干扰lnc4351对牛骨骼肌卫星细胞增殖与成肌分化的影响
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摘要
为探究长链非编码RNA(lncRNA)对牛骨骼肌卫星细胞增殖及分化的影响,本研究以牛骨骼肌卫星细胞及已建立的体外成肌诱导分化模型为基础,以前期高通量测序获得的牛骨骼肌卫星细胞分化前后表达差异倍数较大的一个预测lncRNA为靶标,对其进行生物信息学分析及亚细胞定位,命名为lnc4351。设计合成lnc4351的siRNA,转染牛骨骼肌卫星细胞,采用EdU染色的方法检测干扰lnc4351对细胞增殖的影响;对转染siRNA的牛骨骼肌卫星细胞进行体外成肌诱导分化,观察肌管的形成状态,同时采用实时荧光定量PCR和Western blotting检测分化标志因子MyoG和MHC基因的mRNA及蛋白水平的表达变化,研究干扰lnc4351对细胞分化的影响。结果显示,lnc4351位于牛的14号染色体,不具有蛋白编码潜能,是一个未报道过的lncRNA,在牛骨骼肌卫星细胞的细胞质和细胞核内均有分布,主要存在于细胞核;干扰lnc4351表达后,EdU阳性细胞比率显著下降(P<0.05),说明下调lnc4351表达显著抑制了牛骨骼肌卫星细胞的增殖;下调lnc4351表达后肌卫星细胞经诱导分化产生的肌管量呈现增多趋势,分化标志因子MyoG和MHC的蛋白水平显著或极显著上调(P<0.05;P<0.01),说明干扰lnc4351能够促进牛骨骼肌卫星细胞的成肌分化过程。本研究结果表明,干扰lnc4351表达可以抑制牛骨骼肌卫星细胞的增殖并促进其成肌分化过程,为进一步开展lncRNA对牛骨骼肌发育的调控机制及肌肉发育相关研究提供参考。
Based on the previous established isolation and culture of bovine skeletal muscle satellite cells and in vitro myogenic differentiation model,a high-throughput sequencing predicted lncRNA(lnc4351) with a great differentially expression during myogenic differentiation process was used as a target to explore the effect of lncRNA on the proliferation and differentiation of bovine skeletal muscle satellite cells,and the bioinformatics analysis and subcellular localization were performed in this study.The siRNA of lnc4351 was synthesized and transfected into bovine skeletal muscle satellite cells,the EdU staining was performed to detect the effect of interference with lnc4351 on cell proliferation.Simultaneously,an in vitro induced differentiation experiment was performed to observe the formation of myotubes after transfecting siRNA.To explore effects of interference with lnc4351 on cell differentiation,the mRNA and protein levels of the differentiation markers MyoG and MHC genes were detected using Real-time PCR and Western blotting,respectively.The results showed that lnc4351,located on chromosome 14 in cattle,was an unreported lncRNA with no protein coding potential.lnc4351 was distributed in the cytoplasm and nucleus of bovine muscle satellite cells,mainly in the nucleus.When the expression of lnc4351 was interfered,the ratio of EdU-positive cells was significantly decreased(P<0.05),suggesting that down-regulation of lnc4351 significantly inhibited the proliferation of bovine skeletal muscle satellite cells.In addition,the number of myotubes had an increasing trend and the protein levels of differentiation markers MyoG and MHC were significantly or extremely significantly up-regulated after interfering lnc4351(P<0.05;P<0.01),indicating that down-regulation of lnc4351 could promote the myogenic differentiation process of bovine satellite cells.Down-regulating lnc4351 could inhibit the proliferation of bovine skeletal muscle satellite cells and promote its myogenic differentiation process.This study would provide a reference for further study of lncRNA on the regulation of bovine skeletal muscle development and related studies of muscle development.
Based on the previous established isolation and culture of bovine skeletal muscle satellite cells and in vitro myogenic differentiation model,a high-throughput sequencing predicted lncRNA(lnc4351) with a great differentially expression during myogenic differentiation process was used as a target to explore the effect of lncRNA on the proliferation and differentiation of bovine skeletal muscle satellite cells,and the bioinformatics analysis and subcellular localization were performed in this study.The siRNA of lnc4351 was synthesized and transfected into bovine skeletal muscle satellite cells,the EdU staining was performed to detect the effect of interference with lnc4351 on cell proliferation.Simultaneously,an in vitro induced differentiation experiment was performed to observe the formation of myotubes after transfecting siRNA.To explore effects of interference with lnc4351 on cell differentiation,the mRNA and protein levels of the differentiation markers MyoG and MHC genes were detected using Real-time PCR and Western blotting,respectively.The results showed that lnc4351,located on chromosome 14 in cattle,was an unreported lncRNA with no protein coding potential.lnc4351 was distributed in the cytoplasm and nucleus of bovine muscle satellite cells,mainly in the nucleus.When the expression of lnc4351 was interfered,the ratio of EdU-positive cells was significantly decreased(P<0.05),suggesting that down-regulation of lnc4351 significantly inhibited the proliferation of bovine skeletal muscle satellite cells.In addition,the number of myotubes had an increasing trend and the protein levels of differentiation markers MyoG and MHC were significantly or extremely significantly up-regulated after interfering lnc4351(P<0.05;P<0.01),indicating that down-regulation of lnc4351 could promote the myogenic differentiation process of bovine satellite cells.Down-regulating lnc4351 could inhibit the proliferation of bovine skeletal muscle satellite cells and promote its myogenic differentiation process.This study would provide a reference for further study of lncRNA on the regulation of bovine skeletal muscle development and related studies of muscle development.
引文
[1] WANG K C,CHANG H Y.Molecular mechanisms of long noncoding RNAs[J]. Molecular Cell,2011,43(6):904-914.
[2] ULITSKY I,SHKUMATAVA A,JAN C H,et al.Conserved function of lincRNAs in vertebrate embryonic development despite rapid sequence evolution[J].Cell,2011,147(7):1537-1550.
[3] MERCER T R,DINGER M E,MATTICK J S.Long non-coding RNAs:Insights into functions[J].Nature Reviews Genetics,2009,10(3):155-159.
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[7] YU X,ZHANG Y,LI T,et al.Long non-coding RNA linc-RAM enhances myogenic differentiation by interacting with MyoD[J].Nature Communications,2017,8:14016.
[8] HAN X,YANG F,CAO H,et al.Malat1 regulates serum response factor through miR-133 as a competing endogenous RNA in myogenesis[J].FASEB Journal,2015,29(7):3054-3064.
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[11] ZHU M,LIU J,XIAO J,et al.lnc-mg is a long non-coding RNA that promotes myogenesis[J].Nature Communications,2017,8:14718.
[12] LU L,SUN K,CHEN X,et al.Genome-wide survey by ChIP-Seq reveals YY1 regulation of lincRNAs in skeletal myogenesis[J].EMBO Journal,2013,32(19):2575-2588.
[13] DIMARTINO D,COLANTONI A,BALLARINO M,et al.The long non-coding RNA lnc-31,interacts with rock1,mRNA and mediates its YB-1-dependent translation[J].Cell Reports,2018,23(3):733-740.
[14] KALLEN A N,ZHOU X B,XU J,et al.The imprinted H19 lncRNA antagonizes let-7 microRNAs[J].Molecular Cell,2013,52(1):101-112.
[15] 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[J].Genes & Development,2014,28(5):491-501.
[16] 王轶敏,代阳,刘新峰,等.牛骨骼肌卫星细胞的分离鉴定和诱导分化[J].中国畜牧兽医,2014,41(7):142-147. WANG Y M,DAI Y,LIU X F,et al.Isolation,identification and differentiation of satellite cells from bovine skeletal muscle[J].China Animal Husbandry & Veterinary Medicine,2014,41(7):142-147.(in Chinese)
[17] YIN J,JIN X,BECK S,et al.In vitro myogenic and adipogenic differentiation model of genetically engineered bovine embryonic fibroblast cell lines[J].Biotechnology Letters,2010,32(2):195.
[18] JIN C F,LI Y,DING X B,et al.lnc133b,a novel,long non-coding RNA,regulates bovine skeletal muscle satellite cell proliferation and differentiation by mediating miR-133b[J].Gene,2017,630:35-43.
[19] ZHANG W R,ZHANG H N,WANG Y M,et al.miR-143 regulates proliferation and differentiation of bovine skeletal muscle satellite cells by targeting IGFBP5[J].In Vitro Cellular & Developmental Biology -Animal,2016,53(3):265-271.
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[21] 周瑞,王以鑫,龙科任,等.lncRNA调控骨骼肌发育的分子机制及其在家养动物中的研究进展[J].遗传,2018,40(4):292-304. ZHOU R,WANG Y X,LONG K R,et al.Regulatory mechanism for lncRNAs in skeletal muscle development and progress on its research in domestic ani-mals[J].Hereditas,2018,40(4):292-304.(in Chinese)
[22] CUI M,XIAO Z,WANG Y,et al.Long noncoding RNA HULC modulates abnormal lipid metabolism in hepatoma cells through an miR-9-mediated RXRA signaling pathway[J].Cancer Research,2015,75(5):846-857.
[23] 郭云兰,杨慧翠,连晓宁,等.RMP基因干扰对肝癌细胞周期的影响[J].中国生物工程杂志,2011,31(5):8-14. GUO Y L,YANG H C,LIAN X N,et al.Effects of RPB5-mediating protein (RMP) on cell cycle of hepatoma cell[J].China Biotechnology,2011,31(5):8-14.(in Chinese)
[24] 王红娜,孙洪新,张英杰,等.干扰MSTN对绵羊成肌细胞增殖分化及相关基因表达的影响[J].畜牧兽医学报,2018,49(1):46-54. WANG H N,SUN H X,ZHANG Y J,et al.Effects of interfering MSTN on proliferation and differentiation of sheep myoblasts and expression of related genes[J].Acta Veterinaria et Zootechnica Sinica,2018,49(1):46-54.(in Chinese)
[25] 杨洋,许厚强,陈伟,等.从江香猪PDK4基因干扰载体的构建与检测[J].中国畜牧兽医,2018,45(4):873-880. YANG Y,XU H Q,CHEN W,et al.Construction and detection of the interference vector of PDK4 gene in Congjiang Xiang pig[J]. China Animal Husbandry & Veterinary Medicine,2018,45(4):873-880.(in Chinese)
[26] LI Y,CHEN X,SUN H,et al.Long non-coding RNAs in the regulation of skeletal myogenesis and muscle diseases[J].Cancer Letters,2018,417:58-64.
[27] CAI B,LI Z,MA M,et al.lncRNA-Six1 encodes a micropeptide to activate Six1 in Cis and is involved in cell proliferation and muscle growth[J].Frontiers in Physiology,2017,8:230.
[28] SUN X,LI M,SUN Y,et al.The developmental transcriptome sequencing of bovine skeletal muscle reveals a long noncoding RNA,lncMD,promotes muscle differentiation by sponging miR-125b[J].Biochimica et Biophysica Acta,2016,1863(11):2835-2845.
[29] YUE Y,JIN C,CHEN M,et al.A lncRNA promotes myoblast proliferation by up-regulating GH1[J].In Vitro Cellular & Developmental Biology-Animal, 2017,53(8):699-705.
[2] ULITSKY I,SHKUMATAVA A,JAN C H,et al.Conserved function of lincRNAs in vertebrate embryonic development despite rapid sequence evolution[J].Cell,2011,147(7):1537-1550.
[3] MERCER T R,DINGER M E,MATTICK J S.Long non-coding RNAs:Insights into functions[J].Nature Reviews Genetics,2009,10(3):155-159.
[4] QUAN M,CHEN J,ZHANG D.Exploring the secrets of long noncoding RNAs[J].International Journal of Molecular Sciences,2015,16(3):5467-5496.
[5] LEGNINI I,MORLANDO M,MANGIAVACCHI A,et al.A feedforward regulatory loop between HuR and the long noncoding RNA linc-MD1 controls early phases of myogenesis[J].Molecular Cell,2014,53(3):506-514.
[6] 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.
[7] YU X,ZHANG Y,LI T,et al.Long non-coding RNA linc-RAM enhances myogenic differentiation by interacting with MyoD[J].Nature Communications,2017,8:14016.
[8] HAN X,YANG F,CAO H,et al.Malat1 regulates serum response factor through miR-133 as a competing endogenous RNA in myogenesis[J].FASEB Journal,2015,29(7):3054-3064.
[9] GONG C,LI Z,RAMANUJAN K,et al.A long non-coding RNA,lncMyoD,regulates skeletal muscle differentiation by blocking IMP2-mediated mRNA translation[J].Developmental Cell,2015,34(2):181-191.
[10] CARETTI G,SCHILTZ R L,DILWORTH F J,et al.The RNA Helicases p68/p72 and the noncoding RNA SRA are coregulators of MyoD and skeletal muscle differentiation[J].Developmental Cell,2006,11(4):547-560.
[11] ZHU M,LIU J,XIAO J,et al.lnc-mg is a long non-coding RNA that promotes myogenesis[J].Nature Communications,2017,8:14718.
[12] LU L,SUN K,CHEN X,et al.Genome-wide survey by ChIP-Seq reveals YY1 regulation of lincRNAs in skeletal myogenesis[J].EMBO Journal,2013,32(19):2575-2588.
[13] DIMARTINO D,COLANTONI A,BALLARINO M,et al.The long non-coding RNA lnc-31,interacts with rock1,mRNA and mediates its YB-1-dependent translation[J].Cell Reports,2018,23(3):733-740.
[14] KALLEN A N,ZHOU X B,XU J,et al.The imprinted H19 lncRNA antagonizes let-7 microRNAs[J].Molecular Cell,2013,52(1):101-112.
[15] 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[J].Genes & Development,2014,28(5):491-501.
[16] 王轶敏,代阳,刘新峰,等.牛骨骼肌卫星细胞的分离鉴定和诱导分化[J].中国畜牧兽医,2014,41(7):142-147. WANG Y M,DAI Y,LIU X F,et al.Isolation,identification and differentiation of satellite cells from bovine skeletal muscle[J].China Animal Husbandry & Veterinary Medicine,2014,41(7):142-147.(in Chinese)
[17] YIN J,JIN X,BECK S,et al.In vitro myogenic and adipogenic differentiation model of genetically engineered bovine embryonic fibroblast cell lines[J].Biotechnology Letters,2010,32(2):195.
[18] JIN C F,LI Y,DING X B,et al.lnc133b,a novel,long non-coding RNA,regulates bovine skeletal muscle satellite cell proliferation and differentiation by mediating miR-133b[J].Gene,2017,630:35-43.
[19] ZHANG W R,ZHANG H N,WANG Y M,et al.miR-143 regulates proliferation and differentiation of bovine skeletal muscle satellite cells by targeting IGFBP5[J].In Vitro Cellular & Developmental Biology -Animal,2016,53(3):265-271.
[20] DAI Y,WANG Y M,ZHANG W R,et al.The role of microRNA-1 and microRNA-206 in the proliferation and differentiation of bovine skeletal muscle satellite cells[J].In Vitro Cellular & Developmental Biology-Animal,2016,52(1):27-34.
[21] 周瑞,王以鑫,龙科任,等.lncRNA调控骨骼肌发育的分子机制及其在家养动物中的研究进展[J].遗传,2018,40(4):292-304. ZHOU R,WANG Y X,LONG K R,et al.Regulatory mechanism for lncRNAs in skeletal muscle development and progress on its research in domestic ani-mals[J].Hereditas,2018,40(4):292-304.(in Chinese)
[22] CUI M,XIAO Z,WANG Y,et al.Long noncoding RNA HULC modulates abnormal lipid metabolism in hepatoma cells through an miR-9-mediated RXRA signaling pathway[J].Cancer Research,2015,75(5):846-857.
[23] 郭云兰,杨慧翠,连晓宁,等.RMP基因干扰对肝癌细胞周期的影响[J].中国生物工程杂志,2011,31(5):8-14. GUO Y L,YANG H C,LIAN X N,et al.Effects of RPB5-mediating protein (RMP) on cell cycle of hepatoma cell[J].China Biotechnology,2011,31(5):8-14.(in Chinese)
[24] 王红娜,孙洪新,张英杰,等.干扰MSTN对绵羊成肌细胞增殖分化及相关基因表达的影响[J].畜牧兽医学报,2018,49(1):46-54. WANG H N,SUN H X,ZHANG Y J,et al.Effects of interfering MSTN on proliferation and differentiation of sheep myoblasts and expression of related genes[J].Acta Veterinaria et Zootechnica Sinica,2018,49(1):46-54.(in Chinese)
[25] 杨洋,许厚强,陈伟,等.从江香猪PDK4基因干扰载体的构建与检测[J].中国畜牧兽医,2018,45(4):873-880. YANG Y,XU H Q,CHEN W,et al.Construction and detection of the interference vector of PDK4 gene in Congjiang Xiang pig[J]. China Animal Husbandry & Veterinary Medicine,2018,45(4):873-880.(in Chinese)
[26] LI Y,CHEN X,SUN H,et al.Long non-coding RNAs in the regulation of skeletal myogenesis and muscle diseases[J].Cancer Letters,2018,417:58-64.
[27] CAI B,LI Z,MA M,et al.lncRNA-Six1 encodes a micropeptide to activate Six1 in Cis and is involved in cell proliferation and muscle growth[J].Frontiers in Physiology,2017,8:230.
[28] SUN X,LI M,SUN Y,et al.The developmental transcriptome sequencing of bovine skeletal muscle reveals a long noncoding RNA,lncMD,promotes muscle differentiation by sponging miR-125b[J].Biochimica et Biophysica Acta,2016,1863(11):2835-2845.
[29] YUE Y,JIN C,CHEN M,et al.A lncRNA promotes myoblast proliferation by up-regulating GH1[J].In Vitro Cellular & Developmental Biology-Animal, 2017,53(8):699-705.