Scientia Agricultura Sinica ›› 2019, Vol. 52 ›› Issue (14): 2538-2547.doi: 10.3864/j.issn.0578-1752.2019.14.012

• ANIMAL SCIENCE·VETERINARY SCIENCE·RESOURCE INSECT • Previous Articles     Next Articles

Cloning and Identification of Long-Chain Non-Coding RNA Linc24063 and Its Correlation with the Expression Level of miRNAs in Yak

WANG Hui1,2,CHAI ZhiXin1,2,ZHU JiangJiang1,2,ZHONG JinCheng1,2(),ZHANG ChengFu3,Xin JinWei3   

  1. 1Key Laboratory of Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Utilization (Southwest Minzu University), Ministry of Education, Chengdu, 610041
    2Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Utilization Key Laboratory of Sichuan Province, Southwest Minzu University, Chengdu, 610041
    3State Key Laboratory of Barley and Yak Germplasm Resources and Genetic Improvement, Tibet Academy of Agricultural and Animal Husbandry Sciences(TAAAS), Lhasa 850002;
  • Received:2018-11-05 Accepted:2019-05-09 Online:2019-07-16 Published:2019-07-26
  • Contact: JinCheng ZHONG E-mail:zhongjincheng518@126.com

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Abstract:

【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 3' RACE, 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 Linc24063 had 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.

Key words: Bos grunniens, Linc24063, RACE, prokaryotic expression, milk fat, milk protein

Table 1

Specific primers used for RT-qPCR"

基因名称 Gene name 引物序列（5′-3′） Primer sequence (5′-3′) 产物大小 Product length (bp)
Linc24063 F:ACTGTGAACATAGATGGTGAGG 141
R:CACGGGGTCACAAAGAGTCA
RPS9 F:GTGGTGAACATCCCGTCCTT 130
R:GCCACTGCACCTTGTAACAC
UXT F:CGAGGCTTTCATCTCTGACG 138
R:TCCGAGTGATTAGCTTCCTGG
18S rRNA F:GTGGTGTTGAGGAAAGCAGACA 79
R:CGATCCCTGTCCTCACCTCATC
MiR-200a RT:GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACaacatcgt
F:CTGGtaacactgtctggta 70
MiR-24 RT:GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACctgttcct
F:GGAtggctcagttcagc 69
MiR-27a RT:GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACcggaactt
F:GGCacttcacagtggct 69
MiR-141 RT:GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACccatcttt
F:GGCataacactgtctggt 69
miRNA universal primer GTGCAGGGTCCGAGGT

Fig. 1

The 5′ Race and 3′ Race electrophoresis analysis (A) and the sequence (B) of Linc24063"

Table 2

The coding probability analysis of Linc24063, Meg9 and CSN2"

基因 Gene 编码/非编码 Coding/Noncoding 编码潜能 Coding probability Fickett 分 Fickett score 等电点 Isoelectric point
Linc24063 Noncoding 0.01 0.35 9.52
Meg9 Noncoding 0.17 0.36 7.90
CSN2 coding 1.00 0.45 5.89

Fig. 2

The prokaryotic expression of Linc24063 (A) Identification of prokaryotic expression vector of Linc24063 and CSN2 by restriction endonuclease. (B) The prokaryotic expression of Linc24063 and CSN2"

Fig. 3

The expression of Linc24063 in tissues The lowercase a, b means significant difference, P<0.05"

Table 3

The conserved miRNAs interacting with Linc24063"

Name 成熟体序列（5′-3′）Mature sequence(5′-3′)
bta-miR-137 UUAUUGCUUAAGAAUACGCGUAG
bta-miR-139 UCUACAGUGCACGUGUCUCCAGU
bta-miR-141 UAACACUGUCUGGUAAAGAUGG
bta-miR-154a UAGGUUAUCCGUGUAGCCUUCG
bta-miR-200a UAACACUGUCUGGUAACGAUGUU
bta-miR-221 AGCUACAUUGUCUGCUGGGUUU
bta-miR-223 UGUCAGUUUGUCAAAUACCCCA
bta-miR-24 UGGCUCAGUUCAGCAGGAACAG
bta-miR-27a AGGGCUUAGCUGCUUGUGAGCA
bta-miR-30b-5p UGUAAACAUCCUACACUCAGCU
bta-miR-369-3p AAUAAUACAUGGUUGAUCUUU
bta-miR-374a UUAUAAUACAACCUGAUAAGUG
bta-miR-376b AUCAUAGAGGAAAAUCCAUGUU
bta-miR-378b ACUGGACUUGGAGUCAGAAGGC
bta-miR-378c ACUGGACUUGGAGUCAGAAGU
bta-miR-455-3p GCAGUCCAUGGGCAUAUACACU
bta-miR-487a AAUCAUACAGGGACAUCCAGU
bta-miR-500 UAAUCCUUGCUACCUGGGUGAGA
bta-miR-543 AAACAUUCGCGGUGCACUUCUU
bta-miR-551a GCGACCCAAUCUUGGUUUCCA
bta-miR-655 AUAAUACAUGGUUAACCUCUCU

Table 4

The top ten GO terms of the conserved miRNAs interacting with Linc24063"

GO条目
Go Term		 基因数目
Gene count		 富集倍数
Fold enrichment		 P值
P value
Nucleus细胞核 201 1.63 7.98E-14
Cytoplasm细胞质 171 1.28 2.06E-04
Regulation of transcription from RNA polymerase II promoter
RNA聚合酶II启动子转录的调控		 116 1.98 4.76E-06
Nucleoplasm 核质 83 1.48 2.68E-04
Metal ion binding金属离子结合 71 1.42 0.00231
Cytosol胞液 60 1.37 0.011998
RNA polymerase II core promoter proximal region sequence-specific DNA binding
DNA特异性结合RNA聚合酶II核心启动子近区序列		 52 2.92 3.51E-08
Transcription, DNA-templated DNA为模板的转录 42 1.57 0.004197
Transcriptional activator activity, RNA polymerase II core promoter proximal region
RNA聚合酶II核心启动子近区转录激活活性		 33 3.05 2.15E-06
Sequence-specific binding序列特异性结合
Chromatin binding染色质结合 30 2.22 8.71E-05

Table 5

The KEGG pathway of the conserved miRNAs interacting with Linc24063"

信号通路条目 Pathway term 基因数 Input number P值 P-value 校正后P值 Corrected P-value
TGF-beta signaling pathway TGF-β信号通路 16 1.46E-06 8.22E-05
PI3K-Akt signaling pathway PI3K-Akt信号通路 28 0.000267 0.0063
Axon guidance 轴突导向 18 0.000392 0.00865
ErbB signaling pathway ErbB 信号通路 10 0.002087 0.03393
Chronic myeloid leukemia慢性粒细胞白血病 9 0.002965 0.04313
FoxO signaling pathway FoXO信号通路 13 0.003366 0.04738
Pathways in cancer 癌症通路 27 0.003372 0.04783
Insulin signaling pathway胰岛素信号 13 0.003495 0.04786
Maturity onset diabetes of the young糖尿病 5 0.003572 0.04789
MAPK signaling pathway MAPK信号通路 19 0.003611 0.04952

Fig. 4

The correlation between expression levels of Linc24063 and miRNAs in 12 mammary gland of yaks"

[1] SALMENA L, POLISENO L, TAY Y, KATS L, PANDOLFI P . A ceRNA hypothesis: the Rosetta stone of a hidden RNA language? Cell, 2011,146(3):353-358.
doi: 10.1016/j.cell.2011.07.014
[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.
doi: 10.1016/S0065-2660(02)46006-3
[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.
doi: 10.1093/hmg/ddy235
[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.
doi: 10.1080/17476348.2017.1355241
[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.
doi: 10.1007/s00018-014-1698-9
[7] WÜST S, DRÖSE S, HEIDLER J, WITTIG I, KLOCKNER I, FRANKO A, BONKE E, GÜNTHER S, GÄRTNER U, BOETTGER T, 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.
doi: 10.1016/j.cmet.2018.02.022
[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.
doi: 10.1101/gr.172701
[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.
doi: 10.1074/jbc.M110.131995
[10] STADTFELD M, APOSTOLOU E, FERRARI F, CHOI J, WALSH R M, CHEN T, OOI S, KIM S Y, BESTOR T H, SHIODA T, PARK P J, HOCHEDLINGER K . Ascorbic acid prevents loss of Dlk1-Dio3 imprinting and facilitates generation of all-iPS cell mice from terminally differentiated B cells. Nature Genetics, 2012,44(4):398-405.
doi: 10.1038/ng.1110
[11] ZHONG Z, YE Y, GUO W, HE Y, HU W . Relationship between DLK1 gene promoter region DNA methylation and non-small cell lung cancer biological behavior. Oncology Letters, 2017,13(6):4123-4126.
doi: 10.3892/ol.2017.6019
[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.
doi: 10.11843/j.issn.0366-6964.2017.01.001
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)
doi: 10.11843/j.issn.0366-6964.2017.01.001
[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, DING X 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 lncRNA MEG3 promotes hepatic insulin resistance via increasing FoxO1 expression. Biochemical and Biophysical Research Communications, 2016,469(2):319-325.
doi: 10.1016/j.bbrc.2015.11.048
[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.
doi: 10.1007/s10735-011-9337-3
[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.
doi: 10.1007/s10059-013-2275-z
[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.
doi: 10.11843/j.issn.0366-6964.2016.09.013
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)
doi: 10.11843/j.issn.0366-6964.2016.09.013
[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.
doi: 10.1007/s10709-016-9939-5
[20] LI M, SUN X, CAI H, SUN Y, PLATH M, LI C, LAN X, LEI C, LIN F, 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.
doi: 10.1016/j.bbagrm.2016.05.003
[21] WANG H, SHI H, LUO J, YI Y, YAO D, ZHANG X, MA G, LOOR J J . 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.
doi: 10.1002/jcp.v232.5
[22] CHEN C, RIDZON D A, BROOMER A J, ZHOU Z, LEE D, NGUYEN J, BARBISIN M, XU N, MAHUVAKAR V, ANDERSEN M, LAO K, LIVAK K, GUEGLER K . Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Research, 2005,33(20):e179.
doi: 10.1093/nar/gni178
[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.
doi: 10.1186/1471-2164-13-731
[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.
doi: 10.3168/jds.2016-11461
[25] ROCHA S T D, EDWARDS C A, ITO M, OGATA T, FERGUSON- SMITH A C . Genomic imprinting at the mammalian Dlk1-Dio3 domain. Trends in Genetics, 2008,24(6):306-316.
doi: 10.1016/j.tig.2008.03.011
[26] WAKEFIELD L M, PIEK E, BÖTTINGER E P . TGF-β Signaling in Mammary Gland Development and Tumorigenesis. Journal of Mammary Gland Biology and Neoplasia, 2001,6(1):67-82.
doi: 10.1023/A:1009568532177
[27] RÄDLER 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.
doi: 10.1016/j.mce.2017.04.025
[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 mTOR/SREBP1 Axis in Dairy Goat Mammary Epithelial Cells. Journal of Agricultural and Food Chemistry, 2018,66(5):1197-1205.
doi: 10.1021/acs.jafc.7b05305
[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.
doi: 10.1371/journal.pone.0065127
[30] 林先滋, 罗军, 张犁苹, 朱江江, 石恒波, 苟德明 . miR-200a对奶山羊乳腺上皮细胞乳脂合成相关基因mRNA表达的影响. 畜牧兽医学报, 2012,43(7):1028-1036.
LIN X Z, LUO J, ZHANG L P, ZHU J J, SHI H B, GOU D M . The Effect of miR-200a on Gene mRNA Expression Related to Milk Fat Synthesis in Dairy Goat Mammary Gland Epithelial Cells. Chinese Journal of Animal & Veterinary Sciences, 2012,43(7):1028-1036.(in Chinese)
[31] LIN X Z, LUO J, ZHANG L P, WANG W, SHI H B, ZHU J J . MiR-27a suppresses triglyceride accumulation and affects gene mRNA expression associated with fat metabolism in dairy goat mammary gland epithelial cells. Gene, 2013,521(1):15-23.
doi: 10.1016/j.gene.2013.03.050
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