expression and imprinting analysis of AK044800,a transcript from the Dlk1-Dio3 imprinted gene cluster during mouse embryogenesis.Molecules and Cells,2013,35(4):285-290.
[18]杨文志,张明月,王冠楠,赵宇鹏,张巍巍,李世杰.2个印记的基因间lncRNAs位于牛Dlk1-Dio3印记区域.畜牧兽医学报,2016,47(9):1848-1852.YANG W Z,ZHANG M Y,WANG G N,ZHAO Y P,ZHANG W W,LI S J.Two imprinted long non-coding RNAs located in cattle dlk1-dio3 domain.Acta Veterinaria Et Zootechnica Sinica,2016,47(9):1848-1852.(in Chinese)
[19]ZHANG M,ZHAO Y,WANG G,LI D,CHEN W,ZHANG C,L S.An imprinted long noncoding RNA located between genes Meg8 and Meg9 in the cattle Dlk1-Dio3 domain.Genetica,2017,145(1):1-7.
[20]LI M,SUN X,CAI H,SUN Y,PLATH M,LI C,LAN X,LEI C,LINF,BAI Y,CHEN H.Long non-coding RNA ADNCR suppresses adipogenic differentiation by targeting miR-204.Biochimcia et Biophysica Acta(BBA)-Gene Regulatory Mechanisms,2016,1859(7):871-882.
[21]WANG H,SHI H,LUO J,YI Y,YAO D,ZHANG X,MA G,LOOR JJ.Mir-145 regulates lipogenesis in goat mammary cells via targeting insig1 and epigenetic regulation of lipid-related genes.Journal of Cellular Physiology,2017,232(5):1030-1040.
[22]CHEN C,RIDZON D A,BROOMER A J,ZHOU Z,LEE D,NGUYEN J,BARBISIN M,XU N,MAHUVAKAR V,ANDERSENM,LAO K,LIVAK K,GUEGLER K.Real-time quantification of microRNAs by stem-loop RT-PCR.Nucleic Acids Research,2005,33(20):e179.
[23]LI Z,LIU H,JIN X,LO L,LIU J.Expression profiles of microRNAs from lactating and non-lactating bovine mammary glands and identification of miRNA related to lactation.BMC Genomics,2012,13(1):731.
[24]CAI X,LIU Q,ZHANG X,REN Y,LEI X,LI S,CHEN Q,DENG K,WANG P,ZHANG H,SHI D.Identification and analysis of the expression of microRNA from lactating and nonlactating mammary glands of the Chinese swamp buffalo.Journal of Dairy Science,2017,100(3):1971-1986.
[25]ROCHA S T D,EDWARDS C A,ITO M,OGATA T,FERGUSON-SMITH A C.Genomic imprinting at the mammalian Dlk1-Dio3domain.Trends in Genetics,2008,24(6):306-316.
[26]WAKEFIELD L M,PIEK E,B?TTINGER E P.TGF-βSignaling in Mammary Gland Development and Tumorigenesis.Journal of Mammary Gland Biology and Neoplasia,2001,6(1):67-82.
[27]R?DLER P D,WEHDE B L,WAGNER K U.Crosstalk between STAT5 activation and PI3K/AKT functions in normal and transformed mammary epithelial cells.Molecular and Cellular Endocrinology,2017,451:31-39.
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