CN111690645A - 一种源于鹿茸软骨的microRNA及其应用 - Google Patents
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Abstract
一种源于鹿茸软骨的microRNA及其应用,它涉及一种小RNA(microRNA),本发明提供了来源于东北马鹿(Cervus elaphus)鹿茸软骨并对细胞起抑制作用的因子,其命名为:microRNA PC‑5p‑2869,其序列为CGCAUUGGUGGUUCAGUGG,具有高效下调EEF1A1蛋白表达的作用,可抑制细胞增殖、迁移和侵袭。
Description
技术领域
本发明涉及一种小RNA(microRNA),负调控细胞的增值、迁移和侵袭。
背景技术
在哺乳动物中,只有鹿茸角是目前所知唯一具有表形态再生能力的器官,即在失去后还能完全再生出来。在再生周期里,鹿茸从顶部生长点平均每天延伸1-2cm,又会在近百天的快速生长后最终自动停止,茸性皮肤会慢慢死亡脱落。鹿茸角再生机理复杂,目前仍然是再生生物学的重要研究模型。
MicroRNA(miRNA)是一类内生的、长度为18-24个核苷酸、重要的基因表达的负调控因子,通过调节目标基因mRNA稳定性或翻译效率来表现其功能。其来源于长的具有发卡样二级结构的转录子,并被两种核酸内切酶Drosha和Dicer剪切,形成双链的miRNA。其中一条链形成成熟的miRNA而另一条链降解。成熟miRNA通过与靶标基因mRNA的3’非翻译区(Untranslated Regions,UTR)目标位点结合而对其进行调控。一个miRNA的选择性活性依赖于与这个同源mRNA互补的miRNA的种子序列(seed region)(图1所示)。因为microRNA 种子内的不完美碱基配对代表这个核心5-7核苷酸序列可以潜在识别结合很多的mRNA序列,一个miRNA同时调控几百个目标基因的mRNA。证据表明miRNA在很多细胞的增殖、分化、器官形态发生和肿瘤发生等生物学过程中起非常关键的调节作用。miRNA通过抑制目标基因起到癌基因或肿瘤抑制的作用来参与调节肿瘤发生和发展。目前,越来越多的miRNA 被证明在癌症发生过程中发挥非常重要的作用,这说明miRNA可以作为治疗癌症的潜在新靶标。
真核翻译延伸因子1Alpha1(Eukaryotic Translation Elongation Factor1Alpha 1,EEF1A1) 是参与蛋白质翻译延伸的重要蛋白,具有触发翻译延伸启动的作用。EEF1A1是真核细胞中高度保守的GTP结合蛋白,在蛋白质合成过程中起着重要作用。研究表明,EEF1A1在细胞中发挥重要作用,调节细胞的各种生物学特性,如细胞周期、细胞生长和细胞死亡;EEF1A1在许多肿瘤组织中异常上调,EEF1A1的过度表达与癌症细胞增殖密切相关,下调EEF1A1表达可抑制许多肿瘤细胞增殖。
发明内容
本发明确定了东北马鹿(Cervus elaphus)鹿茸软骨来源的,通过EEF1A1介导抑制细胞增殖、迁移和侵袭的负调控因子,并确定这种因子的名称、序列。本发明从马鹿鹿茸软骨组织的microRNA数据库中筛选得到的microRNA PC-5p-2869(简称miRNA PC-2869),是一种在鹿茸软骨中特异性表达的miRNA,长度19nt。序列如SEQ ID NO:1所示(CGCAUUGGUGGUUCAGUGG)。
本发明经过293T、MG63等细胞的细胞增殖、迁移、侵袭检测和Western Blotting等实验证明miRNA PC-2869下调细胞增殖关键转录因子EEF1A1的表达(图2、3、4所示),并可介导抑制细胞增殖(图5、6所示)、迁移(图7、8所示)和侵袭(图9、10所示)。
本发明包含以下有益效果:
作为基因表达的负调控因子,microRNA在细胞生长、器官发育、个体生长及各种疾病的进程中都起到非常重要的作用。从快速生长的鹿茸顶端软骨组织中分离出来的microRNA对鹿茸的再生生长会起非常重要的调控作用。
因此,本发明从快速生长期的新生马鹿鹿茸顶端软骨组织microRNA测序结果中得到一个 miRNA PC-2869,其转录前体长约88nt,可形成发卡结构,成熟miRNA全长19nt,在miRBase 数据库(Release 22.1)中无同源序列。BLAST结果显示,无论是成熟miRNA全长还是其seed 序列,在人、小鼠、大鼠、狗、鸡、猪、牛等模式物种基因组中均无同源序列。
通过TargetScan预测鹿茸软骨发育的关键性因子EEF1A1是miRNA PC-2869的靶基因之一,通过miRNA PC-2869mimics实验验证了miRNA PC-2869对EEF1A1具有靶向作用,在MG63、293T等细胞中证明了miRNA PC-2869具有高效下调EEF1A1蛋白的作用(实验结果如图2、3、4所示),并抑制细胞增殖(实验结果如图5、6所示)、迁移(实验结果如图7、8所示) 和侵袭(实验结果如图9、10所示)。
本发明筛选的基因序列如下:
名称:microRNA PC-5p-2869
序列:CGCAUUGGUGGUUCAGUGG。
附图说明
图1为多种模式生物EEF1A1mRNA 3’UTR区具有潜在的miRNA PC-2869的seed序列(小写斜体加下划线序列)互补结合保守位点(小写正体序列);Has,人;Mmu,小鼠;Rno,大鼠;Eca,马;Bta,牛。
图2为miRNA PC-2869mimics下调MG63细胞内源EEF1A1蛋白水平图;将PC-2869mimics与NC分别转染MG63细胞,48小时后收蛋白并进行western检测,内参为GAPDH。
图3为miRNA PC-2869mimics下调转染293T细胞的pMIR-REPORT-EEF1A1-3’UTR-WT载体中Firefly luciferase的表达。将PC-2869mimic与下述两种载体分别共转染293T细胞, 48小时后收蛋白并进行western检测,由于pMIR-REPORT-EEF1A1-3’UTR-MUT中miRNAPC-2869结合位点因突变而缺失,所以转染该突变载体的细胞中Firefly luciferase依然表达;实验设NC为对照组,内参为psiCHECK-1中的Renilla luciferase。
图4为MG63中转染si-EEF1A1后EEF1A1的表达受到抑制。将si-EEF1A1转染MG63 细胞,48小时后收蛋白并进行western检测;实验设si-NC为对照组,内参为GAPDH。
图5为CCK-8法检测转染miRNA PC-2869mimics后以24小时为间隔的MG63细胞增殖活性时序图,设miR-NC阴性对照组;实验组中细胞增殖显著受到抑制。其中,横坐标为时间,纵坐标为细胞450nm吸收值;实验独立重复三次,取平均数并计算SD值,t检验统计学差异(*.p<0.05,**.p<0.01,***.p<0.001)。
图6为CCK-8法检测转染si-EEF1A1后以24小时为间隔的MG63细胞增殖活性时序图;设si-NC阴性对照组;实验组中细胞增殖显著受到抑制。其中,横坐标为时间,纵坐标为细胞450nm吸收值;实验独立重复三次,取平均数并计算SD值,t检验统计学差异(*.p<0.05,**.p<0.01,***.p<0.001)。
图7为Trans-well法检测miRNA PC-2869mimics后24小时检测迁移的细胞数;设miR-NC 阴性对照组;实验组中细胞迁移显著受到抑制。实验独立重复三次,取平均数并计算SD值, t检验统计学差异(***.p<0.001)。
图8为Trans-well法检测转染si-EEF1A1后24小时检测迁移的细胞数;si-NC阴性对照组;实验组中细胞迁移显著受到抑制。实验独立重复三次,取平均数并计算SD值,t检验统计学差异(***.p<0.001)。
图9为Trans-well法检测miRNA PC-2869mimics后24小时检测侵袭的细胞数;设miR-NC 阴性对照组;实验组中细胞侵袭显著受到抑制。实验独立重复三次,取平均数并计算SD值, t检验统计学差异(***.p<0.001)。
图10为Trans-well法检测转染si-EEF1A1后24小时检测侵袭的细细胞数;si-NC阴性对照组;实验组中细胞侵袭显著受到抑制。实验独立重复三次,取平均数并计算SD值,t检验统计学差异(***.p<0.001)。
具体实施方式
具体实施方式一:本实施方式的一种源于鹿茸软骨的microRNA,它命名为:microRNA PC-5p-2869,其序列为CGCAUUGGUGGUUCAGUGG(如Seq ID No:1所示)。
具体实施方式二:本实施方式的一种源于鹿茸软骨的microRNA的应用,抑制真核翻译延伸因子EEF1A1表达。
具体实施方式三:本实施方式的一种源于鹿茸软骨的microRNA的应用,它具有通过抑制EEF1A1的表达介导抑制细胞增殖、迁移和侵袭的生物学功能。
本发明内容不仅限于上述各实施方式的内容,其中一个或几个具体实施方式的组合同样也可以实现发明的目的。
通过以下实施例验证本发明的有益效果:
以下的实施例便于更好地理解本发明,但并不限定本发明。下述实施例中的实验方法,如无特殊说明,均为常规方法。下述实施例中所用的试验材料,如无特殊说明,均为自常规生化试剂公司购买得到的。下述实施例中的%,如无特殊说明,均为质量百分含量。以下实施例中的定量试验,均设置三次重复实验,结果取平均值。
实施例1、鹿茸软骨miRNA PC-2869的获取过程
1)材料获取:实验所用材料为处于快速增长期约70天的东北马鹿鹿茸软骨组织。锯茸后立即用消毒的剪刀将鹿茸顶端组织切成厚度小于0.5cm的片状,立即放入装有防止RNA降解的保护液(RNA later)中,置于低温保存箱中并带回实验室保存。
2)mRNA的提取:将实验室保存鹿茸的软骨组织分离后称取30mg,在超净工作台中快速切碎置于无菌1.5mL试管中,利用QIAGEN公司(德国)试剂盒DNeasy Blood&Tissue Kit(Cat.No.69504)提取鹿茸软骨中的总RNA,并将提取的总RNA最终溶解于RNAse-free的水中,提取的总RNA经过分光光度检测均具有较高的质量(OD260nm/OD280nm: 1.76-1.85)。
3)鹿茸软骨small RNA高通量测序:将提取的总RNA递交到美国联川公司(杭州)通过Illumina miRNA Solexa测序平台进行鹿茸软骨组织small RNAs文库高通量测序后,用ACGT101-miR v4.2(LC Sciences)软件进行数据处理。
4)鹿茸软骨新microRNA(miRNA),miRNA PC-2869的获取:对鹿茸软骨组织的smallRNA文库Solexa深度测序后获得的序列原始数据进行综合分析,得到一种新microRNA,miRNA PC-2869,其成熟体序列可以比对到马鹿基因组第20号染色体上,而且延伸的基因组序列可以形成合格的发夹结构。
5)靶基因预测:通过TargetScan预测miRNA PC-2869的靶基因之一为调节翻译的关键因子EEF1A1。
实施例2、miRNA PC-2869在细胞水平上能够下调促细胞增殖、迁移和侵袭的关键性因子EEF1A1表达
1)miRNA PC-2869和si-EEF1A1的合成:将miRNA PC-2869和si-EEF1A1的序列递交到上海吉玛公司合成mimics和si-EEF1A1(Rossella Farra et al,2017)(如SEQ ID NO:2所示)如下:AUGCGGUGGCAUCGACAAATT,及相应的Negative Control(NC)和Si-NC。
2)构建pMIR-REPORT-EEF1A1-3’UTR载体:序列比对分析得知多种模式生物EEF1A1mRNA 3’UTR区具有潜在的miRNA PC-2869seed序列(小写加下划线序列)互补结合保守位点(小写未加下划线序列)(图1所示)。因此,本实施例根据人、小鼠、大鼠、马、牛等物种中保守的EEF1A1的3’UTR区序列合成了64bp的片段(Invtrogene公司,上海)插入到 pMIR-REPORT(Applied Biosystems公司,美国)载体中,构建野生型载体 pMIR-REPORT-EF1A1-3’UTR-WT,其中插入片段包含miRNA PC-2869种子序列的互补序列,插入片段(如Seq ID NO:3所示)如下:
CCATTTAAGTTTAGTAGTAAAAGACTGGTTAATGATAACAATGCATCGTAAAACCTTCAG AAGG;相应地,构建突变型载体pMIR-REPORT-EF1A1-3’UTR-MUT,其中插入片段58bp 中不包含miRNAPC-2869种子序列的互补序列,插入片段(如Seq ID NO:4所示)如下: CCATTTAAGTTTAGTAGTAAAAGACTGGTTAATGATAAATCGTAAAACCTTCAGAAGG。
pMIR-REPORT载体是ABI公司(美国)设计的将一个miRNA的靶序列插入到多克隆位点中,pMIR-REPORT的荧光素酶蛋白Luciferase报告载体可以用来定性和定量衡量miRNA的功能。当miRNA PC-2869mimics和pMIR-REPORT-EF1A1-3’UTR-WT载体共转染细胞时,如果Luciferase蛋白表达下调,说明miRNA PC-2869对EEF1A1表达有下调作用。
3)细胞培养:293T细胞和MG63细胞均购自吉满生物科技(上海)有限公司,培养基为DMEM(10569010,Gibco,美国),均添加10%的胎牛血清(110099141,Gibco,美国)。置于37℃,5%二氧化碳条件下培养。
4)转染与Western检测:细胞以6×105个/孔种于6孔培养板上,培养24小时,细胞汇合度达到60%~70%,以Lipofectmin3000(L3000015,Invitrogen,美国)为介导,将合成的miRNA PC-2869的mimics和EEF1A1-3’UTR-pMIR-REPORT载体对293T细胞、MG63细胞进行转染,转染的重组质粒量为2.0ug/孔。24~36小时收细胞,对蛋白进行变性处理后,利用4%~12%的 PAGE预制胶(NP0335BOX,Invitrogen,美国)电泳。转硝酸纤维膜后,用5%脱脂奶粉的 TBST混合液封闭1小时,然后硝酸纤维膜分别在EEF1A1一抗(ab157455,abcom,英国)(图 2)或Firefly Luciferase一抗(Santa Cruz Biotechnology,美国)(图3)中孵育1小时,荧光标记的二抗(Licor Bioscience,美国)中孵育1小时,以GAPDH(Sc47724,SantaCruz Biotechnology,美国)或Renilla Luciferase(ab185926,abcom,英国)为内参,Odyssey红外激光成像系统扫膜。检测结果表明miRNA PC-2869有效下调EEF1A1的表达(图2、图3、图4所示)。
实施例3、miRNA PC-2869在细胞水平上通过抑制真核生物翻译延伸因子1(EEF1A1) 的表达介导抑制细胞增殖、迁移和侵袭的功能
1)细胞培养与转染:293T细胞和MG63细胞均购自吉满生物科技(上海)有限公司,培养基为DMEM(Gibco,美国),培养方法同实施例2中3)。细胞转染同实施例2中的4)。
2)细胞增殖活性检测:利用CCK-8(Dojindo)试剂盒检测miRNA PC-2869mimics/NC转染MG63细胞后的增殖情况,每隔24小时对转染后的细胞进行计数统计。检测结果表明相比对照组(NC),miRNA PC-2869mimics的MG63细胞增殖受到抑制(图5所示),而这种对细胞增殖的抑制是通过抑制真核生物翻译延伸因子EEF1A1的表达介导的(图6所示)。
3)细胞迁移检测:将悬浮有1.6×105个细胞的200μl无血清DMEM加入到24-well(8.0μm 孔径)的细胞培养插件transwell(353097,Corning,美国)上室中,transwell下室内加入600μl 的无血清DMEM培养基,培养6小时之后,将下室的无血清DMEM培养基换成含10%FBS的DMEM培养基,并对上室的细胞分别转染25pmol的microRNA mimics和mimics NC,或siRNA 和si-NC,细胞转染同实施例2中的4)。转染24小时后,用棉球将膜上层的细胞除去,迁移到膜下层的细胞用4%的多聚甲醛(P0099,碧云天,上海)固定并用0.5%的结晶紫(C0121,碧云天,上海)染色,蒸馏水清洗并晾干,显微镜下(200×倍数)观察,随机选取5个视野拍照并计数。细胞迁移结果表明相比对照组(NC),miRNA PC-2869mimics的MG63细胞迁移受到抑制(图7所示),而这种对细胞迁移的抑制是通过抑制真核生物翻译延伸因子EEF1A1 的表达介导的(图8所示)。
4)细胞侵袭检测:将悬浮有1.6×105个细胞的200μl无血清培养基加入到24-well-Biocat基质胶侵袭室(354480,Corning,美国)transwell上室中,以下步骤同实施例3中的3)。细胞侵袭结果表明相比对照组(NC),miRNA PC-2869mimics的MG63细胞侵袭受到抑制(图9 所示),而这种对细胞侵袭的抑制是通过抑制真核生物翻译延伸因子EEF1A1的表达介导的(图 10所示)。
Claims (3)
1.一种源于鹿茸软骨的microRNA及其应用,其特征在于它命名为:microRNA PC-5p-2869,其序列为CGCAUUGGUGGUUCAGUGG。
2.一种源于鹿茸软骨的microRNA及其应用,其特征在于它抑制细胞增殖、迁移和侵袭。
3.根据权利要求1所述的一种源于鹿茸软骨的microRNA的应用,其特征在于是通过抑制真核生物翻译延伸因子1(EEF1A1)的表达介导抑制细胞增殖、迁移和侵袭生物学功能的完成。
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