干扰lnc23后牛骨骼肌卫星细胞转录组测序的生物信息学分析

doi:10.16431/j.cnki.1671-7236.2020.04.001

摘要/Abstract

摘要： 试验旨在分析lnc23在调控牛骨骼肌卫星细胞分化过程中转录组水平的变化，筛选出可能参与调节骨骼肌卫星细胞生长的调控通路及相关mRNAs。采用第二代测序技术（next-generation sequencing，NGS）基于Illumia hiSeq测序平台对成功构建的lnc23抑制模型及相应对照组的牛骨骼肌卫星细胞进行转录组测序，将数据整理、过滤及评估后，通过生物信息学方法分析样品间基因转录差异表达，对这些差异基因进行GO和KEGG富集分析，并应用实时荧光定量PCR技术对转录组数据结果进行验证。结果显示，转录组测序共鉴定到19 358个基因，筛选到1 297个差异表达基因，其中上调856个，下调441个。差异基因的GO功能分析共包括细胞组分、分子功能和生物学过程3大类222个分支，其中有大量的差异基因与催化活性、分子功能调节、信号转导、生物黏附、新陈代谢相关。KEGG分析显示，差异基因共涉及190个通路，显著富集在PI3K-Akt、P53、TNF、RIG-Ⅰ样受体、神经活性配体受体互作、细胞因子互作等信号通路上，进一步从中筛选出可能参与细胞生长、肌肉发育过程的差异基因，如CCNA2、RRM2、IL-6、MYL2等。实时荧光定量PCR结果显示，所选的11个差异基因中有9个与转录组测序结果变化一致，表明了测序结果的可靠性。本试验完成了干扰lnc23及其对照后牛骨骼肌卫星细胞的转录组测序分析，获取差异基因的功能注释信息，初步揭示lnc23调控牛骨骼肌卫星细胞分化的潜在基因和通路，为深入探究lnc23调控牛骨骼肌卫星细胞的分化机制打下良好的基础。

关键词: lncRNA; 牛骨骼肌; 卫星细胞; 转录组; 信号通路; 差异基因

Abstract: This study was aimed to analyze the changes of transcriptional group level of a muscle highly expressed and functional lncRNA (lnc23),and screen out the regulatory pathways and related mRNAs,which might be involved in regulating the growth of skeletal muscle satellite cells.The second generation sequencing technique (next-generation sequencing,NGS) was used to sequence the successfully constructed lnc23 inhibition model and the corresponding control group of bovine skeletal muscle satellite cells based on Illumia hiSeq sequencing platform.After sorting,filtering and evaluating the data,the transcriptional differentially expressed genes between the samples were analyzed by bioinformatics,and the differential genes were analyzed by GO and KEGG enrichment methods.The results of the transcriptome data were verified by Real-time quantitative PCR.The results showed that 19 358 genes were sequenced and 1 297 differential expression genes were screened,of which 856 genes were up-regulated,and 441 genes were down-regulated.The GO function of the differential genes,including the cellular components,the molecular function and the biological process,were divided into 3 categories (222 branches),among which a large number of differential genes were related to the catalytic activity,the molecular function regulation,the signal transduction,the biological adhesion and the metabolism.KEGG analysis results showed that the differential genes were involved in 190 pathways,which were significantly enriched in PI3K-Akt,P53,TNF,RIG-Ⅰ-like receptors,neuroactive ligand receptor interactions,cytokine interactions and other signaling pathways,and further screened out differential genes,such as CCNA2,RRM2,IL-6 and MYL2,which might be involved in cell growth and muscle development.Real-time quantitative PCR results showed that 9 of 11 differential genes were consistent with the sequencing results of the transcriptional group,indicating the reliability of the sequencing results.This study successfully finished the interference with lnc23 and its control,completed the transcriptional group sequencing analysis of bovine skeletal muscle satellite cells,obtained the functional annotation information of differential genes and preliminarily revealed the potential genes and pathways of lnc23 regulating the differentiation of bovine skeletal muscle satellite cells,which laid a good foundation for further exploring the differentiation mechanism of bovine skeletal muscle satellite cells regulated by lnc23.

Key words: lncRNA; bovine skeletal muscle; satellite cell; transcriptome; signaling pathway; differential genes

中图分类号:

宋瑛燊, 郭益文, 苗曼宁, 张林林, 李新, 丁向彬, 郭宏. 干扰lnc23后牛骨骼肌卫星细胞转录组测序的生物信息学分析[J]. 中国畜牧兽医, 2020, 47(4): 973-983.

SONG Yingshen, GUO Yiwen, MIAO Manning, ZHANG Linlin, LI Xin, DING Xiangbin, GUO Hong. Bioinformatics Analysis of Transcriptome Sequencing of Bovine Skeletal Muscle Satellite Cells After Interference with lnc23[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(4): 973-983.

参考文献

[1] 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.
[2] HITACHI K,NAKATANI M,TSUCHIDA K.Long non-coding RNA myoparr regulates GDF5 expression in denervated mouse skeletal muscle[J].Non-Coding RNA,2019,5(2):33-45.
[3] ANDRESINI O,ROSSI M N,MATTEINI F,et al.The long non-coding RNA Kcnq1ot1 controls maternal p57 expression in muscle cells by promoting H3K27me3 accumulation to an intragenic MyoD-binding region[J].Epigenetics&Chromatin,2019,12(1):8-23.
[4] SUI Y,HAN Y,ZHAO X,et al.Long non-coding RNA Irm enhances myogenic differentiation by interacting with MEF2D[J].Cell Death&Disease,2019,10(3):181-193.
[5] WANG S,ZUO H,JIN J,et al.Long noncoding RNA Neat1 modulates myogenesis by recruiting Ezh2[J].Cell Death&Disease,2019,10(7):505-519.
[6] XIAO H,LIAO Y,TANG C,et al.RNA-Seq analysis of potential lncRNAs and genes for the anti-renal fibrotic effect of norcantharidin[J].Journal of Cellular Biochemistry, 2019,120(10):17354-17367.
[7] SHENG K W,ZONG C H.Single Cell Methods[M].NewYork:Springer,2019.
[8] YU M,SHAN X,LIU Y,et al.RNA-Seq analysis and functional characterization revealed lncRNA NONRATT0075602 regulated cardiomyocytes oxidative stress and apoptosis induced by high glucose[J].Journal of Cellular Biochemistry,2019,120(10):18278-18287.
[9] 贾新平,孙晓波,邓衍明,等.鸟巢蕨转录组高通量测序及分析[J].园艺学报,2014,41(11):2329-2341. JIA X P,SUN X B,DENG Y M,et al.Sequencing and anailsis of the transcriptome of asplenium nidus[J].Acta Horticulturae Sinica,2014,41(11):2329-2341.(in Chinese)
[10] 魏彩虹,吴明明,刘瑞凿,等.肌肉发育相关lncRNA的研究进展[J].中国农业科学,2014,47(20):4078-4085. WEI C H,WU M M,LIU R Z,et al.Research progress in muscular growth and development of long noncoding RNAs[J].Scientia Agricultura Sinica,2014,47(20):4078-4085.(in Chinese)
[11] BHASKAR P T,HAY N.The two TORCs and AKT[J].Developmental Cell,2007,12(4):487-502.
[12] 吴海青.mTOR信号通路对山羊骨骼肌卫星细胞增殖及分化的影响[D].呼和浩特:内蒙古大学,2015. WU H Q.The effects of mammalian of rapamycin signaling pathway on proliferation and differentiation of goat skeletal muscle satellite cells[D].Hohhot:Inner Mongolia University,2015.(in Chinese)
[13] 史新娥,吴国芳,宋子仪,等.阻断PI3K/AKT通路通过激活FoxO1抑制猪骨骼肌卫星细胞分化[J].中国农业科学,2014,47(1):154-160. SHI X E,WU G F,SONG Z Y,et al.Inhibition of P13K/AKT pathway suppressing porcine skeletal muscle satelite differentiation through activation of FoxO1 transcription factor[J].Scientia Agricultura Sinica,2014,47(1):154-160.(in Chinese)
[14] CORNEY D C,FLESKEN-NIKITIN A,GODWIN A K,et al.microRNA-34b and microRNA-34c are targets of p53 and cooperate in control of cell proliferation and adhesion-independent growth[J].Cancer Research,2007,67(18):8433-8438.
[15] FU X,WANG Y,WU G,et al.Long noncoding RNA PURPL promotes cell proliferation in liver cancer by regulating p53[J].Molecular Medicine Reports,2019,19(6):4998-5006.
[16] LI H,HOU L,ZHANG Y,et al.PFN2a suppresses C2C12 myogenic development by inhibiting proliferation and promoting apoptosis via the p53 pathway[J].Cells,2019,8(9):959-978.
[17] KONG F,LI Z,PARKS W M,et al.Cyclic mechanical reinforcement of integrin-ligand interactions[J].Molecular Cell,2013,49(6):1060-1068.
[18] 蒋雅红,杨晓萍.E-钙粘素及相关蛋白的研究进展[J].现代生物医学进展,2011,11(2):375-377. JIANG Y H,YANG X P.Progress on E-cadherin and its associated proteins[J].Progress in Modern Biomedicine,2011,11(2):375-377.(in Chinese)
[19] CHARVILLE G W,CHEUNG T H,YOO B,et al.Ex vivo expansion and in vivo self-renewal of human muscle stem cells[J].Stem Cell Reports,2015,5(4):621-632.
[20] ZHU H,XIAO F,WANG G,et al.STAT3 regulates self-renewal of adult muscle satellite cells during injury-induced muscle regeneration[J].Cell Reports, 2016,16(8):2102-2115.
[21] LI X,MA X L,TIAN F J,et al.Downregulation of CCNA2 disturbs trophoblast migration,proliferation,and apoptosis during the pathogenesis of recurrent miscarriage[J].American Journal of Reproductive Immunology,2019,82(1):e13144.
[22] LIU X,PENG J,ZHOU Y,et al.Silencing RRM2 inhibits multiple myeloma by targeting the Wnt/β-catenin signaling pathway[J].Molecular Medicine Reports, 2019,20(3):2159-2166.
[23] 郑琪,睢梦华,凌英会.骨骼肌卫星细胞增殖与成肌分化过程中关键信号通路的作用[J].畜牧兽医学报,2017,48(11):2005-2014. ZHENG Q,JU M H,LING Y H.The role of key signaling pathways in the proliferation and differentiation of skeletal muscle satellite cells[J].Acta Veterinaria et Zootechnica Sinica,2017,48(11):2005-2014.(in Chinese)