鸡骨骼肌中天然反义lncRNA VGLL2-AS的鉴定及其与VGLL2的关系研究

doi:10.11843/j.issn.0366-6964.2023.01.012

摘要/Abstract

摘要： 旨在探究鸡骨骼肌中VGLL2基因天然反义链转录本VGLL2 AS lncRNA(VGLL2-AS)与VGLL2的关系。本研究首先采用PCR和测序技术验证VGLL2-AS是否存在；之后分别采集不同周龄固始蛋鸡(1日龄、6周龄、16周龄、22周龄、30周龄，每个周龄各6只)组织样,采用荧光定量PCR分析VGLL2基因和其反义链转录本VGLL2-AS的表达谱；采用双向转录试验和核酸酶保护试验检测VGLL2和VGLL2-AS是否可以双向转录且两者之间关系；体外分离培养原代成肌细胞(11胚龄固始鸡胚)，然后用2μg·mL^-1的放线菌素D处理成肌细胞(对照组不做处理)，收取处理不同时间点细胞(0~8h，每组各个时间点各做3个重复)，检测VGLL2-AS与VGLL2的半衰期；分离鸡成肌细胞的细胞核和细胞质，通过RT-qPCR方法确定两者的细胞定位；利用PCR扩增及测序对VGLL2的转录本进行验证；最后利用RT-qPCR检测它们在固始蛋鸡胸肌组织(1日龄、6周龄、16周龄、22周龄、30周龄每个周龄各6只)中的时空表达规律和相关性。结果表明，VGLL2-AS在鸡的转录组中真实存在；VGLL2与VGLL2-AS均在肌肉中特异高表达(P<0.05);VGLL2-AS与VGLL2可以进行双向转录且二者之间可以形成双链RNA；VGLL2-AS半衰期较VGLL2长；在成肌细胞中，VGLL2-AS和VGLL2主要定位于细胞质中(P<0.001)；VGLL2只存在VGLL2-mRNA、VGLL2-X2和VGLL2-X3转录本；VGLL2-mRNA和VGLL2-X3与VGLL2-AS表达趋势一致，且VGLL2-mRNA、X3和VGLL2-AS的表达呈现极强的正相关(r分别为0.943和0.935，P<0.01)。综上所述，VGLL2-AS作为VGLL2的反义链编码的lncRNA定位于细胞质中，可能通过与VGLL2形成的双链RNA之间的相互作用，然后参与调节VGLL2的表达并维持其稳定性，最终在鸡的早期肌肉发育中发挥重要作用。本研究的结果扩展了鸡中关于NATs的研究，并为鸡VGLL2基因与其天然反义链转录本VGLL2-AS在鸡骨骼肌发育中的生物学功能的研究奠定了基础，对于提高禽类的生长发育具有一定的意义。

关键词: NATs, VGLL2-AS, VGLL2基因, 骨骼肌

Abstract: The purpose of the study was to explore the relationship between VGLL2-AS and VGLL2 in chicken skeletal muscle. PCR and sequencing techniques were used to verify the existence of VGLL2-AS; The tissue samples of Gushi layers at different weeks of age (1 day old, 6 weeks old, 16 weeks old, 22 weeks old and 30 weeks old, 6 for per week-old) were collected respectively, and the expression profiles of VGLL2 gene and its antisense chain transcript VGLL2-AS were analyzed by real-time quantitative PCR. Bidirectional transcription test and nuclease protection test were used to detect whether VGLL2 and VGLL2-AS could be transcribed in two directions and the relationship between them. Then, the primary myoblasts of 11 embryo-old Gushi chicken embryos were isolated and cultured in vitro, and then treated with 2 μg·mL^-1 actinomycin D (the control group was not treated), and the cells were collected at different time points (0-8 h, 3 repeats in each time point). The half-life of VGLL2-AS and VGLL2 were measured. The nucleus and cytoplasm of chicken myoblasts were isolated, and their cellular localization was determined by real-time quantitative PCR. The transcripts of VGLL2 were verified by PCR amplification and sequencing; Finally, RT-qPCR was used to detect their spatiotemporal expression and correlation in the breast muscle tissues of Gushi layers (6 birds at 1 day, 6 weeks, 16 weeks, 22 weeks and 30 weeks, respectively). The results showed that VGLL2-AS existed in the chicken transcriptome. VGLL2 and VGLL2-AS were highly expressed in muscle (P<0.05). VGLL2-AS and VGLL2 could conduct bidirectional transcription and form double stranded RNA between them. VGLL2-AS had a longer half-life than VGLL2; In myoblasts, VGLL2-AS and VGLL2 were mainly located in the cytoplasm (P<0.001). VGLL2 only contained VGLL2-mRNA, VGLL2-X2 and VGLL2-X3 transcripts. The expression trend of VGLL2-mRNA and VGLL2-X3 was consistent with that of VGLL2-AS, and the expression of VGLL2-mRNA, VGLL2-X3 and VGLL2-AS showed a strong positive correlation (r=0.943 and 0.935, respectively, P<0.01). To sum up, VGLL2-AS, as the antisense chain encoding lncRNA of VGLL2, is located in the cytoplasm, possibly through the interaction with the double stranded RNA formed by VGLL2, and then participates in regulating the expression of VGLL2 and maintaining its stability, finally playing an important role in the early muscle development of chickens. The results of this study expand the research on NATs in chickens, and lay a foundation for the study of biological function of chicken VGLL2 gene and its natural antisense chain transcript VGLL2-AS in chicken skeletal muscle development, which has certain significance for improving the growth and development of poultry.

Key words: NATs, VGLL2-AS, VGLL2 gene, skeletal muscle

中图分类号:

S831.2

李文雅, 牛欣然, 任团辉, 蔡含芳, 韩瑞丽, 田亚东, 刘小军, 康相涛, 李转见. 鸡骨骼肌中天然反义lncRNA VGLL2-AS的鉴定及其与VGLL2的关系研究[J]. 畜牧兽医学报, 2023, 54(1): 122-132.

LI Wenya, NIU Xinran, REN Tuanhui, CAI Hanfang, HAN Ruili, TIAN Yadong, LIU Xiaojun, KANG Xiangtao, LI Zhuanjian. Identification of Natural Antisense lncRNA VGLL2-AS in Chicken Skeletal Muscle and Its Relationship with VGLL2[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(1): 122-132.

参考文献 42

[1]	CHEN J J,SUN M,KENT W J,et al.Over 20% of human transcripts might form sense-antisense pairs[J].Nucleic Acids Res,2004,32(16):4812-4820.
[2]	CABILI M N,TRAPNELL C,GOFF L,et al.Integrative annotation of human large intergenic noncoding RNAs reveals global properties and specific subclasses[J].Genes Dev,2011,25(18):1915-1927.
[3]	MIHOLA O,FOREJT J,TRACHTULEC Z.Conserved alternative and antisense transcripts at the programmed cell death 2 locus[J].BMC Genomics,2007,8:20.
[4]	FENG Y,ZHANG Q,WANG J,et al.Increased lncRNA AFAP1-AS1 expression predicts poor prognosis and promotes malignant phenotypes in gastric cancer[J].Eur Rev Med Pharmacol Sci,2017,21(17):3842-3849.
[5]	ZHANG C L,ZHU K P,MA X L.Antisense lncRNA FOXC2-AS1 promotes doxorubicin resistance in osteosarcoma by increasing the expression of FOXC2[J].Cancer Lett,2017,396:66-75.
[6]	WATANABE T,TOTOKI Y,TOYODA A,et al.Endogenous siRNAs from naturally formed dsRNAs regulate transcripts in mouse oocytes[J].Nature,2008,453(7194):539-543.
[7]	WANG Y Y,XUE S Y,LIU X R,et al.Analyses of long non-coding RNA and mRNA profiling using RNA sequencing during the pre-implantation phases in pig endometrium[J].Sci Rep,2016,6(1):20238.
[8]	WANG Z,XU H D,LI T,et al.Chicken GHR antisense transcript regulates its sense transcript in hepatocytes[J]. Gene, 2019,682:101-110.
[9]	MAEDA T,CHAPMAN D L,STEWART A F R.Mammalian Vestigial-like 2,a cofactor of TEF-1 and MEF2 transcription factors that promotes skeletal muscle differentiation[J].J Biol Chem,2002,277(50):48889-48898.
[10]	MIELCAREK M,GVNTHER S,KRVGER M,et al.VITO-1,a novel vestigial related protein is predominantly expressed in the skeletal muscle lineage[J].Mech Dev,2002,119 Suppl 1:S269-S274.
[11]	任团辉.天然反义转录本VGLL2-AS在鸡肌肉发育中的作用解析[D].郑州:河南农业大学,2018.REN T H.Effect analysis of natural antisense transcript VGLL2-AS during chicken muscle development[D].Zhengzhou:Henan Agricultural University,2018.(in Chinese)
[12]	戴巍,宋瑞龙,张远浩,等.鸡骨骼肌卫星细胞的分离培养与鉴定[J].畜牧兽医学报,2021,52(3):676-682.DAI W,SONG R L,ZHANG Y H,et al.Isolation,culture and identification of muscle satellite cells of chicken[J].Acta Veterinaria et Zootechnica Sinica,2021,52(3):676-682.(in Chinese)
[13]	YU W Q,GIUS D,ONYANGO P,et al.Epigenetic silencing of tumour suppressor gene p15 by its antisense RNA[J].Nature, 2008,451(7175):202-206.
[14]	MAO Y,XU J,WANG Q,et al.A natural antisense transcript acts as a negative regulator for the maize drought stress response gene ZmNAC48[J].J Exp Bot,2021,72(7):2790-2806.
[15]	CHENG H,ZHANG E S,SHI X,et al.A novel ATM antisense transcript ATM-AS positively regulates ATM expression in normal and breast cancer cells[J].Curr Med Sci,2022,42(4):681-691.
[16]	PARNIGONI A,CAON I,TEO W X,et al.The natural antisense transcript HAS2-AS1 regulates breast cancer cells aggressiveness independently from hyaluronan metabolism[J].Matrix Biol,2022,109:140-161.
[17]	YANG Y Y,SU Z J,SONG X H,et al.Enhancer RNA-driven looping enhances the transcription of the long noncoding RNA DHRS4-AS1,a controller of the DHRS4 gene cluster[J].Sci Rep,2016,6(1):20961.
[18]	YU W Q,GIUS D,ONYANGO P,et al.Epigenetic silencing of tumour suppressor gene p15 by its antisense RNA[J].Nature, 2008,451(7175):202-206.
[19]	ZÚÑIGA A,BORJA M,MEIJERS C,et al.Expression of alternatively spliced bFGF first coding exons and antisense mRNAs during chicken embryogenesis[J].Dev Biol,1993,157(1):110-118.
[20]	TAYLOR E R,SELEIRO E A P,BRICKELL P M.Identification of antisense transcripts of the chicken insulin-like growth factor-II gene[J].J Mol Endocrinol,1991,7(2):145-154.
[21]	FARRELL C M,LUKENS L N.Naturally occurring antisense transcripts are present in chick embryo chondrocytes simultaneously with the down-regulation of the α1(I) collagen gene[J].J Biol Chem,1995,270(7):3400-3408.
[22]	MIHOLA O,FOREJT J,TRACHTULEC Z.Conserved alternative and antisense transcripts at the programmed cell death 2 locus[J].BMC Genomics,2007,8:20.
[23]	SARODAYA N,TYAGI A,KIM H J,et al.Deubiquitinase USP19 enhances phenylalanine hydroxylase protein stability and its enzymatic activity[J].Cell Biol Toxicol,2022,doi:10.1007/s10565-022-09719-z.
[24]	VAN DIJK E L,CHEN C L,D'AUBENTON-CARAFA Y,et al.XUTs are a class of Xrn1-sensitive antisense regulatory non-coding RNA in yeast[J].Nature,2011,475(7354):114-117.
[25]	CLARK M B,JOHNSTON R L,INOSTROZA-PONTA M,et al.Genome-wide analysis of long noncoding RNA stability[J]. Genome Res,2012,22(5):885-898.
[26]	王章.鸡GHR-AS与Let-7b在肝细胞中调控GHR-S的机制研究[D].湛江:广东海洋大学,2018.WANG Z.Chicken GHR-AS and Let-7b Regulate the GHR-S in hepatocytes[D].Zhanjiang:Guangdong Ocean University, 2018.(in Chinese)
[27]	CHEN L L.Linking long noncoding RNA localization and function[J].Trends Biochem Sci,2016,41(9):761-772.
[28]	孟珊,杨阳,李睿霄,等.lncRNA-6617调控猪肌内前体脂肪细胞分化的筛选与功能研究[J].畜牧兽医学报,2022,53(6):1712-1722.MENG S,YANG Y,LI R X,et al.Screening and functional study of lncRNA-6617 regulating porcine intramuscular preadipocytes differentiation[J].Acta Veterinaria et Zootechnica Sinica,2022,53(6):1712-1722.(in Chinese)
[29]	赵学亮,刘海金,萧飒,等.长链非编码RNA——病毒与宿主相互作用中的新型调控因子[J].畜牧兽医学报,2020, 51(9):2059-2067 ZHAO X L,LIU H J,XIAO S,et al.Long Non-coding RNAs:novel regulators of virus-host interactions[J].Acta Veterinaria et Zootechnica Sinica,2020,51(9):2059-2067.(in Chinese)
[30]	李倩倩,李龙,黄子莹,等.猪lncRNA TCONS_00791383对骨骼肌卫星细胞增殖分化的影响[J].畜牧兽医学报,2020, 51(6):1177-1186.LI Q Q,LI L,HUANG Z Y,et al.Effect of Pig lncRNA TCONS_00791383 on the proliferation and differentiation of skeletal muscle satellite cells[J].Acta Veterinaria et Zootechnica Sinica,2020,51(6):1177-1186.(in Chinese)
[31]	CESANA M,CACCHIARELLI D,LEGNINI I,et al.A long noncoding RNA controls muscle differentiation by functioning as a competing endogenous RNA[J].Cell,2011,147(2):358-369.
[32]	PENNY G D,KAY G F,SHEARDOWN S A,et al.Requirement for Xist in X chromosome inactivation[J].Nature,1996, 379(6561):131-137.
[33]	PELECHANO V,STEINMETZ L M.Gene regulation by antisense transcription[J].Nat Rev Genet,2013,14(12):880-893.
[34]	FAGHIHI M A,ZHANG M,HUANG J,et al.Evidence for natural antisense transcript-mediated inhibition of microRNA function[J].Genome Biol,2010,11(5):R56.
[35]	QUINN J J,CHANG H Y.Unique features of long non-coding RNA biogenesis and function[J].Nat Rev Genet,2016,17(1):47-62.
[36]	STAMM S,BEN-ARI S,RAFALSKA I,et al.Function of alternative splicing[J].Gene,2005,344:1-20.
[37]	王晶晶,张培培,郝海生,等.牛体内囊胚全转录组模式解析[J].畜牧兽医学报,2020,51(6):1271-1280.WANG J J,ZHANG P P,HAO H S,et al.Analysis of whole transcriptome pattern of bovine in vivo blastocyst[J].Acta Veterinaria et Zootechnica Sinica,2020,51(6):1271-1280.(in Chinese)
[38]	田威龙,兰干球,张龙超,等.北京黑猪DKK3和CCR1基因多态性检测及其与背膘厚的关联分析[J].畜牧兽医学报,2022, 53(7):2083-2093.TIAN W L,LAN G Q,ZHANG L C,et al.Detection of DKK3 and CCR1 genes polymorphisms and their association with backfat thickness in Beijing black pigs[J].Acta Veterinaria et Zootechnica Sinica,2022,53(7):2083-2093.(in Chinese)
[39]	崔迎迎,芒来,李蓓,等.MEI1基因可变剪切事件对蒙古马精子生成的调控作用[J].畜牧兽医学报,2022, 53(4):1096-1108.CUI Y Y,MANG L,LI B,et al.Regulatory role of MEI1 with alternative splicing event on spermatogenesis in mongolian horse[J].Acta Veterinaria et Zootechnica Sinica,2022,53(4):1096-1108.(in Chinese)
[40]	BELTRAN M,PUIG I,PEÑA C,et al.A natural antisense transcript regulates Zeb2/Sip1 gene expression during Snail1-induced epithelial-mesenchymal transition[J].Genes Dev,2008,22(6):756-769.
[41]	TRINKLEIN N D,ALDRED S F,HARTMAN S J,et al.An abundance of bidirectional promoters in the human genome[J]. Genome Res,2004,14(1):62-66.
[42]	CHEN Y Q,LI H,LI Y Y,et al.Pan-cancer analysis of head-to-head gene pairs in terms of transcriptional activity,Co-expression and regulation[J].Front Genet,2021,11:560997.