影响山羊胎儿肌肉发育mRNA和lncRNA的鉴定与分析

doi:10.11843/j.issn.0366-6964.2023.03.013

摘要/Abstract

摘要： 旨在分析雷州山羊胎儿70、90及120天背最长肌转录组测序中的差异表达mRNAs和lncRNAs及其靶基因，对差异表达基因进行功能富集分析，挖掘影响雷州山羊胎儿骨骼肌发育的关键候选基因。本研究选用年龄约2.5～3.5岁，饲养状况相同，健康无病，体重接近的雷州母山羊，分为妊娠期70、90和120天3组，每组各3只共9只，提取其胎儿背最长肌的RNA构建文库后使用Illumina HiSeq^TM2500进行转录组测序，以Q<0.05为阈值筛选出差异mRNAs和lncRNAs并进行生物学功能富集分析及构建靶向关系网络。最后通过荧光定量PCR鉴定差异基因表达水平，验证测序结果的可靠性。结果发现，在M70 vs. M90组中，总共发现185个差异表达mRNAs，其中170个上调，15个下调，62个差异表达lncRNAs，40个上调，22个下调；在M120 vs. M90组中，共发现1 048个差异表达mRNAs，666个上调，382个下调，352个差异表达lncRNAs，175个上调，177个下调。其中TNNT3、TNNI1、TNNT1、TNNI2、ITGA4、ITGA11、ITGB4等基因参与肌肉发育相关的调控通路如肌钙蛋白复合物、肌丝、粘附斑、ECM-受体相互作用、PI3K-Akt信号通路等。根据lncRNA与mRNA的位置关系进行cis靶基因预测，总共筛选到37 464个靶基因，有10个差异表达mRNAs被差异表达lncRNA靶向，其中NDUFB2、BRAF、METTL7B和ITGA7参与粘着斑以及肌动蛋白细胞骨架的调控，METTL7B和ITGA7还参与PI3K-Akt信号通路，lncRNA TCONS_00214300靶向METTL7B和ITGA7。同时，8个差异表达mRNAs和lncRNAs的RT-PCR结果显示，8个基因的表达水平与转录组测序的表达趋势相似，说明测序结果可靠。结果显示，发现了影响雷州山羊胎儿肌肉发育的mRNA与lncRNA，发现了一些差异表达lncRNA和mRNA可能存在调控关系，对了解雷州山羊胎儿肌肉发育的分子调控机制提供了新的参考，也为雷州山羊分子选育提供了理论借鉴。

关键词: 雷州山羊, mRNA, lncRNA, 背最长肌

Abstract: The study aimed to examine the differentially expressed mRNAs and lncRNAs as well as their target genes in the transcriptome sequencing of Leizhou goat fetuses at 70, 90 and 120 days, and to investigate important candidate genes controlling the development of skeletal muscle in Leizhou goat fetuses, functional enrichment analysis of differentially expressed genes was carried out. Leizhou female goats were separated into 3 groups of 70, 90, and 120 days of gestation, with 3 animals in each group, for a total of 9 animals. The goats were about 2.5-3.5 years old, in the same feeding condition, healthy, and disease-free. The transcriptome was sequenced using Illumina HiSeq^TM2500 after the RNA of fetal longissimus dorsi was extracted in order to establish a library. The differential mRNAs and lncRNAs were screened at a threshold of Q<0.05, enriched for biological functions, and constructed into a target relationship network. In order to confirm the accuracy of the sequencing results, the expression levels of differential genes were lastly detected using fluorescence quantitative PCR. It was found that in the M70 vs. M90 group, a total of 185 differentially expressed mRNAs were identified, of which 170 were upregulated, 15 were downregulated, and 62 differentially expressed lncRNAs, 40 were upregulated, and 22 were downregulated. In the M120 vs. M90 group, we identified 1 048 differentially expressed mRNAs, 666 were upregulated, 382 were downregulated, 352 differentially expressed lncRNAs, 175 were upregulated, and 177 were downregulated. Among them, TNNT3, TNNI1, TNNT1, TNNI2, ITGA4, ITGA11, and ITGB4 genes were involved in regulatory pathways related to muscle development such as troponin complex, myofilament, adhesion plaque, ECM-receptor interaction, PI3K-Akt signaling pathways, etc. A total of 37 464 target genes were screened according to the positional relationship between lncRNA and mRNA for cis target gene prediction. Ten differentially expressed mRNAs were targeted by differentially expressed lncRNAs, among which NDUFB2, BRAF, METTL7B, and ITGA7 regulated adherent spots and the actin cytoskeleton, and METTL7B and ITGA7 were also engaged in PI3K-Akt signaling pathway. lncRNA TCONS_00214300 targeted METTL7B and ITGA7. Meanwhile, RT-PCR results of 8 differentially expressed mRNAs and lncRNAs showed that the expression levels of the 8 genes were similar to the expression trends of transcriptome sequencing, indicating that the sequencing results were reliable. The findings identify the lncRNAs and mRNAs that regulate fetal muscle development in Leizhou goats, as well as the potential regulatory linkages between some lncRNAs and mRNAs with differential expression. In addition, the studys also offer a theoretical foundation for molecular selection and breeding of Leizhou goats, and a novel source for studying the molecular regulatory mechanism of fetal muscle development in Leizhou goats.

Key words: Leizhou goat, mRNA, lncRNA, longissimus dorsi

中图分类号:

S827.2

叶峻宁, 邓铭, 薛慧雯, 柳广斌, 邹娴, 孙宝丽, 郭勇庆, 刘德武, 李耀坤. 影响山羊胎儿肌肉发育mRNA和lncRNA的鉴定与分析[J]. 畜牧兽医学报, 2023, 54(3): 989-1002.

YE Junning, DENG Ming, XUE Huiwen, LIU Guangbin, ZOU Xian, SUN Baoli, GUO Yongqing, LIU Dewu, LI Yaokun. Identification and Analysis of mRNA and lncRNA Affecting Goat Fetal Muscle Development[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(3): 989-1002.

参考文献 45

[1]	TEIXEIRA A,SILVA S,RODRIGUES S.Advances in sheep and goat meat products research[J].Adv Food Nutr Res,2019,87:305-370.
[2]	甘春艳,李军,金海.2021年我国肉羊产业发展概况、未来发展趋势及建议[J].中国畜牧杂志,2022,58(3):258-263.GAN C Y,LI J,JIN H.Overview of the development of the meat sheep industry in China in 2021,future development trends and suggestions[J].Chinese Journal of Animal Science,2022,58(3):258-263.(in Chinese)
[3]	SHI J A,LI W D,LIU A F,et al.MiRNA sequencing of embryonic myogenesis in chengkou mountain chicken[J].BMC Genomics, 2022,23(1):571.
[4]	ANTONIOU A,MASTROYIANNOPOULOS N P,UNEY J B,et al.miR-186 inhibits muscle cell differentiation through myogenin regulation[J]. J Biol Chem,2014,289(7):3923-3935.
[5]	POWNALL M E,GUSTAFSSON M K,EMERSON C P JR.Myogenic regulatory factors and the specification of muscle progenitors in vertebrate embryos[J].Annu Rev Cell Dev Biol,2002,18:747-783.
[6]	李耀坤,许香萍,陆婷婷,等.影响山羊产羔数lncRNA的筛选及功能分析[J].畜牧兽医学报,2020,51(8):1853-1865.LI Y K,XU X P,LU T T,et al.Screening and functional analysis of lncRNA affecting litter size in goats[J].Acta Veterinaria et Zootechnica Sinica,2020,51(8):1853-1865.(in Chinese)
[7]	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)
[8]	WAS N,SAUER M,FISCHER U,et al.lncRNA Malat1 and miR-26 cooperate in the regulation of neuronal progenitor cell proliferation and differentiation[J].RNA,2022,doi:10.1261/rna.079436.122.
[9]	WU W T,CAO L,JIA Y F,et al.Emerging roles of miRNA,lncRNA,circRNA,and their cross-talk in pituitary adenoma[J].Cells, 2022,11(18):2920.
[10]	WU T Y,WANG S H,WANG L H,et al.Long Noncoding RNA (lncRNA) CTTN-IT1 elevates skeletal muscle satellite cell proliferation and differentiation by acting as ceRNA for YAP1 through absorbing miR-29a in Hu sheep[J].Front Genet,2020,11:843.
[11]	ZHANG X J,CHEN M M,LIU X F,et al.A novel lncRNA,lnc403,involved in bovine skeletal muscle myogenesis by mediating KRAS/Myf6[J].Gene,2020,751:144706.
[12]	LIU M,LI B,PENG W W,et al.LncRNA-MEG3 promotes bovine myoblast differentiation by sponging miR‐135[J].J Cell Physiol,2019,234(10):18361-18370.
[13]	HAN H Y,WANG X W,LI W T,et al.Identification and characterization of lncRNAs expression profile related to goat skeletal muscle at different development stages[J].Animals,2022,12(19):2683.
[14]	ZHAN S Y,XUE Y N,YANG L,et al.Transcriptome analysis reveals long non-coding natural antisense transcripts involved in muscle development in fetal goat (Capra hircus)[J].Genomics,2022,114(2):110284.
[15]	SHEN J Y,HAO Z Y,LUO Y Z,et al.Deep small RNA sequencing reveals important miRNAs related to muscle development and intramuscular fat deposition in Longissimus dorsi muscle from different goat breeds[J].Front Vet Sci,2022,9:911166.
[16]	刘恩民,卢增奎,乐祥鹏.我国养羊业现状及未来发展思考[J].中国畜牧业,2018(9):34-35.LIU E M,LU Z K,LE X P.Reflections on the current situation and future development of sheep farming in China[J].China Animal Industry,2018(9):34-35.(in Chinese)
[17]	邱金戈,宋佳杰,刘德武,等.华南地区舍饲川中黑山羊与雷州山羊的生产繁殖性能及死亡状况研究[J].中国畜牧杂志,2021, 57(2):206-208,214.QIU J G,SONG J J,LIU D W,et al.Study on the reproductive performance and mortality of housed Chuanzhong black goats and Leizhou goats in South China[J].Chinese Journal of Animal Science,2021,57(2):206-208,214.(in Chinese)
[18]	ZHU M J,FORD S P,MEANS W J,et al.Maternal nutrient restriction affects properties of skeletal muscle in offspring[J].J Physiol, 2006,575(Pt 1):241-250.
[19]	WANG H,NOULET F,EDOM-VOVARD F,et al.Bmp signaling at the tips of skeletal muscles regulates the number of fetal muscle progenitors and satellite cells during development[J].Dev Cell,2010,18(4):643-654.
[20]	KARUNARATNE J F,ASHTON C J,STICKLAND N C.Fetal programming of fat and collagen in porcine skeletal muscles[J].J Anat,2005,207(6):763-768.
[21]	MUNCIE J M,WEAVER V M.The physical and biochemical properties of the extracellular matrix regulate cell fate[J].Curr Top Dev Biol, 2018,130:1-37.
[22]	FRANTZ C,STEWART K M,WEAVER V M.The extracellular matrix at a glance[J].J Cell Sci,2010,123(Pt 24):4195-4200.
[23]	BIRCH H L.Extracellular matrix and ageing[J].Subcell Biochem,2018,90:169-190.
[24]	ROMER L H,BIRUKOV K G,GARCIA J G N.Focal adhesions:paradigm for a signaling nexus[J].Circ Res,2006,98(5):606-616.
[25]	LASSITER D G,NYLÉN C,SJÖGREN R J O,et al.FAK tyrosine phosphorylation is regulated by AMPK and controls metabolism in human skeletal muscle[J].Diabetologia,2018,61(2):424-432.
[26]	SHEN X X,WEI Y H,YOU G S,et al.Circular PPP1R13B RNA promotes chicken skeletal muscle satellite cell proliferation and differentiation via targeting miR-9-5p[J].Animals (Basel),2021,11(8):2396.
[27]	YOSHIDA T,DELAFONTAINE P.Mechanisms of IGF-1-mediated regulation of skeletal muscle hypertrophy and atrophy[J]. Cells, 2020, 9(9):1970.
[28]	GLASS D J.PI3 kinase regulation of skeletal muscle hypertrophy and atrophy[M]//ROMMEL C,VANHAESEBROECK B,VOGT P K. Phosphoinositide 3-Kinase in Health and Disease.Berlin:Springer,2010:267-278.
[29]	WU P C,WANG Y Y,WU Y J,et al.Expression and prognostic analyses of ITGA11,ITGB4 and ITGB8 in human non-small cell lung cancer[J].PeerJ,2019,7:e8299.
[30]	FANG T Y,YIN X,WANG Y F,et al.Lymph node metastasis-related gene ITGA4 promotes the proliferation,migration,and invasion of gastric cancer cells by regulating tumor immune microenvironment[J].J Oncol,2022,2022:1315677.
[31]	TIGER C F,FOUGEROUSSE F,GRUNDSTRÖM G,et al.α11β1 integrin is a receptor for interstitial collagens involved in cell migration and collagen reorganization on mesenchymal nonmuscle cells[J].Dev Biol,2001,237(1):116-129.
[32]	ZHANG H J,ZHANG L,LU M.Inhibition of integrin subunit alpha 11 restrains gastric cancer progression through phosphatidylinositol 3-kinase/Akt pathway[J].Bioengineered,2021,12(2):11909-11921.
[33]	LI X L,LIU L,LI D D,et al.Integrin β4 promotes cell invasion and epithelial-mesenchymal transition through the modulation of Slug expression in hepatocellular carcinoma[J].Sci Rep,2017,7:40464.
[34]	SHIN J,WATANABE S,HOELPER S,et al.BRAF activates PAX3 to control muscle precursor cell migration during forelimb muscle development[J].eLife,2016,5:e18351.
[35]	MAEDA Y,TIDYMAN W E,ANDER B P,et al.Ras/MAPK dysregulation in development causes a skeletal myopathy in an activating BrafL597V mouse model for cardio-facio-cutaneous syndrome[J].Dev Dyn,2021,250(8):1074-1095.
[36]	WU Z Y,KONG X Y,WANG Z H.Integrin α7 knockdown suppresses cell proliferation,migration,invasion and EMT in hepatocellular carcinoma[J].Exp Ther Med,2021,21(4):309.
[37]	DESGROSELLIER J S,CHERESH D A.Integrins in cancer:biological implications and therapeutic opportunities[J].Nat Rev Cancer,2010,10(1):9-22.
[38]	HELLER K N,MONTGOMERY C L,SHONTZ K M,et al.Human α7 integrin gene (ITGA7) delivered by adeno-associated virus extends survival of severely affected dystrophin/utrophin-deficient mice[J].Hum Gene Ther,2015,26(10):647-656.
[39]	BURKIN D J,WALLACE G Q,MILNER D J,et al.Transgenic expression of α7β1 integrin maintains muscle integrity,increases regenerative capacity,promotes hypertrophy,and reduces cardiomyopathy in dystrophic mice[J].Am J Pathol,2005,166(1):253-263.
[40]	DOMINICI C,RICHARD S.Muscle stem cell polarity requires QKI-mediated alternative splicing of Integrin Alpha-7(Itga7)[J].Life Sci Alliance,2022,5(5):e202101192.
[41]	ROONEY J E,GURPUR P B,YABLONKA-REUVENI Z,et al.Laminin-111 restores regenerative capacity in a mouse model for α7 integrin congenital myopathy[J].Am J Pathol,2009,174(1):256-264.
[42]	JIANG Z P,YIN W,ZHU H C,et al.METTL7B is a novel prognostic biomarker of lower-grade glioma based on pan-cancer analysis[J]. Cancer Cell Int,2021,21(1):383.
[43]	SONG H B,LIU D C,WANG L W,et al.Methyltransferase like 7B is a potential therapeutic target for reversing EGFR-TKIs resistance in lung adenocarcinoma[J].Mol Cancer,2022,21(1):43.
[44]	HENDRICKSON S L,LAUTENBERGER J A,CHINN L W,et al.Genetic variants in nuclear-encoded mitochondrial genes influence AIDS progression[J].PLoS One,2010,5(9):e12862.
[45]	LAUGHLIN T G,BAYNE A N,TREMPE J F,et al.Structure of the complex I-like molecule NDH of oxygenic photosynthesis[J]. Nature,2019,566(7744):411-414.