CN113528522A - miR-339-增强子-靶基因网络激活模型及其应用 - Google Patents
miR-339-增强子-靶基因网络激活模型及其应用 Download PDFInfo
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Abstract
本发明涉及生物技术领域,具体涉及miR‑339‑增强子‑靶基因网络激活模型及其应用。首先公开了一种可激活乳腺癌相关基因的miRNA,所述miRNA的基因座位置与增强子的组蛋白修饰标志H3K4me1或H3K27ac重合。本发明提供了可激活疾病相关基因的miR‑339与乳腺癌相关,为乳腺癌等疾病的诊断、治疗提供新的策略和潜在的药物靶点。并且,本发明首次将“miRNA‑增强子‑靶基因网络激活模型”理论应用于解释乳腺癌的发生发展现象,克服了现有技术的偏见。
Description
技术领域
本发明涉及生物技术领域,具体涉及miR-339-增强子-靶基因网络激活模型及其应用。
背景技术
非编码RNA的研究是近年来逐渐被科学家认识并迅速投入其中的基础科学研究,旨在从本质上认识生命的奥秘。非编码RNA种类繁多,目前研究较多的是microRNA(miRNA)和长非编码RNA(LncRNA)。miRNA是一些长度为21-25个核苷酸,在转录后水平调控基因表达的非编码小RNA。第一个miRNA lin-4是30多年前在研究线虫的发育调控时被意外发现的,并被证明通过结合细胞浆中lin-14mRNA的3’UTR发挥抑制蛋白翻译或降解mRNA的作用。此后,miRNA的负向调控作用机制几乎成为miRNA研究领域“金科玉律”。尽管后来也发现部分miRNA在特殊情况下能够促进基因表达或翻译,但基本都是个例研究,仅限于现象描述和简单的机制研究,致使后续研究者不得要领,缺乏推而广之的意义。另一方面,虽然miRNA参与大多数生命现象和过程,但是miRNA在体内的调控通常被认为是微调控(fine-tune),并没有起到决定性的“开关”作用。
增强子(Enhancer)一直被认为是重要的DNA序列顺式调控元件,它可以近距离或远距离调控基因的表达。H3K4me1以及H3K27ac是增强子重要的表观遗传学标记,其中H3K27ac与增强子的活性密切相关。当多个具有转录活性的增强子高密度富集转录因子后,可以形成超级增强子。增强子尤其是超级增强子通常可以驱动控制细胞身份和细胞命运的基因表达,在细胞分化、组织发育、个体生长和疾病致病过程中发挥很重要的作用。
恶性肿瘤是危害人类健康的全球公共卫生问题,我国作为一个发展中大国,工业化、城镇化和人口老龄化问题日益突出,不良生活方式及环境污染等直接导致国民身体健康指数下降,恶性肿瘤急需等待攻克。
miRNA研究已经被证明在乳腺癌发生发展与迁移中发挥重要的调控作用。相关研究通常是基于传统的miRNA负向调控机制,到目前为止,如何利用miRNA攻克乳腺癌尚未有实质性的突破,尚缺乏有效的治疗药物和治疗方案。miRNA是经典的核酸分子,核酸药物被认为是继抗体药物及细胞免疫疗法之后的下一代药物,其可以达到传统药物无法替代的效果,比如抗体多在细胞表面发挥作用,而核酸药物可以进入细胞内部发挥治疗作用;有的核酸药物甚至可以进入到细胞核发挥精确的靶向治疗作用。比如,2018年美国FDA批准上市的RNAi新药Onpattro(patisiran)是一款核酸药物。主要的治疗方法是把siRNA包裹在脂质纳米颗粒中并将药物直接递送至肝脏,通过靶向和沉默TTR mRNA,阻碍靶向TTR蛋白生成,从而降低周围神经中淀粉样沉积物的积累,最终达到治疗hATTR(遗传性转甲状腺素蛋白)淀粉样变性疾病的目的。目前核酸药物开发日益成熟,在癌症和遗传性疾病以及流感和病毒感染方面取得一些重要成果,但是有待开发的潜力巨大。
发明内容
本发明首次将创造性的“miRNA-增强子-靶基因网络激活模型”理论应用于解释乳腺癌的发生发展现象,即核内miR-339与增强子互作进而正向激活靶基因从而影响肿瘤的增殖生长,这一理论与现有技术“miRNA的负向调控机制”完全不同。
本申请筛选了位于增强子区域的多个miRNA,分别将这些miRNA在不同种类的细胞中过表达,发现在基因组上与这些miRNA邻近的多个基因被转录激活,这些miRNA对基因的转录激活调控依赖于miRNA及其靶向增强子序列的完整性。基于此,本申请将筛选出来的miRNA用于揭示细胞核内miRNA功能,为解析miRNA参与肿瘤等疾病发生发展提供新的视角。
具体地,本申请发现miR-339和靶基因GPER1倾向于在乳腺癌临床组织样本中低表达,进一步验证发现miR-339通过增强子正向调控GPER1基因的表达,进而抑制乳腺癌肿瘤发生发展。如果从核酸药物的角度去开发新型乳腺癌的药物,那么miR-339有可能会为这些疾病患者带来福音。
为实现上述目的,本发明采用如下技术方案:
本发明第一个方面公开了一种可激活乳腺癌相关基因的miRNA,所述miRNA的基因座位置与增强子的组蛋白修饰标志H3K4me1或H3K27ac重合。
优选的,所述乳腺癌为Luminal型乳腺癌和三阴性乳腺癌。
术语“miRNA”即MicroRNAs,其是指在真核生物中发现的一类内源性的具有调控功能的非编码RNA,其大小长约20~25个核苷酸。成熟的miRNAs是由较长的初级转录物经过一系列核酸酶的剪切加工而产生的,随后组装进RNA诱导的沉默复合体,通过碱基互补配对的方式识别靶mRNA,并根据互补程度的不同指导沉默复合体降解靶mRNA或者阻遏靶mRNA的翻译。
优选的,所述miRNA的核苷酸序列如SEQ ID NO:1所示,或
所述miRNA的序列为与所述SEQ ID NO:1具有90%同源性且功能相同的序列。
所述SEQ ID NO:1序列从5’→3’为:UCCCUGUCCUCCAGGAGCUCACG。
优选的,所述乳腺癌相关基因为GPER1(G蛋白偶联雌激素受体1)基因。
本发明第二个方面公开了一种核酸药物,所述核酸药物包括上述的miRNA或其人工模拟物;优选的,所述核酸药物还包括药学上可接受的载体或赋形剂。
更优选的,所述核酸药物剂型包括散剂、片剂、颗粒剂、胶囊剂、溶液剂、气雾剂、注射剂、乳剂或混悬剂。
本发明第四个方面一种与是上述核酸药物相关的生物材料,所述生物材料选自下述A)、B)或C)中的一种:
A)与上述的miRNA的核苷酸序列互补配对的干扰RNA分子或者抑制剂;
B)包含上述miRNA的核苷酸序列的表达载体或重组载体;
C)包含有B)中所述的表达载体或重组载体的细胞或微生物。
优选的,所述表达载体包括pCDH载体、pCMVp-NEO-BAN载体、pEGFP载体、pEGFT-Actin、pSV2载体、pCDNA载体、pLVX载体、pAAV载体、pET载体和pDsRed载体中的至少一种。
优选的,所述重组载体构建步骤包括:1)设计引物,PCR扩增所述miRNA序列;2)将扩增的序列片段和表达载体酶切,连接目的序列片段和表达载体;3)将连接产物转化大肠杆菌,培养;4)鉴定后提取重组质粒并进行包装即得到重组载体。
本发明第五个方面公开了上述的miRNA或上述的核酸药物在诊断、预防和/或治疗乳腺癌中的应用。
本发明第六个方面公开了一种筛选上述的可激活疾病相关基因的miRNA的方法,包括以下步骤:
S1、通过生物信息学分析,筛选基因座位置与H3K4me1或H3K27ac重合的miRNA;
S2、验证步骤S1筛选的miRNA与其周围200kb范围内的靶基因的关系;
S3、获取S2中与所述靶基因具有相关性的miRNA,即为可激活疾病相关基因的miRNA。
优选的,步骤S2中,验证miRNA与其周围200kb范围内的靶基因的关系的方法包括:实时荧光定量PCR检测过表达所述miRNA后所述靶基因的表达量。
本发明第七个方面公开了一种用于研究乳腺癌机制的模型,所述模型为miRNA-增强子-靶基因网络激活模型。
优选的,miRNA与增强子互相作用进而正向激活靶基因从而影响肿瘤的增殖生长,所述miRNA为miR-339;
优选的,所述模型的构建方法包括:筛选位于增强子区域的多个miRNA,分别将所述miRNA在不同种类的细胞中过表达,发现在基因组上与所述miRNA邻近的多个基因被转录激活;得到miRNA-增强子-靶基因网络激活模型。
本发明第八个方面公开了上述的模型在乳腺癌领域中的应用;优选的,所述模型在制备治疗乳腺癌药物中的应用。
发明人前期在不同的组织细胞中,对1594条miRNA前体进行了系统地生物信息学分析,发现300多条miRNA前体在基因组中的位置与增强子的组蛋白修饰标志H3K4me1或H3K27ac高度重叠。并且发现,这些miRNA大多能够定位于细胞核内,进一步研究发现这些miRNA能够与增强子结合,进而在全基因组的水平上激活基因表达。鉴于增强子和miRNA都参与个体发育和疾病发生等过程,发明人将二者联系起来,探究其在肿瘤等疾病发生中的重要作用。
本发明首先通过将miRNA miR-339基因序列过表达,激活周围基因表达水平,影响乳腺癌细胞系的生物学功能。上述筛选及验证对乳腺癌的诊断及检测以及药物开发方面均具有很好的应用价值。本发明所涉及miR-339可以通过增强子激活基因的表达,与乳腺癌密切相关,其人工合成的类似物或者抑制剂可用于核酸药物的开发。
在符合本领域常识的基础上,上述各优选条件,可任意组合,而不超出本发明的构思与保护范围。
本发明相对于现有技术具有如下优点:本发明提供了可激活疾病相关基因的miR-339与乳腺癌相关,为乳腺癌等疾病的诊断、治疗提供新的策略和潜在的药物靶点。并且,本发明首次将“miRNA-增强子-靶基因网络激活模型”理论应用于解释乳腺癌的发生发展现象,即核内miR-339与增强子互作进而正向激活靶基因从而影响肿瘤的增殖生长,这一理论与现有技术“miRNA的负向调控机制”完全不同,克服了现有技术的偏见。
附图说明
图1为本发明实施例2中的乳腺癌细胞系T47D中miR-339和靶基因GPER1的表达量(图中“Relative expression levels”指“相对表达水平”);
图2为本发明实施例2中的乳腺癌细胞系4175中miR-339和靶基因GPER1的表达量;
图3为本发明实施例3中的双荧光素报告基因实验检测增强子序列活性;
图4为本发明实施例3中的双荧光素报告基因实验阐述miR-339的表达可以对增强子活性的影响;
图5为本发明实施例3中的ChIP-seq分析过表达miR-339后,miR-339 DNA locus位置附近H3K27ac富集明显高于对照组;
图6为本发明实施例3中的过表达miR-339可以激活增强子活性。ChIP-QPCR检测过表达miR-339后,miR-339 DNA locus位置附近存在显著的H3K27ac富集(图中“input”指“阳性对照”);
图7为本发明实施例3中的敲除增强子序列后过表达miR-339,无法重激活靶基因GPER1;
图8为本发明实施例3中的miR-339过表达的乳腺癌细胞株4175的增殖能力图;
图9为本发明实施例3中的miR-339过表达的乳腺癌细胞株T47D的增殖能力图;
图10为本发明实施例3中的干扰GPER1表达后的乳腺癌细胞株4175的增殖能力图;
图11为本发明实施例3中的干扰GPER1表达后的乳腺癌细胞株T47D的增殖能力图;
图12为本发明实施例3中的miR-339过表达的乳腺癌细胞株4175的克隆形成图;
图13为本发明实施例3中的miR-339过表达的乳腺癌细胞株T47D的克隆形成图;
图14为本发明实施例3中的干扰GPER1表达后的乳腺癌细胞株4175的克隆形成图;
图15为本发明实施例3中的干扰GPER1表达后的乳腺癌细胞株T47D的克隆形成图;
图16为本发明实施例3中的3组小鼠肿瘤图;
图17为本发明实施例3中的3组小鼠肿瘤重量统计图(图中“Tumor weight(g)”指“肿瘤重量”)。
具体实施方式
下面结合附图和实施例对本发明的技术方案进行详细描述,但并不因此将本发明限制在所述的实施例范围之中。
下列实施例中未注明具体条件的实验方法,按照常规方法和条件,或按照商品说明书选择。本发明所用试剂和原料均市售可得。
本申请早期在研究miRNA自身的表观遗传学调控机制时,在7种不同的组织细胞中,对1594条miRNA前体进行了系统分析,结果发现有300多条miRNA前体在基因组中的位置与增强子的组蛋白修饰标志H3K4me1或H3K27ac高度重叠。进一步应用18种组织细胞的增强子数据库进行比对分析,发现人类的1881条miRNA的60%以上与增强子的组蛋白修饰标志H3K4me1或H3K27ac重合。基于前期的工作,本申请提出miRNA—增强子—靶基因网络激活模型理论,这些miRNA大多能够定位于细胞核内,进一步研究发现这些miRNA能够与增强子结合,进而在全基因组的水平上激活基因表达。进一步地,本申请通过生物信息学数据整合分析,把基因座位置与H3K4me1或H3K27ac重合的miRNA所在位置的200kb范围内的靶基因联系起来,发现更多的miRNA—增强子—靶基因网络可用于解释生命个体的不同生理现象。
目前研究主要集中在miR-339通过负性调控靶基因,参与肿瘤发展和转移过程。发明人首先通过生物信息学分析发现NamiRNA miR-339是乳腺癌发生发展的关键因子。接着,在Luminal型乳腺癌和三阴性乳腺癌细胞系T47D和4175中分别过表达外源性miR-339,结果表明过表达miR-339后,这两个乳腺癌细胞中抑癌基因GPER1的表达均上调,它们的增殖和克隆形成能力均受到明显抑制。最后本研究结果在动物实验中进一步得到验证,提示过表达miR-339可以再激活抑癌基因GPER1从而抑制乳腺癌细胞的生长与增殖。
术语说明
如本发明中所述,术语“蛋白”是25个天然氨基酸以不同组成和排列方式构成的从二肽到复杂的线性、环形结构的不同肽类的总称,是源于蛋白质的多功能化合物。活性肽具有多种人体代谢和生理调节功能,易消化吸收,有促进免疫、激素调节、抗菌、抗病毒、降血压、降血脂等作用,食用安全性极高,是当前国际食品界最热门的研究课题和极具发展前景的功能因子。
如本发明中所述,术语“克隆”是指复制、拷贝和翻倍,就是从原型中产生出同样的复制品,它的外表及遗传基因与原型完全相同。
如本发明中所述,术语“测序”指的是对基因的序列进行判断的一种方法。
实施例1 miRNA过表达载体构建
本实施例为miR-339过表达载体的构建,其步骤主要包括:
1.1、序列获取和引物设计
首先从UCSC数据库和miRbase数据库上获取pri-miR-339的DNA序列信息,利用Primer 5.0软件设计相关引物序列,再用BLAST检测引物适用性。引物委托上海桑尼生物科技有限公司进行合成。最终设计的引物为:PCDH339-EcoRI-F和PCDH339-BamHI-R。
PCDH339-EcoRI-F:GAAGATTCTAGAGCTAGCGAATTCGGCCACAGGCAGGTGCCACC(SEQ IDNO:2);
PCDH339-BamHI-R:GCAGATCCTTCGCGGCCGCGGATCCACACCAGTGGAACATCCCATGC(SEQID NO:3)。
1.2、PCR扩增获取pri-miR-339 DNA序列
使用293T细胞的基因组作为模板以及步骤1.1设计的引物进行PCR扩增。其中,
PCR反应体系:
其中,KODFX高保真酶购于TOYOBO公司,货号KFX-101。
PCR反应条件:
1.3、PCR产物的回收、酶切及纯化
PCR产物的回收:将PCR产物进行1%琼脂糖凝胶电泳检测,并使用普通琼脂糖凝胶DNA回收试剂盒(天根生化科技公司)回收目的片段。
酶切及纯化:酶切过程参照NEB网站酶切体系37℃酶切过夜,并采用PCR产物回收试剂盒(天根生化科技公司)纯化回收。
1.4、连接
利用T4连接酶,将酶切后的PCR产物和酶切后的pCDH载体连接,16℃连接过夜。
连接体系:
1.5、转化、挑取单克隆
(1)将10μl步骤1.4得到的连接产物加入50μl的DH5α感受态细胞中,冰上孵育30min。
(2)42℃热激感受态细胞90s后,立即放到冰上5min。
(3)在超净工作台中加入300μl不含抗生素的LB液体培养基,37℃恒温摇床中摇30min。
(4)菌液1000g离心5min后弃上清,剩50μl菌液,将菌液均匀涂到氨苄抗性的LB固体平板上,37℃恒温培养箱培养过夜。
(5)从过夜培养的平板上挑取适量单克隆菌落,分别放入加有200μl氨苄抗性LB液体培养基的EP管中,37℃恒温摇床中摇菌2h后通过PCR克隆鉴定。
(6)PCR克隆鉴定,使用293T细胞的基因组作为模板进行PCR扩增。
PCR反应体系:
其中,2×Taq Master Mix购于lifetech公司。
PCR反应条件:
取少量菌液接入到LB培养基中,加入相应的抗生素(氨苄或者卡那),以250-300rpm/min的转速,37℃振荡培养过夜。抽提质粒获得过表达miR-339的质粒PCDH-miR339。
实施例2 miR-339激活靶基因GPER1
本实施例在乳腺癌细胞T47D和4175中过表达miR-339序列,对周围基因表达水平进行检测。
2.1、脂质体法包制慢病毒:依照分子克隆,将实施例1得到的质粒PCDH-miR339及病毒包装质粒psPAX2和衣壳质粒pMD2.G-VSVG转入293T细胞。分别于48h和72h后各收取一次上清,将收取的上清于0.45μm滤器过滤细胞碎片后得到慢病毒原液。
2.2、感染细胞:提前将需要被感染的细胞按照~20万每皿铺于6cm培养皿中,待第二天细胞贴壁之后进行第一次感染,第三天再重复感染一次;第四天让细胞恢复一天不加入任何刺激;第五天开始对质粒所带的对应杀药标记进行药物筛选。
2.3、实时荧光定量PCR
(1)总RNA提取
准备约106-107被感染的细胞,用PBS重悬后离心去除上清,加入1ml Trizol室温裂解5min,然后加入0.2ml氯仿,涡旋震荡仪震荡15s,室温静置2min。4℃离心机离心15min,13300rpm。转移上层无色水相于另一EP管中。加入等体积异丙醇,涡旋震荡仪充分混合,入4℃离心机离心10min,13300rpm。倒掉上清,加入DEPC水配制的75%乙醇1ml,上下颠倒至沉淀悬浮起来,入4℃离心机离心5min,13,300rpm。用移液器吸尽上清,室温干燥5-20min,期间观察沉淀形态,待其刚刚变透明时,根据沉淀的量将其溶解于40-100μl DEPC水中。取1μl在Nanodrop上测定其浓度以及OD260/OD280。提取的RNA于-80℃冰箱保存。
(2)逆转录合成cDNA
逆转录使用Takara(D2680A)的反转录PCR试剂盒。
反应体系:
反转录PCR条件:42℃10min,95℃2min。
(3)RT-qPCR
使用Takara实时荧光定量PCR试剂盒检测目的基因转录水平上的表达情况。
反应体系:
通过分析试验结果发现,通过克隆入miR-339序列,乳腺癌细胞T47D和4175中过表达miR-339分别为10.56倍和3.79倍。且乳腺癌细胞T47D和4175中的GPER1均被激活。结果如图1和图2所示。图1中,乳腺癌细胞系4175中miR-339过表达3.79倍,靶基因GPER1被激活2.80倍;图2中,乳腺癌细胞系T47D中miR-339过表达10.56倍,靶基因GPER1被激活3.54倍。上述结果表明,乳腺癌细胞系中miRNA-339可以激活抑癌基因GPER1mRNA转录水平。
2.3、双荧光素报告基因实验
(1)将miR-339 DNA locus位置的上下游588bp DNA序列扩增并插入到报告基因载体PGL3-promoter上,验证miR-339 DNA locus的这一段序列是否具有增强子活性。我们在293T细胞中转染插入miR-339 DNA locus序列的报告基因载体(pGL3-miR-339),和空载的对照组相比较(pGL3),24小时后收集细胞检测荧光强度,结果显示实验组的活性更高(图3),提示插入的miR-339 locus序列具有增强子活性,可以增强报告基因的活性。
(2)接下来,本发明想进一步探讨miR-339是否可以促进增强子的活性。将miR-339的表达质粒(PSR-339)和插入miR-339 locus的pGL 3-p-339载体同时转染到293T细胞中进行双荧光素报告基因实验。结果表示,在共转入miR-339的表达质粒(PSR-339)和插入miR-339 locus的pGL3-p-339载体后,报告基因活性明显比只转染pGL3-p-339载体的对照组活性更高,提示增强子活性被miR-339提高(图4-a,b)。
(3)突变结合位点。通过构建miR-339表达质粒的突变质粒(mut-PSR-339)以及增强子序列质粒pGL3-p-339的突变质粒(mut-pGL3-p-339),结合位点突变后的报告基因活性不能继续被激活(图4-a,b,c,d);如果结合位点按照界碱基互补配对的方式突变,报告基因的活性能继续被激活(图4-a,c,d,e)。提示miR-339激活增强子依赖于miR-339和增强子结合序列的完整性,也就是种子序列结合位点的互补性。
2.4、染色质免疫共沉淀实验
对293T细胞过表达miR-339的实验组和对照组H3K27ac ChIP纯化的DNA进行文库构建,质检合格的文库进行高通量测序分析,以确定miR-339附近的增强子是否被过表达的miR-339激活。H3K27ac ChIP-seq数据分析结果(图5)以及QPCR结果(图6)显示,与对照组细胞相比,位于GPER1基因上游大约60kb位置的一段DNA序列在miR-339过表达后出现明显的H3K27ac富集,提示该区域为一个被miR-339所激活的增强子,而这个位置正好是和miR-339locus重合。
2.5、CRISPR敲除实验
(1)首先设计增强子区域miR-339结合位点的gRNA,构建了CRISPR敲除质粒(CRISPR-339/GFP)并转染到293T细胞中;
(2)通过96孔板稀释法,结合快速克隆验证和Sanger测序等方法我们筛选获得了miR-339结合位点缺失的单克隆细胞株;
(3)对敲除增强子序列后的细胞再进行过表达miR-339,利用QPCR实验进一步研究增强子序列的完整性对miR-339激活作用的影响。敲除增强子序列后过表达miR-339,24小时后收集细胞进行QPCR,实验结果表明,如图7所示,miR-339成功过表达,但是GPER1的表达却明显下调,提示即使在敲除增强子序列中的细胞中再过表达miR-339也不能激活靶基因GPER1。
实施例3 miR-339通过靶向激活靶基因GPER1抑制乳腺癌细胞增殖
3.1、在实施例2的基础上进一步通过CCK8的增殖检测并评估慢病毒构建的稳定表达miR-339的乳腺癌稳转细胞株4175和T47D的增殖能力。并且,每组设立对照组为:转染空载病毒液的乳腺癌细胞株4175和T47D。
结果如图8和9所示。与对照组相比,miR-339过表达的乳腺癌细胞株4175和T47D的增殖能力均下降。
3.2、进一步研究GPER1对乳腺癌细胞系增殖能力的影响。通过转染shGPER1干扰乳腺癌细胞系4175和T47D中GPER1的表达,通过CCK8的增殖检测GPER1对两个乳腺癌细胞系的增殖影响。并且,每组设立对照组为:转染空载病毒液的乳腺癌细胞株4175和T47D。
结果如图10和11所示,两组乳腺癌细胞4175和T47D中,干扰GPER1表达后的实验组比对照组细胞数目明显多,提示GPER1对乳腺癌细胞系的增殖有抑制作用。
3.3、在实施例2的基础上进一步通过克隆形成的增殖检测并评估慢病毒构建的稳定表达miR-339的乳腺癌稳转细胞株4175和T47D的增殖能力。并且,每组设立对照组为:转染空载病毒液的乳腺癌细胞株4175和T47D。
结果如图12和13所示。与对照组相比,miR-339过表达的乳腺癌细胞株4175和T47D的增殖能力均下降。
3.4、进一步研究GPER1对乳腺癌细胞系增殖能力的影响。通过转染shGPER1干扰乳腺癌细胞系4175和T47D中GPER1的表达,通过克隆形成实验的增殖检测GPER1对两个乳腺癌细胞系的增殖影响。并且,每组设立对照组为:转染空载病毒液的乳腺癌细胞株4175和T47D。
结果如图14和15所示,两组乳腺癌细胞4175和T47D中,干扰GPER1表达后的实验组比对照组形成克隆数目明显多,提示GPER1对乳腺癌细胞系的增殖有抑制作用。
3.5、将稳定表达miR-339和相应空载的对照组稳转细胞株及过表达miR-339后敲低GPER1的乳腺癌细胞株,3组细胞株分别注射到裸鼠的后背,饲养并观察每组裸鼠后背成瘤状况。注射细胞4周后裸鼠背上开始成瘤,记录为d0天,在d0后的第28天,部分成瘤裸鼠出现死亡,立即将所有裸鼠引颈处死,收集瘤子,统计肿瘤大小和重量。
收集的肿瘤如图16和17所示,图16为各组肿瘤图,图17为肿瘤重量统计图。过表达miR-339的实验组裸鼠瘤子明显比对照组要小,提示miR-339有抑制乳腺癌肿瘤的发生发展的潜力;而在过表达miR-339后重新敲低GPER1的裸鼠成瘤大小要比过表达miR-339的实验组裸鼠瘤子大,说明GPER1在裸鼠中可以抑制肿瘤生长。
综合实施例1-3,miR-339位于人类基因组7号染色体,目前研究主要集中在miR-339通过负性调控靶基因,参与肿瘤发展和转移过程。本申请首先通过生物信息学分析发现miR-339是乳腺癌发生发展的关键因子。并且,在Luminal型乳腺癌和三阴性乳腺癌细胞系T47D和4175中分别过表达外源性miR-339,结果表明过表达miR-339后,这两个乳腺癌细胞中抑癌基因GPER1的表达均上调,它们的增殖和克隆形成能力均受到明显抑制。最后申请的实验结果在动物实验中进一步得到验证,提示过表达miR-339可以再激活抑癌基因GPER1从而抑制乳腺癌细胞的生长与增殖。GPER1是G蛋白偶联受体,多次被报道作为抑癌基因在与雌激素相关的癌症中表达下调或者失活,并与肿瘤的不良预后相关。本申请的研究结果提示miR-339的类似物或者模拟物可以作为三阴性乳腺癌治疗的潜在策略。
以上对本发明的具体实施例进行了详细描述,但其只作为范例,本发明并不限制于以上描述的具体实施例。对于本领域技术人员而言,任何对本发明进行的等同修改和替代也都在本发明的范畴之中。因此,在不脱离本发明的精神和范围下所作的均等变换和修改,都应涵盖在本发明的范围内。
序列表
<110> 复旦大学
<120> miR-339-增强子-靶基因网络激活模型及其应用
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 23
<212> RNA
<213> 人工序列(Artificial Sequence)
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<210> 2
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
gaagattcta gagctagcga attcggccac aggcaggtgc cacc 44
<210> 3
<211> 47
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
gcagatcctt cgcggccgcg gatccacacc agtggaacat cccatgc 47
Claims (11)
1.一种可激活乳腺癌相关基因的miRNA,其特征在于,所述miRNA的基因座位置与增强子的组蛋白修饰标志H3K4me1或H3K27ac重合。
2.根据权利要求1所述的miRNA,其特征在于,所述miRNA的核苷酸序列如SEQ ID NO:1所示,或
所述miRNA的序列为与所述SEQ ID NO:1具有90%同源性且功能相同的序列。
3.根据权利要求1所述的miRNA,其特征在于,所述乳腺癌相关基因为GPER1基因。
4.一种核酸药物,其特征在于,所述核酸药物包括权利要求1-3任一项所述的miRNA或其人工模拟物;优选的,所述核酸药物还包括药学上可接受的载体或赋形剂。
5.一种与权利要求4所述的核酸药物相关的生物材料,其特征在于,所述生物材料选自下述A)、B)或C)中的一种:
A)与权利要求1-3任一项所述的miRNA的核苷酸序列互补配对的干扰RNA分子或者抑制剂;
B)包含有权利要求1-3任一项所述miRNA的核苷酸序列的表达载体或重组载体;
C)包含有B)中所述的表达载体或重组载体的细胞或微生物。
优选的,所述表达载体包括pCDH载体、pCMVp-NEO-BAN载体、pEGFP载体、pEGFT-Actin、pSV2载体、pCDNA载体、pLVX载体、pAAV载体、pET载体和pDsRed载体中的至少一种。
优选的,所述重组载体构建步骤包括:1)设计引物,PCR扩增所述miRNA序列;2)将扩增的序列片段和表达载体酶切,连接目的序列片段和表达载体;3)将连接产物转化大肠杆菌,培养;4)鉴定后提取重组质粒并进行包装即得到重组载体。
6.权利要求1-3任一项所述的miRNA或权利要求4所述的核酸药物在诊断、预防和/或治疗乳腺癌中的应用。
7.一种筛选权利要求1-3任一项所述的可激活疾病相关基因的miRNA的方法,其特征在于,包括以下步骤:
S1、通过生物信息学分析,筛选基因座位置与H3K4me1或H3K27ac重合的miRNA;
S2、验证步骤S1筛选的miRNA与其周围200kb范围内的靶基因的关系;
S3、获取S2中与所述靶基因具有相关性的miRNA,即为可激活疾病相关基因的miRNA。
8.根据权利要求7所述的方法,其特征在于,步骤S2中,验证miRNA与其周围200kb范围内的靶基因的关系的方法包括:实时荧光定量PCR检测过表达所述miRNA后所述靶基因的表达量。
9.一种用于研究乳腺癌机制的模型,其特征在于,所述模型为miRNA-增强子-靶基因网络激活模型。
10.根据权利要求9所述的模型,其特征在于,miRNA与增强子互相作用进而正向激活靶基因从而影响肿瘤的增殖生长,所述miRNA为miR-339;
优选的,所述模型的构建方法包括:筛选位于增强子区域的多个miRNA,分别将所述miRNA在不同种类的细胞中过表达,发现在基因组上与所述miRNA邻近的多个基因被转录激活;得到miRNA-增强子-靶基因网络激活模型。
11.根据权利要求9-10所述的模型在乳腺癌领域中的应用;优选的,所述模型在制备治疗乳腺癌药物中的应用。
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CN107488735A (zh) * | 2017-10-10 | 2017-12-19 | 广州医科大学附属第二医院 | miR‑339‑5p在抑制前列腺癌骨转移及TGF‑β信号通路中的应用 |
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