CN102703456B - DAPK3基因hsa-miR-20a的作用靶位点 - Google Patents
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Abstract
本发明公开了一种致癌性hsa-miR-20a在抑癌基因DAPK3上基因hsa-miR-20a的作用靶位点，该靶位点位于DAPK3编码区内的第二个外显子上，其核苷酸序列如SEQIDNo.1所示。本发明公开了hsa-miR-20a的寡核苷酸抑制剂，该抑制剂可以有效阻止hsa-miR-20a对DAPK3的抑制作用，导致人类癌症细胞的死亡，寡核苷酸抑制剂序列如SEQIDNo.2所示。对抑癌基因DAPK3上的hsa-miR-20a靶位点进行突变可以导致DAPK3的蛋白表达量提高并因此引起人类癌症细胞的存活率降低。本发明还提供了所述突变序列，以及包含该突变序列的DAPK3。可以根据本发明的靶位点及其突变序列，以及hsa-miR-20a寡核苷酸抑制剂开发相关的基因治疗药物，有着重要的医疗应用前景。
Description
技术领域
本发明涉及分子生物学及医药领域，具体涉及微小RNA（miRNAs）hsa-miR-20a对抑癌基因DAPK3的表达抑制的靶位点（靶分子），本发明还涉及利用该通路解除DAPK3所受到的表达抑制，从而激活其抑制人类癌症细胞生长的方法以及相关药物。
背景技术
微小RNA(miRNAs)是迄今发现的最重要的一类小RNA。它是由20-25个核苷酸组成的非编码调控RNA，主要在RNA水平上对基因表达进行调控，在细胞增殖和死亡、脂肪代谢、神经元定位、造血细胞系分化和各种发育过程中均起着至关重要的调控作用（Kim,V.N.MicroRNA Biogenesis:Coordinated Cropping and Dicing.Molecular Cell Biology:Nature Reviews.6:376-385;2005，Bartel,D.P.MicroRNAs:genomics,biogenesis,mechanism,and function.Cell 116,281-297;2004.）。目前在人体内所发现的miRNA已有1921个，这些miRNAs的表达谱在正常组织和癌症组织中有着极大的不同，这提示miRNA在癌症的发生、侵染、转移等过程中发挥着重要的作用。
2005年来，miRNA在癌症发生中起作用已成为生命科学领域的一个热点，美国科学家当年首次将位于人类13号染色体上的mir-17-92cluster（簇）命名为“oncomiR1”（Lin He,J.Michael Thomson,Michael T.Hemann,Eva Hernando-Monge,David Mu,Summer Goodson,Scott Powers,Carlos Cordon-Cardo,Scott W.Lowe,Gregory J.Hannon and Scott M.Hammond.A microRNA polycistron as a potential human oncogene.Nature 435,828-833）。同期发现几乎所有被研究的癌症都有特定的标签miRNA，大部分的标签miRNA水平被上调，也有相当部分的miRNA水平被下调（Stefano Volinia,George A. Calin,Chang-Gong Liu,Stefan Ambs,Amelia Cimmino,Fabio Petrocca,Rosa Visone,Marilena Iorio,Claudia Roldo,Manuela Ferracin,Robyn L.Prueitt,Nozumu Yanaihara,Giovanni Lanza,Aldo Scarpa,Andrea Vecchione,Massimo Negrini,Curtis C.Harris,and Carlo M.Croce.A microRNA expression signature of human solid tumors defines cancer gene targets.Proc Natl Acad Sci.103,2257-2261）。同年后期发现大约50%被注解的miRNAs都定位在基因组上与肿瘤相关的脆性位点。mir-17-92簇包括miR-17,miR-18,miR-19,miR-20a,and miR-92。人类miR-20a是一个原癌基因，在直肠癌、胰腺癌和前列腺癌均呈现高表达。研究表明高表达miR-20a可以大幅降低c-Myc转基因小鼠癌变细胞的凋亡，从而导致癌症发生的高速度和高致死率，提示miR-20a在体内可能起抗凋亡的作用。到目前为止被验证的miR-20a的作用靶标只有E2F1，miR-20a可能通过降低E2F1的蛋白表达而抑制细胞凋亡（Yannick Sylvestre,Vincent De Guire,Emmanuelle Querido,Utpal K.Mukhopadhyay，Véronique Bourdeau, 
Major,Gerardo Ferbeyre,and Pascal Chartrand.An E2F/miR-20a Autoregulatory Feedback Loop.J.Biol.Chem.282,2135-2143，Kathryn A.O′Donnell,Erik A.Wentzel,Karen I.Zeller,Chi V.Dang,and Joshua T.Mendell.c-Myc-regulated microRNAs modulate E2F1 expression.Nature 435,839-843）。但是当在c-Myc转基因小鼠中敲除E2F1后并没有引起细胞凋亡的降低。这说明miR-20a并不是通过降低E2F1蛋白的表达来抑制细胞凋亡的（Troy A.Baudino,Kirsteen H.Maclean,Jennifer Brennan,Evan Parganas 1 ,,Chunying Yang,Aaron Aslanian,Jacqueline A.Lees,Charles J.Sherr，Martine F.Roussel and John L.Cleveland.Myc-Mediated Proliferation and Lymphomagenesis,but Not Apoptosis,Are Compromised by E2f1 Loss.Molecular Cell,11,905-914）。到目前为止miR-20a抑制凋亡的作用机制仍然不清楚。
发明内容
本发明的目的在于提供抑癌基因DAPK3上被hsa-miR-20a抑制表达的靶位点序列；
本发明另一个目的在于提供一种hsa-miR-20a的抑制剂序列，该抑制剂可以去除hsa-miR-20a对抑癌基因DAPK3的抑制作用；
本发明又一个目的在于提供上述DAPK3靶位点序列的突变序列，该序列保持了DAPK3的氨基酸序列和蛋白质活性，但是逃避了hsa-miR-20a的抑制作用；
本发明的再一个目的在于提供两种抗癌药物。
为实现上述目的，本发明的技术方案如下：
本发明证明了原癌基因hsa-miR-20a通过对抑癌基因DAPK3的表达抑制促进人类癌细胞的存活率。本发明发现了DAPK3上被hsa-miR-20a抑制表达的靶位点序列，该序列位于抑癌基因DAPK3在其第二个外显子上，其核苷酸序列如SEQIDNo.1所示。
本发明研究发现，可以通过一个寡核苷酸序列去除hsa-miR-20a通过该靶点对DAPK3的抑制作用，导致DAPK3表达量提高，其序列如SEQIDNo.2所示。该序列可用于开发抗癌药物。
本发明研究发现通过对上述靶位点序列的突变，可以去除hsa-miR-20a的抑制作用，可以导致DAPK3的蛋白表达量提高并因此而引起人类癌症细胞的存活率降低。进而本发明提供所述靶位点的同意突变序列，其只干扰has-miR-20a对DAPK3的抑制，但不改变DAPK3的氨基酸序列。优选，所述突变序列的核苷酸序列为：TCCTGGACGGCGTTCATTATCTCCATTCAAAACGGATCGCACACTTTGAC。
本发明还进一步提供基于上述靶位点开发的抗癌药物，该药物通过使上述靶位点的序列突变去除hsa-miR-20a对抑癌基因DAPK3的表达抑制，提高癌症细胞内DAPK3的表达水平，以降低癌症细胞的存活率，抑制癌症组织的生长。
本发明进一步还提供含有上述突变序列的抑癌基因DAPK3，由于该基因的hsa-miR-20a靶位点发生了突变，因而其表达不会受 hsa-miR-20a的影响。本发明还提供含有所述基因的表达载体，通过将所述表达载体导入癌细胞内，可以使抑癌基因DAPK3在癌细胞中有效表达，从而有效降低癌细胞存活率。
本发明具有如下有益效果：
本发明提供的对抑癌基因DAPK3上的hsa-miR-20a靶分子，对其进行突变，或对hsa-miR-20a进行抑制，均可以导致DAPK3的蛋白表达量提高并因此引起人类癌症细胞的存活率降低。基于此可以开发出针对该靶分子的基因治疗药物，有着重要的医疗应用前景。
附图说明
图1显示的是hsa-miR-20a对DAPK3表达的抑制作用，其中NC是阴性对照的寡核苷酸序列，图中“-/+/+++”表示在实验中“不加/加入低浓度/加入高浓度/或者加所示的寡核苷酸序列；
图2显示的是hsa-miR-20a抑制剂（anti-miR-20a）对DAPK3表达的促进作用。其中scramble为随机寡核苷酸序列，anti-miR-20a是hsa-miR-20a抑制剂寡核苷酸，其序列为SEQ ID No.2所示。图中“-/+”表示在实验中不加或者加入所示的寡核苷酸序列；
图3显示的是突变型DAPK3和野生型DAPK3在不同情形下的表达情况，其中D3表示野生型，D3M表示突变型，SC是添加了hsa-miR-20a抑制剂的结果，anti-a20是添加了Flag标签抗体的结果；
图4是hsa-miR-20a和DAPK3野生型/突变型表达载体共转染人类宫颈癌细胞系Hela的显微照片，其中Mock是空白对照（以等体积的水代替hsa-miR-20a和DAPK3野生型/突变型表达载体溶液）。
具体实施方式
下面结合具体实施例，进一步阐述本发明。应当理解，这些实施例仅用于说明本发明而不用于限制本发明要求保护的范围，下列实施例中未注明具体实验条件和方法，通常按照常规条件如：J.萨姆布鲁克等主编，科学出版社，1992，分子克隆实验指南（第二版）；D.L.斯佩克特等，科学出版社，2001，细胞实验指南；吕鸿声，科学出版 社，1982，或接照制造厂商所建议的条件。
以下实施例中，hsa-mir-20a表示人类miR-20a，hsa-mir-20a mimics表示人工合成的hsa-mir-20a寡聚核糖核苷酸（RNA）序列。图中的miR-20a指hsa-mir-20a mimics。
实施例1 hsa-miR-20a对DAPK3的表达抑制
（1）采用反式转染的方法将人工合成的hsa-mir-20a mimics转染进人类宫颈癌细胞系Hela，具体步骤为：将Hela细胞用胰酶消化并铺入24孔板中，细胞密度为30%。配置转染复合物，体系如下：
表1 转染混合物的配置（溶解于50μl Opti-mem培养基中）
| NC | + | +++ | |
| NC | 4,5μl | 3μl | 0μl |
| hsa-mir-20a | 0μl | 1.5μl | 4.5μl |
【NC是负对照，是随机的寡聚核糖核苷酸】
将4.5μl Lipo2000溶解于50μl Opti-mem培养基中，室温静置5分钟；滴加入各核酸混合物中，混匀后室温静置20分钟。再将配置好的转染混合物滴加到准备好的24孔板。
（2）48小时后，每孔细胞用PBS洗两次；每孔细胞加入200μl 1×SDS上样缓冲液，室温静置10分钟裂解细胞，吹打均匀后移至1.5ml离心管。
（3）样品于1000C煮沸，后置于冰上5分钟。
（4）10%SDS PAGE分离蛋白样品，10%SDS PAGE分离胶和积层胶分别按表2和表3配方如下：
表2 分离胶的配置
| 蒸馏水 | 1.3ml |
| 30%acrylamide | 1.7ml |
| 1.5M Tris（pH8.8） | 1.9ml |
| 10%SDS | 0.05ml |
| 10%APS | 0.05ml |
| TEMED | 0.002ml |
表3 积层胶配置
| 蒸馏水 | 2.1ml |
| 30%acrylamide | 0.5ml |
| 1.5M Tris（pH8.8） | 0.38ml |
| 10%SDS | 0.03ml |
| 10%APS | 0.03ml |
| TEMED | 0.002ml |
（5）将蛋白在100V电压1小时的条件下转移至合适大小的硝酸纤维素膜上。
（6）将硝酸纤维素膜置于5%脱脂奶粉的TBST阻断液中室温孵育1小时；DAPK3抗体和内参β-actin抗体室温孵育1小时，TBST溶液洗三次；辣根过氧化酶标记的二抗缓冲液室温孵育1小时，TBST溶液洗三次
（7）使用辣根过氧化酶的底物显色试剂盒对硝酸纤维素膜显色处理，并与暗室内用X光片进行压片洗片处理。
hsa-miR-20a mimics购买于广州瑞博生物有限公司，Lipo2000购买于invitrogene公司，DAPK3抗体购买于武汉三鹰生物有限公司，β-actin抗体购买于sigma公司。
结果如图1所示，hsa-miR-20a在50nM（+）的低浓度下对DAPK3的抑制作用不明显，在150nM（+++）的高浓度下对DAPK3的表达有着强烈的抑制作用。
实施例2 hsa-miR-20a抑制剂对DAPK3表达的促进
在本实施例中，设计合成了hsa-miR-20a抑制剂（anti-miR-20a）（SEQ ID No.2）作为抑制DAPK3表达的抑制剂（由广州锐博公司合成）。将该抑制剂转染人类宫颈癌细胞系Hela，48小时后对转染细胞进行裂解收集，用特异性的抗体检测细胞内DAPK3和内参β-actin的蛋白表达水平。方法内容和实施例1中相同，在此不再赘述。
表4 转染混合物的配置（溶解于50μl Opti-mem培养基中）
结果如图2所示，hsa-miR-20a抑制剂在50nM浓度下对DAPK3 的蛋白表达即有明显的促进作用。
实施例3 hsa-miR-20a在DAPK3第二个外显子上靶位点的确立
（1）以Trizol法提取人类宫颈细胞系Hela的总RNA,主要步骤为：
将细胞铺于24孔板，待24小时后细胞生长至80%密度
a)一孔细胞加600μl Trizol，室温静置5分钟，使细胞裂解
b)将裂解的细胞用移液枪转移到1.5ml离心管中，加入120μl氯仿，上下颠倒混匀。
c)在高速离心机上，以12,000的转速，4℃的条件下离心20分钟。
d)吸取上清至新鲜离心管，加入300μl异丙醇，冰上放置20分钟。
e)在高速离心机上，以12,000的转速，4℃的条件下离心10分钟。
f)用移液枪吸走上清，用75%乙醇沉淀洗涤，室温静置5分钟晾干，彻底清除残留的液体
g)用20μl DEPC处理水溶解RNA
h)用Nanovue一起测定RNA的浓度及其纯度，样品保存于-80℃。
Trizol试剂购买于invitrogen公司，氯仿、异丙醇、乙醇均购买于国药集团。
（2）逆转录合成cDNA
取2μg总RNA样品用于逆转录反应，首先去除痕量基因组DNA污染，在0.2ml离心管仲配置如下体系：
RNA 10μl
RQ1 DNA酶 2μl
10X酶反应缓冲液 2μl
DEPC处理水 6μl
置于37℃反应30分钟，然后加入2μl stop solution，65℃孵育 10分钟失活DNase的活性。RQ1DNase酶购于Promega公司。
在上一步反应体系中加入下列成分：
42℃反应1小时，90℃加热5分钟失活MMLV逆转录酶。
（3）克隆DAPK3编码区序列到真核高表达载体pCDNA3配置如下PCR扩增体系：
95℃变性1分钟，60℃退火1分钟，72℃延伸2分钟。35个循环扩增。
扩增DAPK3编码区的引物：
正向引物GAAGATCTACCATGGACTACAAGGACGACGATGACAAGTCCACGTTCAGGCAGGAGGA，反向引物CGGAATTCCCGCTCGAGCTAGCGCAGCCCGCACTCCA。
以上设计的PCR引物由invitrogen公司合成，KOD plus DNA聚合酶购买于TOYOBO公司。
在上步反应体系中加入3μl EcoRI和XbaI限制性内切酶，37℃水浴反应1小时。
1%琼脂糖纯化回收DAPK3的扩增片段，连接到用同样酶切处理的载体片段上，得到质粒pC DAPK3，经验证正确后备用。
（4）突变DAPK3表达载体的构建
PCR反应体系如下配置：
95°C变性2分钟，60°C退火1分钟，72°C延伸8分钟，扩增35个循环。
在如上反应体系中加入1μlDpnI，置于37°C水浴中反应2小时。
取1μl反应后体系，转化后涂平板得到突变型的DAPK3表达载体pCDAPK3M，经验证正确后备用。
突变引物的序列为：
TCCTGGACGGCGTTCATTATCTCCATTCAAAACGGATCGCACACTTTGAC
正义链TCCTGGACGGCGTTCATTATCTCCATTCAAAACGGATCGCACACTTTGAC
反义链GTCAAAGTGTGCGATCCGTTTTGAATGGAGATAATGAACGCCGTCCAGGA
以上设计的PCR引物由invitrogen公司合成，DpnI限制性内切酶购于TAKARA公司。
（5）hsa-miR-20a抑制剂（anti-miR-20a）和DAPK3表达载体共表达
采用顺式转染的方法将人工合成的hsa-mir-20a抑制剂和DAPK3表达载体共转染进人类宫颈癌细胞系Hela，具体步骤为：将生长旺盛的细胞用胰酶消化，铺于24孔板中，24小时后细胞长至80%的密度，使用Lipo2000脂质体转染试剂将hsa-miR-20a和DAPK3野生型（突变型）表达载体转染人类宫颈癌细胞系Hela。转染条件如下表所示：
表5 转染条件
转染方法如实施例1中所述，在此不再赘述。
转染48小时候，收集样品并用Flag标签抗体（购于美国sigma公司）和内参β-actin抗体检测细胞内DAPK3和内参的蛋白表达量。注明：因为在质粒中DAPK3与Flag标签形成融合蛋白，因此可以通过Flag标签的抗体抗体检测胞内的DAPK3的表达量。通过比较DAPK3于内参的表达比例，确定DAPK3的表达调控趋势。Westernblot的方法内容如实施例1中所述，在此不再赘述。
结果如图3所示，突变型DAPK3因不再受到miR-20a的抑制而比野生型表达量高。在共转染hsa-miR-20a抑制剂后，miR-20a不再抑制野生型DAPK3的表达，野生型DAPK3和突变型DAPK3的表达量回复一致。
实施例4 解除hsa-miR-20a对DAPK3的表达抑制作用以抑制人类癌症细胞生长
将突变型和野生型DAPK3表达载体或其对照空载体转染进人类宫颈癌细胞系Hela。
在本实施例中，采用顺式转染的方法将hsa-mir-20a的表达载体和DAPK3表达载体共转染进人类宫颈癌细胞系Hela，具体步骤为：将生长旺盛的细胞用胰酶消化，铺于24孔板中，24小时后细胞长至80%的密度，使用Lipo2000脂质体转染试剂将hsa-miR-20a和DAPK3野生型（突变型）表达载体转染人类宫颈癌细胞系Hela。转染条件如下表：
转染方法如实施例1中所述，在此不再赘述。
转染48小时后，肉眼在倒是显微镜下观察转然后细胞的存活状况，对细胞拍照生成图像，并定性检测细胞的存活率。
结果如图4所示，DAPK3野生型和表达载体可以降低细胞存活率，而转染了DAPK3突变型后癌细胞的存活率更低。暗示突变型DAPK3可以有效逃逸癌细胞内hsa-miR-20a的抑制作用，从而更有效抑制癌细胞的生长。当hsa-miR-20a和野生型DAPK3共表达时，DAPK3的表达受到抑制而无法抑制细胞生长导致癌细胞存活率较高。而hsa-miR-20a和突变型DAPK3共表达时，突变后的DAPK3因不受到该原癌microRNA的抑制而回复了对细胞生长的抑制，导致细胞存活率的降低。该结果有力证明突变型DAPK3表达载体的转染可以逃逸hsa-miR-20a的抑制作用，从而高效抑制癌细胞生长。
Claims (4)
1.抑癌基因DAPK3上被原癌基因hsa-miR-20a抑制表达的靶位点，其特征在于，该靶位点位于DAPK3编码区的第二个外显子上，其核苷酸序列为TCCTGGACGGCGTTCACTACCTGCACTCTAAGCGCATCGCACACTTTGAC。
2.一种DNA，其核苷酸序列为权利要求1所述靶位点的核苷酸序列的突变序列，所述突变序列的核苷酸序列为：TCCTGGACGGCGTTCATTATCTCCATTCAAAACGGATCGCACACTTTGAC。
3.含有权利要求2所述DNA的抑癌基因DAPK3。
4.含有权利要求3所述抑癌基因DAPK3的表达载体。
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