牛LncRNA-133a对骨骼肌卫星细胞增殖分化的影响

doi:10.3864/j.issn.0578-1752.2019.01.013

摘要/Abstract

摘要：

【目的】探讨长链非编码RNA LncRNA-133a对牛骨骼肌卫星细胞增殖分化过程的影响。【方法】利用测序样品3、6、9月龄胎牛及24月龄成年和牛骨骼肌肌肉组织,qRT-PCR法检测LncRNA-133a的组织时序表达谱。构建牛骨骼肌卫星细胞的体外成肌诱导分化模型,模拟牛骨骼肌的生长发育过程,qRT-PCR法检测LncRNA-133a和肌细胞分化标记因子MyoG、MHC的细胞时序表达谱。利用过表达LncRNA-133载体（pCDNA3.1-EGFP-LncRNA-133a）或LncRNA-133a抑制物（si-LncRNA 133a）转染牛骨骼肌卫星细胞,qRT-PCR法检测转染效率以及各转染处理组LncRNA-133a、MyoD、MyoG及MHC基因mRNA的表达水平,Western blotting检测MHC 基因的蛋白表达水平;同时,通过EdU细胞增殖检测、免疫荧光蛋白染色技术检测牛骨骼肌卫星细胞增殖阶段的细胞增殖量和分化阶段的肌管融合程度。【结果】组织表达谱分析发现LncRNA-133a在3月龄胎牛肌肉组织中表达量最高,6月龄胎牛肌肉组织中次之,9月龄胎牛及成年牛肌肉组织中表达量最低,时序表达呈下降趋势;利用成功构建的牛骨骼肌卫星细胞体外诱导分化模型,进行LncRNA-133a、MyoG、MHC的细胞时序表达谱分析,结果发现在牛骨骼肌卫星细胞分化过程中（D0-D3）,肌分化标记因子MyoG、MHC的表达水平逐渐升高,LncRNA-133a的表达在分化阶段呈上升趋势,且分化48 h时（D2）表达量最高;成功构建的过表达LncRNA-133a或抑制LncRNA-133a的牛骨骼肌卫星细胞模型,在增殖期（D0）：与对照组相比,过表达LncRNA-133a处理组EdU增殖染色检测得到EdU阳性细胞数显著增加（P<0.01）,而LncRNA-133a抑制处理组EdU阳性细胞数显著减少（P<0.01）;在分化48 h时（D2）：与对照组相比,LncRNA-133a过表达处理组肌细胞分化标记因子MyoD、MyoG及MHC的mRNA表达水平显著升高（P<0.05）,Western blotting检测MHC蛋白表达量显著增加（P<0.01）,且MHC蛋白的免疫荧光蛋白染色检测观察到融合肌管的体积占比更大;而LncRNA-133a抑制处理组MyoD、MyoG及MHC的mRNA表达水平均降低,其中MyoG显著降低（P<0.05）, MHC蛋白表达量显著减少（P<0.01）,同时MHC蛋白融合肌管的体积占比也降低。【结论】研究证实LncRNA-133a具有促进牛骨骼肌卫星细胞增殖及分化的作用,为进一步挖掘LncRNA-133a调节牛骨骼肌卫星细胞增殖分化调控网络机制奠定了基础。

关键词: LncRNA-133a, 牛, 骨骼肌卫星细胞, 增殖, 分化

Abstract:

【Objective】The objective of this paper was to investigate the effects of long non-coding RNA LncRNA-133a on the proliferation and differentiation of bovine skeletal muscle satellite cells. 【Method】 This study used qRT-PCR to detect the expression level of LncRNA-133a in the skeletal muscle tissues of 3, 6 and 9 months old fetal cattle and 24 months old adult bovine skeletal muscle, and obtained the tissue temporal expression profile of LncRNA-133a. The in vitro induced myoblast differentiation model of bovine skeletal muscle satellite cells was constructed to simulate the growth and development of bovine skeletal muscle. The qRT-PCR was used to detect the cells temporal expression profiles of LncRNA-133a and myocyte differentiation markers MyoG and MHC. The bovine skeletal muscle satellite cells were transfected with LncRNA-133a overexpression vector (pCDNA3.1-EGFP- LncRNA-133a) or LncRNA-133a inhibitor (si-LncRNA 133a), and the transfection efficiency and the mRNA expression levels of LncRNA-133a, MyoD, MyoG and MHC were detected by qRT-PCR in each transfection treatment group, then the protein expression level of MHC gene was detected by western blotting. In addition, the cell proliferation of the bovine skeletal muscle satellite cells and the extent of myotube fusion at the differentiation stage were detected by EdU cell proliferation assay and immunofluorescence protein staining, respectively. 【Result】 Tissue expression profiling revealed that LncRNA-133a had the highest expression in the muscle tissue of 3 months old fetal bovine, followed by the 6-month-old fetal bovine muscle tissue, and the lowest expression in the 9-month-old fetus and adult bovine muscle tissue, which demonstrated that the time expression showed a downward trend. Cell-time expression profiles of LncRNA-133a, MyoG, and MHC were analyzed by a successfully constructed bovine skeletal muscle satellite cell differentiation model in vitro, and the results showed that the expression levels of myogenic differentiation markers MyoG and MHC gradually increased during the differentiation of bovine skeletal muscle satellite cells (D0-D3). The expression of LncRNA- 133a increased in the differentiation stage, and the expression level reached the highest at 48 h of differentiation (D2). The bovine skeletal muscle satellite cell model of overexpressing LncRNA-133a or inhibiting LncRNA-133a was constructed successfully, and in the proliferative phase (D0): the number of EdU positive cells in the overexpressed LncRNA-133a-treated group was significantly increased (P<0.01), and the number of EdU positive cells in the LncRNA-133a inhibition treatment group was significantly decreased (P<0.01), compared with the control group. At 48 h of differentiation (D2): compared with the control group, the results of LncRNA-133a overexpression treatment showed that mRNA expression levels of myocyte differentiation markers MyoD, MyoG and MHC were significantly increased (P<0.05). Western blotting showed that the expression of MHC protein was also significantly increased (P<0.01), and the immunofluorescence protein staining of MHC protein showed that the volume of fusion myotubes was larger. On the contrary, in the LncRNA-133a inhibition treatment group, the mRNA expression levels of MyoD, MyoG and MHC were decreased, and MyoG was significantly decreased (P<0.05). Meanwhile, the expression of MHC protein was significantly decreased (P<0.01), and the volume fraction of MHC protein fusion myotubes was also decreased. 【Conclusion】Thus, this study confirmed that LncRNA-133a promoted the proliferation and differentiation of bovine skeletal muscle satellite cells, which laid a foundation for further research on the regulatory network mechanism of LncRNA-133a regulating the proliferation and differentiation of bovine skeletal muscle satellite cells.

Key words: LncRNA-133a, bovine, skeletal muscle satellite cells, proliferation, differentiation

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[J]. 中国农业科学, 2019, 52(1): 143-153.

LI Yan,CHEN MingMing,ZHANG JunXing,ZHANG LinLin,LI Xin,GUO Hong,DING XiangBin,LIU XinFeng. Effects of Bovine LncRNA-133a on the Proliferation and Differentiation of Skeletal Muscle Satellite Cells[J]. Scientia Agricultura Sinica, 2019, 52(1): 143-153.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2019.01.013

https://www.chinaagrisci.com/CN/Y2019/V52/I1/143

图/表 9

表1

图1

图2

图3

图4

图5

图6

图7

图8

参考文献 36

[1]	李伯江, 李平华, 吴望军, 李齐发, 黄瑞华, 刘红林 . 骨骼肌肌纤维形成机制的研究进展. 中国农业科学, 2014,47(6):1200-1207. doi: 10.3864/j.issn.0578-1752.2014.06.016
	LI B J, LI P H, WU W J, LI Q F, HUANG R H, LIU H L . Progresses in Research of the Mechanisms of Skeletal Muscle Fiber Formation. Scientia Agricultura Sinica, 2014, 47(6):1200-1207. (in Chinese) doi: 10.3864/j.issn.0578-1752.2014.06.016
[2]	Margaret Buckingham, Stéphane D Vincent . Distinct and dynamic myogenic populations in the vertebrate embryo. Current Opinion in Genetics and Development, 2009,19(5):444-453. doi: 10.1016/j.gde.2009.08.001 pmid: 19762225
[3]	魏彩虹, 吴明明, 刘瑞凿, 赵福平, 张莉, 杜立新 . 肌肉发育相关LncRNA的研究进展. 中国农业科学, 2014,47(20):4078-4085. doi: 10.3864/j.issn.0578-1752.2014.20.016
	WEI C H, WU M M, LIU R Z, ZHAO F P, ZHANG L, DU L X . Research progress in muscular growth and development of long noncoding RNAs. Scientia Agricultura Sinica, 2014,47(20):4078-4085. (in Chinese) doi: 10.3864/j.issn.0578-1752.2014.20.016
[4]	ZHANG H J, YU Y H, JIAKE C . Expression signatures oflncRNAsin skeletalmusclesat the early flow phase revealed by microarray in burned rats. Ulusal Travma ve Acil Cerrahi Dergisi, 2016,22(3):224-232. doi: 10.5505/tjtes.2015.04831 pmid: 27598585
[5]	FATICA A, BOZZONI I . Longnon-codingRNAs: new players in cell differentiation and development. Nature Reviews Genetics, 2014, 15(1):7-21.
[6]	CESANA M, CACCHIARELLI D, LEGNINI I, SANTINI T, STHANDIER O, CHINAPPI M, TRAMONTANO A, BOZZONI I . A long noncoding RNA controls muscle differentiation by functioning as a competing endogenous RNA. Cell, 2011, 147:358-369. doi: 10.1016/j.cell.2011.09.028 pmid: 22000014
[7]	ZHU M, LIU J, XIAO J, YANG L, CAI M, SHEN H, CHEN X, MA Y, HU S, WANG Z, HONG A, LI Y, SUN Y, WANG X . Lnc-mg is a long non-coding RNA that promotes myogenesis. Nature Communications, 2017, 8:14718. doi: 10.1038/ncomms14718 pmid: 28281528
[8]	ZHANG Z K, LI J, GUAN D, LIANG C, ZHUO Z, LIU J, LU A, ZHANG G , ZHANG B T. A newly identified lncRNA MAR1 acts as a miR-487b sponge to promote skeletal muscle differentiation and regeneration. Journal of Cachexia SarcopeniaMuscle, 2018,
[9]	ZHOU L, SUN K, ZHAO Y, ZHANG S, WANG X, LI Y, LU L, CHEN X, CHEN F, BAO X, ZHU X, WANG L, TANG L Y, ESTEBAN M A, WANG C C, JAUCH R, SUN H, WANG H . Linc-YY1 promotes myogenic differentiation and muscle regeneration through an interaction with the transcription factor YY1. Nature Communications, 2015,6:10026. doi: 10.1038/ncomms10026 pmid: 26658965
[10]	吴明明 . Lnc-SEMT促进绵羊肌肉分化生成的功能研究[D]. 北京:中国农业大学, 2016.
	WU M M . Research of Lnc-SEMT function in the process of enhancing sheep muscle differentiation and generation[D]. Beijing: China Agricultural University, 2016. ( in Chinese)
[11]	KALLEN A N, ZHOU X B, XU J, QIAO C, MA J, YAN L, LU L, LIU C, YI J S, ZHANG H, MIN W, BENNETT A M, GREGORY R I, DING Y, HUANG Y . The Imprinted H19 LncRNA Antagonizes Let-7 MicroRNAs. Molecular Cell, 2013, 52(1):101-112. doi: 10.1016/j.molcel.2013.08.027 pmid: 24055342
[12]	DEY B K, PFEIFER K, DUTTA A . The H19 long noncoding RNA gives rise to microRNAs miR-675-3p and miR-675-5p to promote skeletal muscle differentiation and regeneration. Genes and Development, 2014,28(5):491-501. doi: 10.1101/gad.234419.113
[13]	LIU X F, DING X B, LI X, JIN C F, YUE Y W, LI G P, GUO H . An atlas and analysis of bovine skeletal muscle long noncoding RNAs. Animal Genetics, 2017,48(3):278-286. doi: 10.1111/age.12539 pmid: 28262958
[14]	SUN X, LI M, SUN Y, CAI H, LAN X, HUANG Y, BAI Y, QI X, CHEN H . The developmental transcriptome sequencing of bovine skeletal muscle reveals a long noncoding RNA, lncMD, promotes muscle differentiation by sponging miR-125b. Biochim Biophys Acta, 2016,1863(11):2835-2845. doi: 10.1016/j.bbamcr.2016.08.014 pmid: 27589905
[15]	GUO Y, WANG J, ZHU M, ZENG R, XU Z, LI G, ZUO B . Identification of myod-responsive transcripts reveals a novel long non-coding rna (lncrna-ak143003) that negatively regulates myoblast differentiation. Scientific Reports, 2017,7(1):2828. doi: 10.1038/s41598-017-03071-7 pmid: 28588232
[16]	丁向彬, 张蔚然, 张俊星, 王轶敏, 刘新峰, 郭宏 . lncRNA-HZ5对牛骨骼肌卫星细胞成肌分化的调控作用研究. 天津农学院学报, 2017(3):64-68. doi: 10.3969/j.issn.1008-5394.2017.03.015
	DING X B, ZHANG W R, ZHANG J X, WANG Y M, LIU X F, GUO H . Effects of lnc RNA-HZ5 on myogenic differentiation process of bovine skeletal muscle satellite cells.Journal of Tianjin Agricultural University, 2017(3):64-68. (in Chinese) doi: 10.3969/j.issn.1008-5394.2017.03.015
[17]	XU X, JI S, LI W, YI B, LI H, ZHANG H, MA W . LncRNA H19 promotes the differentiation of bovine skeletal muscle satellite cells by suppressing Sirt1/FoxO1. Cellular & Molecular Biology Letters, 2017,22:10. doi: 10.1186/s11658-017-0040-6 pmid: 5481879
[18]	代阳 . microRNA-128对牛骨骼肌卫星细胞增殖和成肌分化的调控机制研究[D]. 天津: 天津农学院, 2016.
	DAI Y . Study on the regulation mechanism of microRNA-128 in the proliferation and myogenic differentiation process of bovine skeletal muscle satellite cells[D]. Tianjin: Tianjin Agricultural University, 2016. (in Chinese)
[19]	PALLAFACCHINA G, FRANOIS S, REGNAULT B, CZARNY B, DIVE V, CUMANO A, MONTARRAS D, BUCKINGHAM M . An adult tissue-specific stem cell in its niche: A gene profiling analysis of in vivo quiescent and activated muscle satellite cells. Stem Cell Research, 2010,4(2):77-91. doi: 10.1016/j.scr.2009.10.003 pmid: 19962952
[20]	王禹 . Sirt1 AS IncRNA通过抑制miR-34a的作用促进C2C12细胞增殖[D]. 陕西: 西北农林科技大学, 2015.
	WANG Y . Sirt1 AS IncRNA Promotes Proliferation of C2C12 Cells by Inhibiting miR-34a[D]. Shaanxi: Northwest A&F University, 2015. (in Chinese)
[21]	LIANG T, ZHOU B, SHI L, WANG H, CHU Q, XU F, LI Y, CHEN R, SHEN C , SCHINCKEL A P. lncRNA AK017368 promotes proliferation and suppresses differentiation of myoblasts in skeletal muscle development by attenuating the function of miR-30c.FASEB Journal, 2017, 32(1):377-389. doi: 10.1096/fj.201700560RR pmid: 28904016
[22]	王子帅 . 长非编码RNA-GTL2对C2C12细胞增殖的影响及机制的研究[D]. 北京: 中国农业科学院, 2015.
	WANG Z S . Research on impact mechanism long noncoding RNA- GTL2 on proliferation of C2C12 cells[D]. Beijing: Chinese Academy of Agricultural Sciences, 2015. (in Chinese)
[23]	王红娜, 孙洪新, 张英杰, 刘月琴, 谷振慧, 史秀芬 . 干扰MSTN对绵羊成肌细胞增殖分化及相关基因表达的影响. 畜牧兽医学报, 2018,49(01):46-54.
	WANG H N, SUN H X, ZHANG Y J, LIU Y Q, GU ZH, SHI X F . Effects of interfering MSTN on proliferation and differentiation of sheep myoblasts and expression of related genes. Chinese Journal of Animal and Veterinary Sciences, 2018, 49(01):46-54. (in Chinese)
[24]	DOUCET C, GUTIERREZ G J, LINDON C, LORCA T, LLEDO G, PINSET C, COUX O . Multiple phospho-rylationeventscontrolmitoticdegradation of the muscle transcription factor Myf5. BMC Biochemistry, 2005,6:27. doi: 10.1186/1471-2091-6-27 pmid: 1322219
[25]	TINTIGNAC L A, LEIBOVITCH M P, KITZMANN M, FERNANDEZ A, DUCOMMUN B, MEIJER L, LEIBOVITCH S A . Cyclin E-cdk2 phosphorylation promotes late G1-phase degradation of MyoD in muscle cells. Experimental Cell Research, 2000,259(1):300-307. doi: 10.1006/excr.2000.4973 pmid: 10942602
[26]	BUCKINGHAM M, RIGBY P W . Gene regulatory networks and transcriptional mechanisms that control myogenesis. Developmental Cell, 2014, 28(3):225-238. doi: 10.1016/j.devcel.2013.12.020 pmid: 24525185
[27]	WIGMORE P M , EVANS D J R. Molecular and cellular mechanisms involved in the generation of fiber diversity during myogenesis.International Review of Cytology, 2002, 216(216) : 175-232. doi: 10.1016/S0074-7696(02)16006-2 pmid: 9551857
[28]	TAKAGAKI Y, YAMAGISHI H, MATSUOKA R . Factors involved in signal transduction during vertebrate myogenesis. International Review of Cell and Molecular Biology, 2012, 296:187-272. doi: 10.1016/B978-0-12-394307-1.00004-7 pmid: 22559940
[29]	CUSELLA DE AMG, MOLINARI S, LE DONNE A, COLETTA M, VIVARELLI E, BOUCHE M, MOLINARO M, FERRARI S, COSSU G . Differential response of embryonic and fetal myoblasts to TGF beta: A possible regulatory mechanism of skeletal muscle histogenesis. Development( Cambridge, England), 1994,120(4):925-933. doi: 10.1111/j.1365-2303.1994.tb00538.x pmid: 7600968
[30]	SABOURIN L A, RUDNICKI M A . The molecular regulation of myogenesis. Clinical Genetics, 2000, 5(1):16-25. doi: 10.1034/j.1399-0004.2000.570103.x pmid: 10733231
[31]	WANG L, ZHAO Y, BAO X, ZHU X, KWOK YK, SUN K, CHEN X, HUANG Y, JAUCH R, ESTEBAN MA, SUN H, WANG H . LncRNA Dum interacts with Dnmts to regulate Dppa2 expression during myogenic differentiation and muscle regeneration. Cell Research, 2015,25(3):335-350. doi: 10.1038/cr.2015.21
[32]	GONG C, LI Z, RAMANUJAN K, CLAY I, ZHANG Y, LEMIRE-BRACHAT S, GLASS D J . Long non-coding RNA, LncMyoD, regulates skeletal muscle differentiation by blocking IMP2-mediated mRNA translation. Developmental Cell, 2015,34(2):181-191. doi: 10.1016/j.devcel.2015.05.009 pmid: 26143994
[33]	LEGNINI I, MORLANDO M, MANGIAVACCHI A, FATICA A, BOZZONI I . A feedforward regulatory loop between HuR and the long noncoding RNA linc-MD1 controls early phases of myogenesis. Molecular Cell, 2013, 53:506-514. doi: 10.1016/j.molcel.2013.12.012 pmid: 3919156
[34]	ALBRECHT E, LEMBCKE C, WEGNER J, MAAK S . Prenatal muscle fiber development and bundle structure in beef and dairy cattle. Journal of Animal Science, 2013, 91(8):3666-3673. doi: 10.2527/jas.2013-6258 pmid: 23658343
[35]	YUE Y W, JIN C F, CHEN M M, ZHANG L L, LIU X F, MA W Z, GUO H . A lncRNA promotes myoblast proliferation by up-regulating GH1. In Vitro Cellular & Developmental Biology - Animal, 2017, 53(8):699-705. doi: 10.1007/s11626-017-0180-z pmid: 28726188
[36]	ZHOU W, YE X L, XU J, CAO M G, FANG Z Y, LI L Y, GUAN G H, LIU Q, QIAN Y H, XIE D . The lncRNA H19 mediates breast cancer cell plasticity during EMT and MET plasticity by differentially sponging miR- 200b/c and let-7b. Science Signaling, 2017, 10(483). DOI: 10. 1126/ scisignal. aak9557 doi: 10.1126/scisignal.aak9557 pmid: 28611183

基因 ID Gene ID	引物序列(5′-3′) Primers sequence (5′-3′)		产物长度 Product length (bp)
LncRNA-133a	Forward	GCATAGCCGGTGTCTGAGAG	108
LncRNA-133a	Reverse	CGGCCGCTTGTATATTGTCC	108
MHC	Forward	GTGGAATCCGGAGGCAGAA	105
MHC	Reverse	TTTTCGAAGGTAGGGAGCGG	105
MyoG	Forward	GGCTGACAAATGCCAGACTATCC	140
MyoG	Reverse	TGGTCCCTTGCTTTATCTCCCT	140
MyoD	Forward	GACGGCTCTCTCTGCAACTT	101
MyoD	Reverse	CGGCGCGGATCCAGGT	101
GAPDH-1	Forward	ACAGTCAAGGCAGAGAACGG	98
GAPDH-1	Reverse	CCAGCATCACCCCACTTGAT	98
GAPDH-2	Forward	CCTGCCCGTTCGACAGATAG	153
GAPDH-2	Reverse	ATGGCGACGATGTCCACTTT	153