CN103013997B - 抑制透明质酸酶活性的miRNA及其应用 - Google Patents
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Abstract
抑制透明质酸酶活性的miRNA及其应用,序列如SEQIDNo.1所示。该miRNA可用于制备抑制透明质酸酶活性药物。本发明是天然植物中特异性分离提取的微小核苷酸,简单易行,成本相对低。本发明水解溶液可低温保存,无其他有机溶剂残留,安全无污染。
Description
技术领域
本发明涉及微小核苷酸技术领域,尤其涉及一类抑制透明质酸酶活性的miRNA及其应用。
背景技术
微小RNA(microRNAs;miRNA,又称小分子RNA)是真核生物中广泛存在的一种长约16到26个核苷酸的RNA分子,可调节其他基因的表达。miRNA从一些DNA转录而来,但无法进一步转译成蛋白质的RNA(属于非编码RNA)。这些RNA是从初级转录本(primarytranscript),也就是pri-miRNA,转变成为称为pre-miRNA的茎环结构,最后成为具有功能的成熟miRNA。pri-miRNA长度大约为300~1000个碱基,pri-miRNA经过一次加工后,成为pre-miRNA即microRNA前体,长度大约为70~90个碱基;pre-miRNA再经过Dicer酶酶切后,成为长约16~26nt的成熟miRNA。其特点有:(1)位于基因组非编码区;(2)进化上高度保守;(3)可在转录后水平对基因表达进行调节;(4)参与多种生命过程,调控多达30%的蛋白的表达。成熟miRNA通过RNA诱导的沉默复合体(RISC)与靶信使核糖核酸(mRNA)特异结合,从而抑制转录后基因表达,在调控基因表达、细胞周期、生物体发育时序等方面起重要作用。尤其微小RNA从5’端起第2-8位的核苷酸在相同微小RNA家族中高度保守,被称为种子序列(seed sequence),其在转录后水平抑制靶信使核糖核酸(mRNA)翻译过程中起至关重要作用。另外,目前已有研究表明植物的微小RNA可以跨界调控动物的基因表达,这为本发明的最初设想提供了重要依据。
透明质酸(Hyaluronan、hyaluronic acid,又称糖醛酸、玻尿酸、琉璃糖碳基酸),其基本结构是由双糖单位D-葡萄糖醛酸及N-乙酰葡糖胺组成的高级多醣类。与其它粘多糖不同,它不含硫。透明质酸为组织基质中具有限制水分及其它细胞外物质扩散作用的成分,它的透明质分子能携带500倍以上的水分,为目前所公认的最佳保湿成分,目前广泛的应用在保养品和化妆品中。作为一种多功能基质,透明质酸广泛分布于人体各部位,其中皮肤也含有大量的透明质酸。人类皮肤成熟和老化过程也随着透明质酸的含量和新陈代谢而变化,它可以改善皮肤营养代谢,使皮肤柔嫩、光滑、去皱、增加弹性、防止衰老,在保湿的同时又是良好的透皮吸收促进剂。而透明质酸酶(Hyaluronidase,HYAL)作为一种能水解透明质酸的酶,在生物体皮肤、口腔和关节中的透明质酸酶会随着机体的衰老或处于病态而分泌增加,随之引起透明质酸的减少,出现皮肤粗糙干燥、弹性下降、皱褶增加等衰老症状,所以通过抑制作为透明质酸水解酶的表达,可有助于维持机体内透明质酸酶的含量。
另外,透明质酸酶也是I型过敏反应(如荨麻疹、特意反应性皮炎等)和肿瘤细胞增殖迁移的参与者,因此,抑制透明质酸酶的活性,既能使透明质酸不被分解以维持其正常生理功能,又能达到抗炎抗过敏的功效。
目前,已经报道的透明质酸酶抑制剂按其化学组成可分为蛋白质类、多糖类、生物碱和萜类等,而且大批量制备所需成本较高。迄今,没有核酸类制剂对于透明质酸酶活性的抑制作用的报道,最接近的现有技术方案为从植物中提取分离纯化1,2,3,4,6-五-O-没食子酰基-β-D-葡萄糖(PGG)对透明质酸酶活性的抑制剂,但是其分离纯化过程繁琐,得率低。
发明内容
解决的技术问题:本发明提供一种核酸类透明质酸酶活性抑制剂及其应用,具体的说,所涉及抑制透明质酸酶活性的miRNA,可从多种天然植物中特异性分离提取,纯度高、得率高、成本低,抑制效果好。
技术方案:抑制透明质酸酶活性的miRNA,序列如SEQ ID No.1所示。
序列如SEQ ID No.1所示的miRNA在抑制透明质酸酶活性的应用。
序列如SEQ ID No.1所示的miRNA在制备抑制透明质酸酶活性药物上的应用。
有益效果:
1)本发明是天然植物中特异性分离提取的微小核苷酸,简单易行,成本相对低。
2)本发明水解溶液可低温保存,无其他有机溶剂残留,安全无污染。
附图说明
图1为生物信息学软件RNAhybrid计算得出的上述miR164与所述透明质酸酶(HYAL1)的mRNA3’-非编码区(3’-UTR)相互作用从而产生转录后抑制作用机制关系;
图2为载体图谱:pMIR-REPORTTM miRNA表达报告基因载体;
图3为过表达miR-164与过表达ncRNA对荧光素酶活性的抑制效果图;
图4为miR164抑制HYAL1的蛋白印记(western blotting)结果图。
具体实施方式
本发明是一种透明质酸酶抑制剂,该透明质酸酶抑制剂中含有微小核苷酸miR164。此处,所述“miR164”是指下面的核酸序列:UGGAGAAGCAGGGCACGUGCA。
以下,通过实施例进一步详细说明本发明,但本发明并不限于这些实施例。
实施例1:
1、序列比对
对所述miR164不特别限定植物物种,在对其进行物种间序列保守性对比发现miR164在葡萄(Vitis vinifera,vvi-miR164a/c/d),苹果(Malus domestica,mdm-miR164b/c/d/e/f),甜瓜(Cucumis melo,cme-miR164c/d)等22种植物中高度保守,序列比对结果如表1。
2、序列合成及提取方法
对于获得miR164的方法没有特别的限定,可以通过合成得到,也可以通过从植物中提取得到。另外,在从植物中提取的情况,对作为原料的植物没有特别的限定。另外,可以只使用一种所述植物来提取miR164,也可以合用2种以上植物来提取miR164。对于葡萄这种植物,优选原料组织为根,叶子,花和未成熟果实。
2.1从植物中提取miR164方法步骤
1).匀浆处理
取50-100mg葡萄植物组织葡萄籽于液氮中研磨,保持研钵中有液氮(如果没有液氮也可以直接加1mL的裂解液PL后匀浆。组织样品容积不能超过PL容积的10%)。
2).转移样品至1.5mL RNase free离心管中,加入1mL的Trizol颠倒混匀,在65℃条件下孵育10分钟(期间颠倒混匀几次)以使核蛋白体完全分解。
3).室温条件下12,000rpm离心5分钟,取上清转入一个新的2mL RNase free离心管中。
4).加入等体积事先预冷的异丙醇,颠倒混匀,4℃12,000rpm 离心5分钟。
5).去除上清,加入100μL RNase-free H2O重新溶解沉淀。
6).加入200μL氯仿,涡旋混匀,放置2-3分钟。
7).于4℃12,000rpm 离心10分钟,样品会分成三层:下层有机相,中间层和上层无色的水相,RNA存在于水相中。把水相(约700μL)转移到新的1.5mL RNase free离心管中,进行下一步操作。
8).加入0.6倍体积70%wt乙醇,颠倒混匀。
9).于4℃13,000rpm离心5分钟。
10).弃上清将沉淀晾干直至变透明。
11).用50μLDEPC H2O溶解沉淀。
12).测定OD值确定总RNA浓度。
13).用过滤杂交方法亲和纯化总RNA中的miR164。
3、生物信息学预测靶位点相互作用
图1显示用生物信息学软件RNAhybrid计算得出的上述miR164与所述透明质酸酶(HYAL1)的mRNA3’-非编码区(3’-UTR)相互作用从而产生转录后抑制作用机制关系。
4、细胞培养
将293T细胞(中国科学院上海生命科学研究院细胞资源中心)在含有10%wt胎牛血清(FBS)的DMEM中、于37℃下培养。
5、萤光素酶分析
我们用PCR的方法从小鼠基因组上克隆出HYAL 3’-UTR区域与miR164相互作用的长约140个核苷酸的片段:5’-TTGCCCAAGGTTGCACAGCAAGAAAAGGGAGAAGTTGAGATTCAAACCCAGGCTGTCTAGCTCCGGGGGTACAGCCCTTGCACTCCTACTGAGTTTGTGGTAACCAGCCCTGCACGACCCCTGAATCTGCTGAGAGGCAC-3’,然后此产物被嵌入一个荧光素酶的报告基因p-MIR-report(Ambion公司)的3’-UTR端(图3)。测序结果显示质粒构建是成功的。
具体步骤为:
(1)构建荧光素酶报告质粒
合成一段miR164靶位点序列,然后通过DNA酶连接反应将其插入到荧光素酶报告基因的3’非编码区,之后把构建得到的荧光素酶报告质粒转化进入感受态大肠杆菌中,再挑选单克隆菌落进行测序验证构建得到的荧光素酶报告质粒的正确性。具体操作为:
I)利用HindIII和SpelI内切酶酶切(37℃,4小时)荧光素酶报告质粒的3’非编码区,再从琼脂糖中回收目的酶切产物;
II)通过T4DNA连接酶分别催化miR164靶位点与酶切产物的连接(16℃,过夜)。miR164靶位点序列是由invitrogen公司合成提供,其140个核苷酸的片段为:5’-TTGCCCAAGGTTGCACAGCAAGAAAAGGGAGAAGTTGAGATTCAAACCCAGGCTGTCTAGCTCCGGGGGTACAGCCCTTGCACTCCTACTGAGTTTGTGGTAACCAGCCCTGCACGACCCCTGAATCTGCTGAGAGGCAC-3’;
III)把构建好的荧光素酶报告质粒转化进入感受态大肠杆菌中然后铺在含有100μg/mL抗生素(Amp)固体培养基板上37℃过夜培养。
IV)挑选单克隆菌落,扩大培养,再从中提取重组质粒进行测序验证。
实验中所用质粒用Endo-free Midi Kit(OMEGA BIO-TEK)试剂盒提取,提取步骤按照说明书步骤进行,最后一步以相同体积三蒸水代替缓冲液洗下质粒。
(2)质粒转染
利用lipofectamine2000(invitrogen公司)及按照厂商提供的标准试验流程将荧光素酶报告质粒转染到细胞膜内。具体操作为:
分别将293T细胞种入24孔板中,以含有10%(v/v)牛血清和1%(v/v)的双抗培育。在其生长密度达到70%时,给细胞换液,将正常细胞培养液换为Opti-MEM I培养基(每孔400μL),再加入100μL的miR164-荧光素酶报告质粒(0.5 μg),β-gal(0.3 μg)质粒和lipofectamine2000(2μL)混合液;往对照孔内加入等量的miR164荧光素酶报告质粒,β-gal质粒和lipofectamine2000混合液。
(3)收集细胞及荧光素酶活性检测
主要分为两个部分:(1)目的蛋白-荧光素酶活性检测(2)内参-β-gal的测定。具体检测方法为:
(a)荧光素酶活性检测:每孔加入100μL 1X Cell CμLture Lysis Reagent(由5×稀释而来),剧烈摇动10min。用枪头或刮板尽可能多地将细胞残余物与溶液一起收集到2mL离心管中,液氮冻融两次。取20μL细胞溶液到1.5mL离心管中,加入100μL荧光底物(luciferaseassay system中溶剂与荧光物质混合而成),涡旋振荡数秒后,放入检测仪ModμLus single tubemμLtimode reader检测,读数记录。
(b)β-gal测定:溶液体系组成为1.5μL 100×Mg2+溶液,33μL 1×ONPG,100.5μL磷酸钠溶液,15μL细胞溶液。将混合溶液在37℃放置30min,至黄色出现,加入250μL 1M碳酸钠溶液终止反应。吸取100μL溶液加入96孔细胞培养板,在420nm处测定吸光度。
(4)数据分析
数据分析为相对比较方法,β-gal为内参。实验结果证明miR164直接识别并作用于HYAL1的3’-UTR:为证明miR164通过直接识别HYAL的3’-UTR使蛋白的表达受抑制,我们将HYAL1的3’-UTR构建到荧光素酶报告基因的3’端,然后在细胞系中过表达miR164,若荧光素酶活性受抑制,说明miRNA是直接作用于HYAL1的3’-UTR的。由下图可知,过表达miR-164可以显著抑制荧光素酶活性,而过表达ncRNA没什么作用,因此miR-164可以特异性地识别HYAL1的3’-UTR并调控HYAL1的表达,具体见图3。
6、蛋白印记法(Western blotting)检测miR164对透明质酸酶(HYAL1)蛋白表达水平的影响
实验步骤为:(1)总蛋白的提取。按照6孔板每孔加入150-250微升裂解液的比例分别向转染了miR164和ncRNA的MCF7细胞(中国科学院上海生命科学研究院细胞资源中心)中加入细胞裂解液(50mM Tris-Cl,150mMNaCl,0.02%wtNaN3,1%wtNP-40,使用前加入0.5ug/mL蛋白酶抑制剂),充分吹打并转移至Eppendorf管中,离心去除不溶物,把上清分装成小份保存于-80℃备用。注意细胞裂解液加入量不宜过多,否则造成蛋白浓度过低,一般细胞加入100μL,组织大于200μL,线粒体50μL。(2)用BCA方法测定总蛋白或线粒体蛋白的浓度。(3)取100μg总蛋白进行常规Western blotting分析。堆积胶配方:H2O 2.92mL,0.5M Tris-HCl(pH 6.8)1.25mL,10%wtSDS 50μL,Acryl/Bis 0.8mL,10%wtAPS 25μL,TEMED2.5μL。10%分离胶配方:H2O 4mL,1.5M Tris-HCl(pH 8.8)2.5mL,10%wtSDS 100μL,Acryl/Bis3.3mL,10%wtAPS 66.7μL,TEMED 5μL。样品煮沸变性后上样,20mA电泳至loading buffer的染料前沿到达底部,小心剥胶,蛋白条带电转移至PVDF膜上,5% wtBSA封闭一小时,按体积比1:1000的一抗孵育过夜,第二天按体积比1:2000孵育二抗,再加入荧光底物,置暗房内用胶片显影、定影。这里使用的一抗为小鼠抗HYAL1单克隆抗体(Santa Cruz公司)。二抗为偶联了HRP的羊抗小鼠IgG(Santa Cruz公司)。
图4为miR164抑制HYAL1的蛋白印记(western blotting)结果,用ImageJ软件分析测定转染ncRNA灰度值为101292;转染miR164灰度值为50509,即HYAL1表达量下降为0.499倍,证实miR164的确可以降低HYAL1的表达,从而抑制HYAL1的活性。
7.制成潜在药物剂型说明
可将亲和纯化的miR164或者人工合成的miR164mimic与不含RNA水解酶高压灭菌后的甘油混合,辅以其他芳香剂,制成液剂、乳剂、乳膏剂、固融体油膏剂及固融体棒型剂等。
上述具体实施方式不以任何形式限制本发明的技术方案,凡是采用等同替换或等效变换的方式所获得的技术方案均落在本发明的保护范围。
SEQUENCE LISTING
<110> 南京大学
<120> 抑制透明质酸酶活性的miRNA及其应用
<130>
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 21
<212> RNA
<213> 人工序列
<400> 1
uggagaagca gggcacgugc a 21
<210> 2
<211> 140
<212> DNA
<213> 人工序列
<400> 2
ttgcccaagg ttgcacagca agaaaaggga gaagttgaga ttcaaaccca ggctgtctag 60
ctccgggggt acagcccttg cactcctact gagtttgtgg taaccagccc tgcacgaccc 120
ctgaatctgc tgagaggcac 140
Claims (1)
1.序列如SEQ ID No.1所示的miRNA在制备抑制透明质酸酶活性药物上的应用。
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siRNA沉默HYAL1基因表达对人乳腺癌细胞生长和增殖的影响;谭金祥等;《癌症》;20061231;第25卷(第7期);844-848 * |
谭金祥等.siRNA沉默HYAL1基因表达对人乳腺癌细胞生长和增殖的影响.《癌症》.2006,第25卷(第7期),844-848. |
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