CN114807136A - 长链非编码RNA Gm10561在调控成肌细胞增殖分化中的应用 - Google Patents
长链非编码RNA Gm10561在调控成肌细胞增殖分化中的应用 Download PDFInfo
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Abstract
本发明公开了长链非编码RNA Gm10561在调控成肌细胞增殖分化中的应用。本发明通过研究表明,lncRNA Gm10561与成肌细胞增殖分化相关,通过超表达载体pcDNA3.1‑Gm10561转染进成肌细胞,上调lncRNA Gm10561表达可以促进小鼠和猪成肌细胞的增殖分化功能,同时,经过siRNA下调lncRNA Gm10561表达会抑制小鼠C2C12细胞的增殖分化功能。lncRNA Gm10561对成肌细胞增殖分化具有重要调控作用:lncRNA Gm10561能够显著促进成肌细胞增殖分化,为今后进一步研究骨骼肌发育机制打下基础,并且为提高家畜产肉性能提供新思路。
Description
技术领域
本发明属于分子生物学技术领域。更具体地,涉及长链非编码RNA Gm10561在调控成肌细胞增殖分化中的应用。
背景技术
骨骼肌作为哺乳动物机体的重要组成部分,在调节动物代谢和体内平衡中起关键作用。研究骨骼肌生长发育机制,探究提高家畜肉产量及品质的方法,对畜牧业发展具有重要意义。
骨骼肌生长发育是一个受多种转录因子及肌细胞特异基因等调控的复杂生物学过程,起源于轴旁中胚层,胚胎期轴旁中胚层发育成体节,体节逐渐分化为生肌节后定向分化为成肌细胞,成肌细胞经过迁移和不断增殖,彼此融合形成多核细胞并构成肌管,肌管分化形成肌纤维,最后肌纤维经过生长达到成熟。出生后骨骼肌生长发育主要依赖于肌纤维肥大或类型转化。目前发现调控骨骼肌生成的主要分子机制包括生肌调节因子家族、钙离子信号通路和胰岛素样生长因子等。此外,表观遗传调控如DNA甲基化、组蛋白修饰和非编码RNA在骨骼肌生长发育中也起着至关重要的作用。
长链非编码RNA(lncRNA)是一类长度大于200个核苷酸,没有蛋白编码能力(或只能编码微肽)的RNA分子,在X染色体失活、基因印记、胚胎发育、免疫功能、肿瘤发生等多种生物学过程中发挥重要调控作用。目前通过高通量测序鉴定出的数以万计的lncRNA中仅少数被报道在骨骼肌生长发育中具有重要功能,大多数lncRNA对骨骼肌发育的调控功能尚不明确。已有的研究结果说明在成肌分化过程lncRNA确实发挥了重要的调控作用。
但到目前为止,绝大部分的lncRNA的功能还是未知的,只有小部分的lncRNA的功能被研究,例如:LncRNA HZ-5、LncRNA H19、lncMD等,现有技术公开了牛lncRNA-133a及在牛骨骼肌卫星细胞增殖分化调控中的应用和验证方法。但是,这些lncRNA参与的是牛骨骼肌肌细胞分化发育的调节,相较其他动物的成肌细胞生长发育的相关LncRNA在骨骼肌生长发育中的具体作用尚不清楚,参与调节成肌细胞增殖分化的LncRNA的相关报道仍然较少。因此,为了明确lncRNA对骨骼肌生长发育的调控作用,有必要研究和开发出更多的、能用于调节成肌细胞增殖分化的LncRNA,有利于为提高家畜肉产量及畜牧产业经济效益提供新思路。
发明内容
本发明要解决的技术问题是克服上述问题的缺陷和不足,提供长链非编码RNAGm10561在调控成肌细胞增殖分化中的应用。
本发明的第一个目的是提供SEQ ID NO:1所示的长链非编码RNA Gm10561的应用。
本发明另一目的是提供一种促进成肌细胞增殖和/或分化的方法。
本发明上述目的通过以下技术方案实现:
本发明前期通过RNA测序发现长链非编码RNA Gm10561在小鼠C2C12细胞分化过程中差异表达,为验证其调控骨骼肌生长发育的功能,根据SEQ ID NO:1所示的lncRNAGm10561序列,设计并合成siRNA干涉片段,转染小鼠C2C12细胞,通过EdU增殖活性检测发现干扰lncRNA Gm10561表达抑制小鼠C2C12细胞的增殖;经过qRT-PCR检测,显示干扰lncRNAGm10561表达同样抑制小鼠C2C12细胞的分化。
然后,根据lncRNA Gm10561转录本序列SEQ ID NO:1构建超表达载体pcDNA3.1-Gm10561,转染小鼠C2C12细胞和猪骨骼肌卫星细胞,通过EdU增殖活性检测发现过表达lncRNA Gm10561显著促进小鼠C2C12细胞和猪骨骼肌卫星细胞增殖;再通过细胞免疫荧光等结果显示过表达lncRNA Gm10561促进小鼠C2C12细胞和猪骨骼肌卫星细胞分化。上述结果均证明lncRNA Gm10561对成肌细胞具有正调控作用。
因此,本发明提供SEQ ID NO:1所示的长链非编码RNA Gm10561在调控动物骨骼肌生长发育、或其表达促进剂在促进动物骨骼肌生长发育、在调控动物成肌细胞增殖和/或分化、或其表达促进剂在促进动物成肌细胞的增殖和/或分化、在调控动物骨骼肌卫星细胞的增殖和/或分化、或其表达促进剂在促进动物骨骼肌卫星细胞增殖和/或分化中的应用。
优选地,所述动物成肌细胞为小鼠C2C12细胞,所述动物骨骼肌卫星细胞为猪骨骼肌卫星细胞。
本发明提供一种促进成肌细胞增殖和/或分化的方法,在动物体内过表达长链非编码RNA Gm10561或利用其表达促进剂,来促进成肌细胞增殖和/或分化。
优选地,构建含SEQ ID NO:1所示的长链非编码RNA Gm10561的超表达载体,转染成肌细胞。
优选地,所述动物为小鼠或猪。
本发明具有以下有益效果:
本发明研究表明,lncRNA Gm10561与成肌细胞增殖分化相关,通过超表达载体pcDNA3.1-Gm10561转染进成肌细胞,上调lncRNA Gm10561表达可以促进小鼠和猪成肌细胞的增殖分化功能,同时,经过siRNA下调lncRNA Gm10561表达会抑制小鼠C2C12细胞的增殖分化功能。表明lncRNA Gm10561对成肌细胞增殖分化具有重要调控作用,为今后进一步研究骨骼肌发育机制打下基础,并且为提高家畜产肉性能提供新思路。
附图说明
图1为lncRNA Gm10561全长PCR扩增结果,其中M1为Trans2k DNA Marker,M2为Trans5k DNA Marker,扩增片段大小为1539bp;
图2为lncRNA Gm10561及分化标志基因MyHC在小鼠C2C12细胞增殖分化中的表达趋势图;
图3为siRNA干扰lncRNA Gm10561后抑制小鼠C2C12细胞增殖(A)分化(B)的效果图,其中图A为EdU增殖检测成像结果及阳性率统计图,标尺为100μm;图B为qRT-PCR检测小鼠C2C12细胞分化标志基因(MyHC、MyoD、MyoG)的表达水平情况;
图4为注射干扰慢病毒LV3-sh-Gm10561后抑制小鼠体内骨骼肌分化标志基因MyHC、MyoG蛋白表达水平的效果图;
图5为超表达载体lncRNA Gm10561双酶切鉴定结果,其中上面的条带为pcDNA3.1(+)载体骨架,大小为5428bp,下面的条带为插入的lncRNA Gm10561片段,大小为1539bp,M为Trans5K DNA Marker;
图6为超表达lncRNA Gm10561后促进小鼠C2C12细胞增殖的效果图,其中图A为EdU增殖检测成像结果图,图B为EdU阳性率统计图,标尺为100μm;
图7为超表达lncRNA Gm10561后促进小鼠C2C12细胞分化的效果图,其中图A为细胞免疫荧光结果图,图B为细胞核MyoG(标尺为100μm)、MyHC(标尺为200μm)阳性率统计图;
图8为超表达lncRNA Gm10561后促进猪骨骼肌卫星细胞增殖的效果图,其中图A为EdU增殖检测成像结果图,图B为EdU阳性率统计图,标尺为100μm;
图9为超表达lncRNA Gm10561后促进猪骨骼肌卫星细胞分化的效果图,其中图A为细胞免疫荧光结果图,图B为细胞核MyHC阳性率统计图,标尺为200μm。
具体实施方式
以下结合说明书附图和具体实施例来进一步说明本发明,但实施例并不对本发明做任何形式的限定。除非特别说明,本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
除非特别说明,以下实施例所用试剂和材料均为市购。
小鼠C2C12细胞购自于中国科学院细胞库。
原代猪骨骼肌卫星细胞来源于本发明课题组提取组织自行分离培养。
实施例1 lncRNA Gm10561的获得
通过前期研究RNA测序发现lncRNA Gm10561在小鼠C2C12细胞分化过程中差异表达。lncRNA Gm10561的序列如SEQ ID NO:1所示。使用Trizol试剂(Invitrogen)提取小鼠C2C12细胞总RNA,根据制造商说明书进行提取,并用PrimeScriptTM RT reagent Kit withgDNA Eraser试剂盒(Takara)进行反转录获得cDNA。
1、引物设计
根据lncRNA Gm10561序列设计引物对,序列如表1所示。
表1 lncRNA Gm10561扩增引物
注:F代表上游引物,R代表下游引物;下划线部分为酶切位点。
2、PCR扩增反应
用上述提取的质量检测合格的cDNA作为模板,用Tks GflexTM DNA高保真聚合酶(Takara)特异性扩增lncRNA Gm10561转录本,PCR扩增反应体系和反应条件见下表。
表2 PCR反应体系
表3 PCR反应条件
PCR扩增结果如图1所示,扩增片段大小为1539bp。将PCR扩增产物经过1.5%琼脂糖凝胶电泳检测后利用Omega公司的Gel Exteaction kit试剂盒进行纯化回收,并置于-20℃冰箱存放备用。
实施例2成肌细胞的培养、转染
1、小鼠C2C12细胞培养
(1)将常规复苏冻存的小鼠C2C12细胞,置于37℃、5%CO2潮湿环境的细胞培养箱中培养;
(2)增殖培养,向步骤(1)中培养的细胞提供含10%胎牛血清(FBS;Gibco)的高葡萄糖培养基(DMEM;Gibco)进行细胞增殖培养;
(3)诱导分化,培养一段时间待细胞融合度达到80%-90%,将步骤(3)培养基更换为含有2%马血清(HS;Gibco)、1%青霉素-链霉素的DMEM,进行诱导分化。
2、猪骨骼肌卫星细胞的培养
(1)将常规复苏冻存的原代猪骨骼肌卫星细胞,置于37℃、5%CO2潮湿环境的细胞培养箱中培养;
(2)增殖培养,向步骤(1)中培养的细胞提供含20%胎牛血清、1%非必需氨基酸(NEAA;Gibco)、1%GlutaMax(Gibco)、0.5%鸡胚提取物(CEE;Absin)、1%青霉素-链霉素(PS;Gibco)及2.5ul/100ml细胞生长因子(bFGF;Life)的RPMI 1640培养基(Gibco)进行细胞增殖培养;
(3)诱导分化,将培养基更换为含有2%马血清、1%青霉素-链霉素的DMEM,进行诱导分化。
3、细胞转染
待增殖期细胞融合度达到60%-70%,使用Lipofectamine 3000试剂(Invitrogen)根据生产商说明书分别将超表达载体质粒及siRNA转染至小鼠C2C12细胞及猪骨骼肌卫星细胞中。
实施例3 lncRNA Gm10561的表达
将小鼠C2C12细胞接种于12孔板中于37℃、5%CO2的培养箱中培养,分别在增殖期(D0)、分化第2天、第4天和第6天收取细胞,根据制造商说明书,使用Trizol试剂提取总RNA,并用PrimeScriptTM RT reagent Kit with gDNA Eraser试剂盒(Takara)进行反转录获得cDNA。然后再进行qRT-PCR检测lncRNA Gm10561的表达量,每个样本技术重复3次,qRT-PCR检测使用引物序列、反应体系和反应条件如下表所示。
表4 qPCR引物序列
表5 qPCR反应体系
表6 qPCR反应条件
检测结果应用2-Δ-CT法进行分析,结果如图2所示,Gm10561在C2C12分化过程中上调,与肌源性标志物MyHC基因的表达模式一致,表明Gm10561可能在肌生成过程中具有调节作用。
实施例4 siRNA干扰lncRNA Gm10561表达后抑制小鼠C2C12细胞增殖分化
1、siRNA的设计、合成
根据lncRNA Gm10561序列设计siRNA及阴性对照siRNA-NC,序列如表7所示,序列由苏州吉玛基因有限公司合成。
表7 siRNA序列
2、EdU细胞的增殖检测
实验前,将小鼠C2C12细胞接种于24孔板中,并置于37℃、5%CO2培养箱培养,待细胞融合度达到60%-80%,转染重组质粒,转染24h后利用BeyoClickTM EdU细胞增殖试剂盒(Beyotime)按照制造商说明书进行细胞增殖检测,其具体操作步骤如下:
(1)使用细胞完全培养基按1000:1的比例稀释EdU溶液,制备适量50μMEdU培养基;
(2)每孔加入400μL 50μM EdU培养基于37℃、5%CO2的培养箱中孵育2h,弃培养基;
(3)采用DPBS清洗细胞1-2次,每次5min;
(4)每孔加入200μL 4%多聚甲醛固定液(Biosharp)室温孵育30min,弃固定液;
(5)每孔加入200μL 2mg/mL甘氨酸,脱色摇床孵育5min,弃甘氨酸溶液;
(6)每孔加入500μL DPBS,脱色摇床清洗2次,每次5min,弃DPBS;
(7)每孔加入400μL渗透剂(含0.5%TritonX-100的DPBS),脱色摇床孵育10min,DPBS清洗5min;
(8)每孔加入200μL的1×Apollo染色反应液,避光,室温下脱色摇床孵育30min,弃染色反应液;
(9)每孔加入400μL渗透剂(含0.5%TritonX-100的DPBS),避光,室温下脱色摇床清洗2次,每次10min,弃渗透剂;
(10)DPBS清洗细胞1-2次,每次5min;
(11)每孔加入200μL DAPI染料(Beyotime),避光室温脱色摇床孵育5min,弃反应液;
(12)DPBS清洗细胞1-3次,每次10min,用荧光显微镜进行成像检测。
成像检测结果如图3A所示,在转染siRNA组中细胞EdU阳性率显著低于NC组,表明下调lncRNA Gm10561的表达,能抑制小鼠C2C12细胞增殖。
3、mRNA水平检测
实验前,将小鼠C2C12细胞接种于12孔板,37℃、5%CO2的培养箱中培养,待细胞融合度达到60%-80%,转染重组质粒,继续培养。在诱导分化3天时,收集si-Gm10561组和对照组细胞,采用Trizol法裂解并提取总RNA,并用PrimeScriptTM RT reagent Kit withgDNA Eraser试剂盒(Takara)进行反转录获得cDNA,使用qRT-PCR法检测各分化标志基因(MyHC、MyoD、MyoG)的表达。qPCR使用引物序列、反应体系及反应条件同实施例3中表4-6所示。重复40个循环,每个样本技术重复3次,应用2-Δ-CT法分析检测结果。
结果如图3B所示,转染si-Gm10561组的细胞分化标志基因MyHC、MyoD、MyoG的mRNA水平表达量低于对照组,表明下调lncRNA Gm10561的表达,抑制了小鼠C2C12细胞的分化。
实施例5抑制lncRNA Gm10561表达后影响小鼠体内骨骼肌生长发育
采用吉玛公司生产的干扰慢病毒LV3-sh-Gm10561和LV3-sh-NC,滴度为1×109TU/mL,注射前用PBS稀释至最终滴度为5×108TU/mL。分别在6周龄C57BL/6小鼠(购自广东省医学实验动物中心)后腿肌肉中注射终体积100μLLV3-sh-Gm10561或LV3-sh-NC对照慢病毒,每周进行一次,持续一个月。最后一次注射后一周处死小鼠,采集腿肌样品,在含有1%PMSF(Beyotime)的RIPA缓冲液(Beyotime)中裂解,并在6×蛋白上样缓冲液中于95℃加热5分钟,进行Western blotting实验,使用的抗体如下表8所示。
表8小鼠蛋白抗体
结果如图4所示,Gm10561敲低组细胞分化标志基因MyoG和MyHC蛋白表达水平显著低于对照组,表明下调lncRNA Gm10561的表达,能抑制小鼠体内骨骼肌发育。
实施例6超表达载体pcDNA3.1-Gm10561的构建
使用Bam HⅠ与HindⅢ对pcDNA3.1(+)真核表达载体进行双酶切,具体酶切反应体系如下表9所示,并于37℃酶切2h,再用1.5%琼脂糖凝胶电泳检测酶切结果并回收目的片段。
表9 pcDNA3.1(+)双酶切反应体系
将回收的片段按照表10反应体系进行配制,于16℃反应3min(此步骤过程按照T4DNA连接酶试剂盒(Takara)操作)。
表10反应体系
重组连接产物转化采用Trans5α感受态细胞(TransGen)进行转化,其具体操作步骤如下:
(1)取50μL冰浴上融化的感受态细胞,加入目的DNA,轻轻混匀,在冰浴中放置30min;
(2)置于42℃水浴中热激45s,然后快速将其转移至冰浴中2min,该过程不要摇动离心管;
(3)向离心管中加入500μL无菌的LB培养基(不含抗生素),混匀后置于37℃,200rpm摇床中复苏1h;
(4)根据实验要求,吸取适量体积已转化的感受态细胞加到含氨苄抗性的LB琼脂培养基上,将细胞均匀涂开;
(5)将平板置于37℃培养箱中正放培养30min,倒置平板,过夜培养。
在超净工作台中用灭菌枪头从抗性平板中挑取单克隆菌落到500μL含有氨苄青霉素的LB液体培养基中,置于37℃恒温摇床扩大培养4-6h,取培养后的菌液1μL为模板,以实施例1中表1所示特异性引物进行PCR扩增,琼脂糖凝胶电泳检测,确定阳性克隆后测序。利用NCBI网站中的BLASTn比对测序结果,鉴定序列是否正确。使用Endo-free Plasmid MiniKitⅡ试剂盒(OMGEA)提取去内毒素质粒,利用BamHⅠ与HindⅢ双酶切鉴定抽提的质粒,酶切体系如表11所示,37℃酶切2h,置于-20℃冰箱备用。采用1.5%琼脂糖凝胶电泳进行检测,检测结果如图5所示,其中pcDNA3.1(+)载体骨架扩增出的条带大小为5428bp,插入的lncRNA Gm10561片段扩增出的条带大小为1539bp。
表11克隆载体双酶切反应体系
实施例7超表达载体pcDNA3.1-Gm10561在小鼠成肌细胞增殖分化中的作用
1、EdU细胞增殖检测
实验前,将小鼠C2C12细胞接种于24孔板中,于37℃、5%CO2培养箱培养,待细胞融合度达到60%-80%,转染重组质粒,转染24h后利用BeyoClickTM EdU细胞增殖试剂盒(Beyotime)按照制造商说明书进行细胞增殖检测,具体操作步骤同实施例4。
检测结果如图6所示,转染pcDNA3.1-Gm10561组细胞EdU阳性率高于对照组,表明上调lncRNA Gm10561的表达,促进了小鼠C2C12细胞的增殖。
2、细胞免疫荧光表型水平检测
实验前,将小鼠C2C12细胞接种于24孔板中,于37℃、5%CO2的培养箱中培养,待细胞融合度达到60%-80%,转染重组质粒,继续培养。在诱导分化2天后进行细胞免疫荧光实验,采用的细胞免疫荧光抗体如表12所示,具体步骤如下:
(1)DPBS清洗细胞2次,每次5min;
(2)每孔加入200μL 4%多聚甲醛固定细胞,室温脱色摇床孵育30min,弃固定液;
(3)每孔加入500μL DPBS,脱色摇床清洗3次,每次5min,弃DPBS;
(4)每孔加入200μL透化液(0.5%Triton X-100),室温脱色摇床孵育30min,弃透液;
(5)每孔加入500μL DPBS,脱色摇床清洗3次,每次10min,弃DPBS;
(6)每孔加入200μL快速封闭液(Beyotime),37℃脱色摇床封闭2h;
(7)每孔加入200μL一抗(具体见表12),4℃过夜;
(8)每孔加入500μL DPBS,脱色摇床清洗3次,每次10min,弃DPBS;
(9)每孔加入200μL荧光标记二抗(具体见表12),避光,37℃脱色摇床孵育1h;
(10)每孔加入500μL DPBS,脱色摇床清洗3次,每次10min,弃DPBS;
(11)每孔加入200μL DAPI试剂(Beyotime)用于染色细胞核,避光室温脱色摇床孵育5min;
(12)弃反应液,DPBS清洗细胞3次,每次10min,用荧光显微镜进行成像检测。
表12细胞免疫荧光抗体
结果如图7所示,转染pcDNA3.1-Gm10561组细胞核MyoG、MyHC阳性的细胞数量高于对照组,说明上调lncRNA Gm10561的表达,促进了小鼠C2C12细胞的分化。
实施例8超表达载体pcDNA3.1-Gm10561在猪骨骼肌卫星细胞增殖分化中的作用
1、EdU细胞增殖检测
实验前,将猪骨骼肌卫星细胞接种于24孔板中,于37℃、5%CO2的培养箱中培养,待细胞融合度达到60%-80%,转染重组质粒,转染24h后利用BeyoClickTM EdU细胞增殖试剂盒(Beyotime)按照制造商说明书进行细胞增殖检测,其具体操作步骤同实施例4。
检测结果如图8所示,转染pcDNA3.1-Gm10561组细胞EdU阳性率高于对照组,说明上调lncRNA Gm10561的表达,促进了猪骨骼肌卫星细胞的增殖。
2、细胞免疫荧光表型水平检测
实验前,将猪骨骼肌卫星细胞接种于24孔板中,于37℃、5%CO2的培养箱中培养,待细胞融合度达到60%-80%,转染重组质粒,继续培养。在诱导分化2天后进行细胞免疫荧光实验,具体步骤如实施例7。
结果如图9所示,转染pcDNA3.1-Gm10561组细胞核MyHC阳性的细胞数量高于对照组,说明上调lncRNA Gm10561的表达,促进了猪骨骼肌卫星细胞的分化。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
序列表
<110> 华南农业大学
<120> 长链非编码RNA Gm10561在调控成肌细胞增殖分化中的应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1539
<212> DNA
<213> 长链非编码RNA Gm10561(SIPOSequenceListing 1.0)
<400> 1
gtctcaccca gttggctgaa tggaaaagag gtgtccagaa acggaattaa agacaaaagt 60
gcaactctgg tgtgattatc tacggataga agataaagtc agtcaaagtg atgcggttct 120
ttctgtttct tctgtgggac atctgccaca ggatggcttt caagactggg agagaactgg 180
tgttctgccc tagcacccgg cctgcagaga gcaatgctcc tatgagacag agctggcgtc 240
aggattggaa ggaagtgatt tcggcaccac ttcaactatt tacaaaatca accctgatct 300
cctttgagaa cctggagagt gccagtgaat ggactgttgt gctgttatac agtagcaatg 360
gaatctttgc tggtcactgg caccaattac ggtattggag agttgtttat taagtatcca 420
ctacctttat caagtgcctg ccacttccag gtacttaaaa agcactggat acaaaagatg 480
actcaacagg aacatatggg acgatatcct ggatggtttc gaggcatcgt ttcctgagcc 540
ctgaggtttg atgaagatgt cccatttagg actgagtgtt ccacagcctc tcatcctctg 600
cacactgttc actcatggct atctatacaa gctctcatct gtaggaggac acttttctga 660
tgctggctca gtgagacact gatctatgca tacagcagaa tgttgttagg cgtcatttta 720
ttgttattgt ccttctagca gaacagtagt gtttggtttg ctcctgggtc catgcctgtc 780
tagtctcagg ttcttggcca actgagaagt gtcagggact ggctttattt catggagtgg 840
tccctaaatc caaccagaca gtggttggtt actcccacat cttgtgtggc attgttgtac 900
cagcatatca tgcagagagg ccatcattgc ataaggaaag gtttgtaact ggcttggtgt 960
ttagctttat cctctagtca gtgcagagta ccttccagga ccatgaatgc tagtgagcag 1020
acaaccctct acttttagtt ttatctactt tctacttgcc ttttgcaaat ttgcagatca 1080
actagaacac acagactgac tacgccgtgg acgtttgttt caccttctgt agttcctgta 1140
gagaacgcca tcagtctatc ttctaaatca tcccaaaaag catatccaac tcaccacaag 1200
tctttacaca ccatgcagaa gtcaaaagga atgaacaata tggttgaatc ttaggaagaa 1260
aatgttgaat tagaaagtga caactgccct gctcttctgc tacctgctga gaagctgaac 1320
ccgttttcct gagaatttcc ggaataagcg gccagcctgg ccaccgagtg ctgacgaaat 1380
ggctccaggc accaagtgtg gagccggcag actagtgcta acaacttggc aacaatacat 1440
caagaagcct ggcgggggtg ggtggggggg gtgggggggc gatgggcctt cccccttata 1500
agcacggtct cttaataaac tcgtgggcct tgatcagga 1539
Claims (10)
1.SEQ ID NO:1所示的长链非编码RNA Gm10561在调控动物骨骼肌生长发育中的应用。
2.SEQ ID NO:1所示的长链非编码RNA Gm10561或其表达促进剂在促进动物骨骼肌生长发育中的应用。
3.SEQ ID NO:1所示的长链非编码RNA Gm10561在调控动物成肌细胞增殖和/或分化中的应用。
4.SEQ ID NO:1所示的长链非编码RNA Gm10561或其表达促进剂在促进动物成肌细胞的增殖和/或分化中的应用。
5.SEQ ID NO:1所示的长链非编码RNA Gm10561在调控动物骨骼肌卫星细胞的增殖和/或分化中的应用。
6.SEQ ID NO:1所示的长链非编码RNA Gm10561或其表达促进剂在促进动物骨骼肌卫星细胞增殖中的应用。
7.SEQ ID NO:1所示的长链非编码RNA Gm10561或其表达促进剂在促进动物骨骼肌卫星细胞分化中的应用。
8.一种促进成肌细胞增殖和/或分化的方法,其特征在于,在动物体内过表达长链非编码RNA Gm10561或利用其表达促进剂,来促进成肌细胞增殖和/或分化。
9.根据权利要求8所述方法,其特征在于,构建含有权利要求1所述长链非编码RNAGm10561的超表达载体,转染成肌细胞。
10.根据权利要求1-7任一所述应用或权利要求8所述方法,其特征在于,所述动物为小鼠或猪。
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