微小RNA（microRNA）以及RNA结合蛋白对参与心肌重构重要基因调节作用的研究
摘要
体内自然存在的微小RNA (miRNA)是小的转录后调节的非编码RNA,它能够通过影响翻译或者靶mRNA稳定性从而调节基因表达。miRNA参与多种基本生物学过程,如细胞增殖、发育、分化、凋亡、病毒感染和癌症等。在生命活动中发挥了重要调控作用。将是继siRNA之后新的研究热点之一。超过三分之一的人类基因都受miRNA的控制。美国《科学》杂志把miRNA列为2002年世界十大科技突破之首。
由于miRNA天然存在于动植物体内,探索miRNA对肿瘤抑制基因和疾病相关基因的作用无疑将会揭开这些疾病治疗的新篇章。目前只有少数miRNA的部分功能和靶点被鉴定。组织金属蛋白酶抑制因子3基因和miR-1,miR-2-(microRNA-21)均在心血管紊乱疾病和癌症中异常表达。miR-1广泛分布于心肌,骨骼肌。TIMP3(组织金属蛋白酶抑制因子3基因)在心脏中大量存在,在缺陷的心脏中表达下调。冠状动脉硬化的个体内miR-1过度表达。在正常或心肌梗塞的大鼠心脏里过表达miR-1会加速心律不齐。用反义的抑制剂除去心肌梗塞大鼠心脏内过表达的miR-1会减轻这种症状。miR-1表达低于正常水平可引起心肌肥大。miRNAs的失调成为心脏病的起因之一。未来的任务是确认这些失调miRNA的靶mRNA,因为miRNA通常有许多个靶mRNAs,每个miRNA对心肌生长和功能的作用反映了许多mRNA表达的改变。进一步分析这些miRNA的靶mRNA会为心血管疾病提供新的诊断,预后和治疗靶标。
心力衰竭高发病率、患病率和死亡率已经成为21世纪发达和发展中国家的重要公共卫生问题。目前的心衰治疗方案均有一定的副作用,因此继续研究疾病的基本病因以便选择特异性的对因治疗方案和靶点具有特殊的重要性和迫切性。几个不同的研究小组发现miR-21在心肌肥厚模型中表达持续增高。而且用反义核苷酸敲减miR-21的表达可以抑制心肌细胞肥大的表型。miRNA-21在心力衰竭模型鼠的成纤维细胞内大大增加。很重要的是,心力衰竭患者心肌亦有同样结果。心肌重构在心衰进展中起重要作用,目前尚未有人证明miR-21在心肌细胞内调节参与心肌重构通路基因的表达。正常情况下,MMPs(基质金属蛋白酶)和TIMPs(组织金属蛋白酶抑制剂)之间处于平衡状态。MMPs和TIMPs的失衡与心室扩张和重构有关,并将最终导致心力衰竭。终末期心衰患者心室肌中TIMP-3减少程度与心室基质重构程度平行。据文献报道MMPs和ADAMs(分离整和素金属蛋白酶)的有力抑制剂TIMP-3将会是一个很有希望的治疗靶点。近年来的研究已经明确导致心衰发生发展的基本机制是心肌重构,慢性心力衰竭治疗的关键还是逆转或者延缓心肌重构。心肌成纤维细胞分泌的MMPs／TIMPs(组织基质金属蛋白酶／组织基质金属蛋白酶抑制剂)的平衡在心室重构中起着十分重要的作用,因而成为心力衰竭治疗的一个新靶标。临床上已经开始应用多种药物增加组织会属蛋白酶抑制因子3基因表达减少MMPs水平来改善心室重构。基于这些研究基础,本课题以在心力衰竭中异常表达的mi R-21为靶点的反义核苷酸(antagomiR-21)对心肌成纤维细胞表达的TIMP3／MMPs进行调控,从而达到改善心肌重构,避免心脏纤维化,治疗心力衰竭的目的。本项目证明miR-21参与调控心肌重构重要基因,miR-21调控组织金属蛋白酶抑制因子3基因,MMP9在心肌细胞内未见相关报道。从而根据调控通路推断miR-21反义核苷酸同时可以抑制MMPs等参与心肌重构的重要基因在心力衰竭中的异常表达。进而可能抑制心力衰竭。
更好地理解转录后调节不仅需要研究mRNA中的顺式作用元件,而且需要确定反式作用因子(以mRNA中特殊基序为靶标的RNA结合蛋白)。
真核生物mRNA 3’非翻译区可以调节转录本的稳定性、亚细胞定位和翻译水平,决定某一特定mRNA的命运,是许多基因表达所必需的一个调节区。3’非翻译区介导的功能的修饰可影响一个或多个基因的表达,从而导致疾病的发生。对相关mRNA 3’非翻译区的调节序列和与这些序列特异结合的蛋白质等具体信息的认识,将成为药物设计的新的分子靶。
本实验用生物信息学工具,分子克隆等生物学手段探讨了是否micoRNA以及其反义核苷酸调节心肌重构重要基因表达水平,明确了调控其表达的顺式作用元件和反式作用因子。为心力衰竭和肿瘤治疗的临床前期研究打下了基础。
实验方法
一、生物信息学预测
用targetscan, pictar,microinspector等程序预测最可能以组织金属蛋白酶抑制因子3基因为靶基因的miRNAs.
二、细胞培养
三、转染
转染293细胞用siportneofx,根据Ambion的操作规程。
HuR siRNA转染试验
HuR siRNA终浓度为20nM,同时设置正常对照组和20nM终浓度的Ctrl.siRNA阴性转染对照组。oligoFectamine转染原代培养的美国ATCC人心脏成纤维细胞株HCF。
四、western blot
1、弃细胞培养液,冷PBS洗两次,细胞裂解液中加入蛋白酶抑制剂,每孔加100μl细胞裂解液。在4℃冷室内摇床上摇15分钟。细胞擦子刮细胞及细胞外基质,收集。15000 rpm,10分钟,4℃冷室内离心收集上清.
2、定量蛋白：吸取上清后,以BCA试剂做蛋白定量。
3、制胶：分离胶浓度为12%,浓缩胶浓度为4%。浓缩胶内加8M尿素。
4、SDS-PAGE凝胶电泳：用微量加样器分别吸取待分析样品,根据蛋白质浓度和加样孔体积决定加样量,BIORAD电泳板。浓缩胶80V，分离胶100V
5、转印
6、封闭
7、一抗孵育
8、二抗孵育
9、ECL显色
10、扫描胶片
五、RNA提取
按照操作说明用SV40 total RNA分离试剂盒提取转染后293细胞和美国ATCC人心脏成纤维细胞株HCF的mRNA。用nanodrop分光光度计进行定量确定mRNA质量和浓度后进行反转录。
六、反转录
向总量为～200 ng RNA加入随机引物(Invitrogen) 2μl,去离子水定容至11μl进行反转录。将样品在70℃恒温10分钟后,取出冰块骤冷5分钟,加入4μl10×第一链缓冲液和酶(250mM Tris-Hcl, PH8.3室温下375mM KCL;15mM MgCl2),1μl 10mM dNTP混合物(Roche),1μl RNAse抑制剂(40单位／μlPromega), lμl superscriptⅡ反转录酶(20u/μl Invitrogen),2μl 0.1M DTT(Invitrogen),42℃恒温1小时得产物。第一链产物cDNA将通过PCR扩增。
七、实时荧光定量PCR
用已知浓度的样品作标准曲线,将已知浓度的样品2μl分别加入8μl水中。其它样品为等体积含5ng cDNA的溶液。空白组则加入去离子水至10μl,加入151μl通用Taqman mastermix(1×Taqman buffer A,5.5mM Mgcl2,8%甘油,dATP,dCTP,dGTP各200μM, dUTP 400μM,0.01 units/μl of Amperase Uracil N Glycosylase (UNG), AmpliTaq GoldTM DNA多聚酶0.01 units/μl) 8.33μl,探针100nM及正向引物200nM,反向引物200nM,加水使总体积达到251μl。将反应混合液放入实时荧光定量PCR仪进行循环反应,起始温度设为50℃,保持2分钟。每个循环控制条件为95℃10分钟(cDNA链变性),95℃15秒,60℃60秒。此循环进行40次,约2小时完成全部循环。RNA／cDNA相对拷贝数用标准曲线表示出来并且除以各样品的18 SrRNA得到mRNA的相对数量
八、PCR扩增
以反转录的cDNA为模板,围绕miR-1在组织金属蛋白酶抑制因子3基因内的靶点用Pfx高保真聚合酶扩增。
九、双酶切
十、快速连接
用Promega快速连接系统将纯化的PCR片段与pGL3载体或pGEMT载体连接。0.8μl pGL3载体与3'UTR片段混合：3.2 or 3μl 5μl 2×加入酶的快速连接缓冲液,1μl快速连接酶.。将3'UTR片段与pGL3载体以1：3(or>3：1)的比例混合,在连接反应之前可以用电泳法大致测量。或者用分光光度计准确测量。在与pGEMT载体连接之前用Taq-多聚酶(Roche)为PCR片段加尾。设立连接反应的阴性对照实验。将连接反应置于室温下一小时。
十一、转化
建立阳性对照来检测转入活性细胞的效率。每份连接反应都取2μl加入50μl的Topo 10细胞(invitrogen)或JM109细胞.将转化物置冰上30分钟,然后在42℃下进行30秒钟(JM109细胞45秒)的热休克,接着再放入冰中冷却2分钟。在每份转化物种加入250μl的SOC medium,然后在震荡保温箱培养一个小时。每份转化物取100μl涂平板。
十二、琼脂糖凝胶电泳分离提纯DNA片段
1g琼脂糖溶于100 ml的1×Tris-Acetate/EDTA缓冲液,然后在微波炉中加热大约1分钟(至澄清),冷却之后加入5μl EB溶液(10mg／ml)并充分混合。取20μl PCR产物用1%琼脂糖凝胶电泳分析以确定PCR是否成功,分离切割正确的电泳带。凝胶电泳也可用来检测是否片断成功插入pGL3载体上。每20μl样品加入2μ1 10×上样液,同时加1kb plus ladder (invitrogen)或lkb ladder (invitrogen and promega)。将凝胶倒入封好的槽中,插入梳子并且放置40分钟凝固。连上电极后DNA会向阳极泳动。在凝胶电泳系统下100U电泳1小时后,在微弱的紫外光下显影,以防DNA发生突变。
十三、DNA测序分析
测序引物被设计成正好从pGL3载体上的插入片断之前开始测序。M13forward被用于pGEMT克隆测序。每个插入片断的测序反应都在正向引物或者反向引物的引导下完成。
DNA的痕迹文件可以用软件程序Chromos打开。最后用clustal和GeneDoc把这些序列与NCBI中的mRNA序列比对。
十四、质粒大提
按照质粒纯化作用手册的详细说明进行质粒DNA的大提纯化。在4℃15min6000×g离心后收集细菌细胞,沉淀物在缓冲溶液P1中重新悬浮,加入缓冲液P2,然后倒置3-4次使其混合,室温下放置5分钟。取10 ml冷冻了的缓冲液buffer P3加入溶菌产物,迅速倒置4-6次将其混合。将溶菌产物置于Qiafilter管中在室温(25℃)下放置10分钟。之后将其从QIAfilter滤过,用50ml falcon tube收集,加入2.5ml缓冲液ER倒置10次使其混合,在冰中放置30分钟。QIAGEN-tip 500用缓冲液QBT平衡后,将溶菌产物滤过。用缓冲液QC清洗QIAGEN-tip 500。用QIAGEN-tip 500和缓冲液QN洗脱DNA,用10.5 ml异丙醇沉淀并以15000×g离心30分钟。用70%的消毒酒精清洗片状沉淀物,以15000×g离心10分钟。风干片状沉淀物并在300-500μl TE中重新悬浮。用分光光度计测出DNA样品的浓度和纯度。
十五、用stratagene的Quick change变变试剂盒定点突变插入片断
stratagene的Quick change变变试剂盒能够一次性在多位点引发突变,首先用PCR扩增合成大量突变载体。热循环反应成份包括一个超螺旋的双链DNA模板,两个或更多含有所需突变的合成寡核苷酸引物,还有试剂盒提供的具有PfuTurbo(?)DNA多聚酶混合物。首先突变的引物结合到载体上(注意所有的寡核苷酸都设计成与模板DNA的同一条链结合)然后PfuTurbo DNA多聚酶高保真扩增,产生双链DNA分子,其中一条链包含多个突变位点并包含缺刻。缺口被酶混合物封住。突变引物包含突变的hsa-miR-1靶序列。按照产品说明进行聚合酶链式反应。在操作过程的第二步,用限制性内切酶Dpn I处理热循环反应产物。Dpn I对甲基化和半甲基化的DNA具有特异性,用于消化亲代DNA的模板。几乎从所有的大肠杆菌株中分离出的DNA都是甲基化的,因此对消化具有敏感性。在第三步,含有大量复制的突变单链DNA的反应混合物被转入XL10-Gold(?)超感受态细胞,在细胞内形成突变的闭合环状双链DNA。然后可以从转化细胞中制备出双链质粒DNA并用适当的方法分析鉴定包含所需突变的克隆。从大多数普遍使用的大肠杆菌株(dam+)中分离的质粒DNA都是甲基化的,但是从dam大肠杆菌株中分离的质粒DNA,包括JM110和SCS110,不适合这个试剂盒。
十六、双亮荧光酶测定法
将生长液从培养细胞中移除,轻轻地将lml PBS加至24孔板的表面。移除漂浮的细胞和残留的生长液。在用细胞裂解剂之前完全移除PBS。在每个培养孔中加入100μlX被动细胞裂解液(Promega)盖住单层细胞。将培养皿放在摇床上轻轻晃动以确保单层细胞完全被等量的1X被动细胞裂解液覆盖。在室温下摇培养皿15分钟。将细胞裂解液移入1.5ml eppendorf管。在冷冻微量离心机中以最高速度离心溶菌产物30秒。去上清液。在一个孔中连续测定荧火虫荧光素酶和海肾荧光素酶的活性。预先配制LARⅡ和Stop & Glo溶液。小心地将20μl细胞裂解液转移至盛有100ul LARⅡ测定板中,通过2或3次吸液将其混合。将测定板放入荧光分光光度计开始读数。然后加入100μl Stop&Glo(?)反应物并迅速晃动混合,迅速开始读数。在转染36小时后用双重荧光素酶测定法(Promega)连续测量荧火虫荧光素酶和Renilla荧光素酶的活力。
十七、免疫沉淀实验
(一)制备培养细胞1nRNP (mRNA蛋白质复合体)裂解物
1、培养原代培养的美国ATCC人心脏成纤维细胞株HCF细胞于10cm培养皿中,收集细胞前用冰PBS洗细胞两次。
2、4℃／2000rpm／5min离心得到细胞沉淀物。
3、轻弹离心管的底部加入大约和细胞沉淀物相等体积的含RNA酶和蛋白酶抑制剂的冰PLB缓冲液。
4、用移液器吹打混匀细胞,切勿用漩涡震荡器。置于冰上10分钟。
5、30 min,14,000 rpm(20,000 x g)／4℃离心,转移上清到新的Ependorf管中。
6、可以置于-80℃冰箱中(最好直接用于下面的IP实验中)
7、14,000 rpm／4℃离心,保留上清,做BCA实验测蛋白质浓度。我们通常得到蛋白质浓度为15-30μg/μl的裂解物。对于IP实验我们需要50-100μl的细胞裂解物。
(二)抗体包被ProteinA-Sepharose微球
1、在50ml的Falcon管内用5%BSA 4℃溶涨ProteinA-Sepharose微球(sigma),加3-4倍多余的溶液直到15ml.早上倒掉多余的溶液,加0.1%叠氮化钠储存于4℃。
2、加30μg的抗HuR抗体(Santa Cruz Biotech.)或IgG1 (BD Pharmingen)于100μl的ProteinA-Sepharose微球内。用免RNA酶的Eppendorf管,加大约100-200μl的NT-2缓冲液,4℃温和混合过夜。
3、第二天清晨,用1ml冰NT2洗五次,在最后一次离心后弃NT2,微球可以用了。
(三)免疫沉淀mRNP(mRNA蛋白质复合体)
1、用1.5ml Eppendorf管预包被ProteinA-Sepharose抗体微球,首先加入700μl NT-2缓冲液。然后加入10μl 0.1 M DTT(不要直接加到细胞沉淀上,这会让IP不工作)。10μl RNA酶抑制剂,33μl 0.5 M EDTA.加100μl细胞沉淀物并且加NT-2到1ml.4℃孵育1-2小时。5000 g,5 mins离心。用1ml冰NT-2缓冲液洗细胞沉淀物5次。
2、最后一次清洗后,加含5ul DNase I (2U/ul)的100μlNT2 buffer,37℃孵育5-10 mins.加1 ml NT2缓冲液,5000 g,5 mins离心,弃上清。然后加5μl蛋白激酶K,1μl 10% SDS and 100μl NT-2.55℃孵育15-30 min,混匀。
3、5000 g,5 mins离心,收集上清100μl。
4、加200ul NT2缓冲液于微球,5000g/2 mins离心收集上清,弃微球。
5、结合两次收集的上清(100μl和200μl),加300μl底层的苯磺酰氯酸。
6、于室温震荡混悬1分钟,最大速度离心一分钟。
7、收集上层250ul,加25ul pH 5.2的叠氮化钠,625 ul 100%的乙醇和5ulglycoblue,混匀,置于-20℃。
8、第二天,上下颠倒3-5次混匀试管,14.000rpm/4C/30 mins离心,弃上清。
9、加70%乙醇于蓝色的细胞沉淀,混匀,14.000rpm/4℃/2min离心。
10、弃上清,1min/14.000rpm/4℃离心沉淀物,加70%乙醇,室温空气风干5分钟,重悬于20-40 ul水中。
实验结果
1、Targetscan, pictar等生物信息学工具预测表明,hsa-miR-1,hsa-miR-20a, hsa-miR-106b, hsa-miR-30e-5p, hsa-miR-206, hsa-miR-144, hsa-miR-181, hsa-miR-30d, hsa-miR-221, hsa-miR-21, hsa-miR-30b, hsa-miR-101, hsa-miR-22等均可能以组织金属蛋白酶抑制因子3基因为靶基因,其中miR-1最为可能,因为它被许多miRNA预测程序列为前三个最有可能以组织金属蛋白酶抑制因子3基因为靶基因的miRNA。
2、Western blot证实has-miR-1(人microRNA-1)下调组织金属蛋白酶抑制因子3基因蛋白表达水平。
3、实时荧光定量PCR证明相对于阴性转染对照组,hsa-miR-1下调组织金属蛋白酶抑制因子3基因mRNA水平。
4、应用生物信息学技术预测hsa-miR-1在组织金属蛋白酶抑制因子3基因基因中的靶点,PCR技术扩增niR-1预测靶点附近组织金属蛋白酶抑制因子3基因,克隆入荧光素酶表达载体pGL3构建重组质粒pGL3-TIMP3,并用定点突变技术成功构建突变载体,电泳鉴定重组质粒表达及测序验证；
5、hsa-miR-1野生型与突变型载体与renilla和hsa-miR-1共转染粒转染293细胞,双荧光素酶实验证实了hsa-miR-1在组织金属蛋白酶抑制因子3基因内的第一个预测靶点为ACATTCCA。hsa-miR-1对组织金属蛋白酶抑制因子3基因的调控是直接的。从而确定了参与组织金属蛋白酶抑制因子3基因的顺式调控序列。用克隆,定点突变和双亮荧光酶测定技术证实了hsa-miR-1直接打靶组织金属蛋白酶抑制因子3基因基因,在组织金属蛋白酶抑制因子3基因3’非翻译区内存在靶点。对于hsa-miR-1在组织金属蛋白酶抑制因子3基因内的第一个预测靶点,突变型载体的荧光强度以海肾荧光素酶的荧光强度作内参优化后,明显高于野生型。T-test统计分析具有显著性p<0.001。而miRNA前体阴性对照转染组野生型和突变型的荧光素酶的活力以海参荧光素酶作内参优化后则呈不规则变化。对于miR-1在组织金属蛋白酶抑制因子3基因内的第二个预测靶点,在突变四个碱基的情况下,这种变化不明显,无统计学意义。
6、通过转染和实时荧光定量PCR实验证实了miR-21可以下调组织金属蛋白酶抑制因子3基因,增加MMP9的表达,同时证明了antagomiR-21可以增加TIMP3降低MMP9的表达。TIMP3在心肌肥大和心力衰竭中表达降低,而miR-21在这些心血管疾病中异常升高。TIMP3和MMP9是参与心肌重构的重要分子。而心力衰竭的基础就是心肌重构。实验结果提示了通过抑制miR-21在心肌肥大和心力衰竭中的异常表达,有望调控心肌重构途径。通过此试验证明了TIMP3受另外一个反式作用因子miR-21的调节。
7、通过IP和-RT-PCR实验证实HuR蛋白质结合组织金属蛋白酶抑制因子3基因3’未翻译区,通过siRNA knock down HuR从而下调组织金属蛋白酶抑制因子3基因mRNA水平证实HuR参与组织金属蛋白酶抑制因子3基因的转录后调节。其可能结合的区域由生物信息学方法预测。通过此试验证明了组织金属蛋白酶抑制因子3基因的另外一个反式作用因子：ARE结合蛋白HuR。
结论
实验证明了在HEK293细胞内hsa-miR-1直接调控肿瘤抑制基因组织金属蛋白酶抑制因子3基因的转录后调节。下调其mRNA和蛋白质表达水平。在组织金属蛋白酶抑制因子3基因3’未翻译区内存在组织金属蛋白酶抑制因子3基因的靶点。hsa-miR-1通过组织金属蛋白酶抑制因子3基因3'UTR调节的顺式作用元件得以确定。通过IP和RT-PCR实验证实ARE结合蛋白HuR结合组织金属蛋白酶抑制因子3基因3’未翻译区,通过siRNA敲减HuR基因表达从而下调组织金属蛋白酶抑制因子3基因mRNA水平证实HuR参与组织金属蛋白酶抑制因子3基因的转录后调节。从而确定了HuR为参与组织金属蛋白酶抑制因子3基因转录后调节的反式作用元件。通过转染和实时荧光定量PCR实验证实了miR-21可以下调组织金属蛋白酶抑制因子3基因,增加MMP9的表达,同时证明了antagomiR-21可以增加组织金属蛋白酶抑制因子3基因降低MMP9的表达。通过此试验证明了组织金属蛋白酶抑制因子3基因受另外一个反式作用元件miR-21的调节。
组织金属蛋白酶抑制因子3基因和hsa-miR-1在关节炎,癌症,以及心脏病中均异常表达,这为探索其致病机理,以及基因治疗这些疾病提供了线索。组织金属蛋白酶抑制因子3基因在心肌肥大和心力衰竭中表达降低,而miR-21在这些心血管疾病中异常升高。TIMP3和MMP9是参与心肌重构的重要分子。而心力衰竭的基础就是心肌重构。实验结果提示了通过抑制miR-21在心肌肥大和心力衰竭中的异常表达,有望调控心肌重构途径。由于miR-21在多种癌症中表达上调,而组织金属蛋白酶抑制因子3基因在这些癌症中表达沉默。以往的观点认为这可能是由于组织金属蛋白酶抑制因子3基因启动子甲基化引起,但是并非所有癌症都发现了组织金属蛋白酶抑制因子3基因启动子的甲基化,我们的研究为合理解释这一现象提供了开创性的提议和验证,就是miRNAs有可能是组织金属蛋白酶抑制因子3基因在癌症和心血管疾病中经常沉默的原因。
Objective
Naturally occurring microRNAs (miRNAs) are small posttranscriptional regulatory noncoding RNAs that regulate gene expression by affecting the translation or stability of target mRNAs.
Tissue inhibitors of matrix metalloproteinases (TIMPs) maintain a balance in the metabolism of the extracellular matrix.Disruption of this balance may result in diseases associated with uncontrolled turnover of matrix, such as arthritis, cancer, cardiovascular diseases, nephritis, and acute lung injury.
Research in the past five years has put miRNA into a prominent role in development and disease. MiRNAs have been discovered to be involved in tumorigenesis, development, control of cell proliferation, cell death,,fat metabolism,neuronal patterning in nematode, control of leaf and flower development in plants, insulin secretion, B-cell development, and neural stem cell fate, and they may be important for proper immune function.
As miRNAs naturally exist in plants and animals to investigate whether miRNA target the tumor suppressor gene and disease related gene will provide clue to cure these diseases. Currently only the function and target site of a few miRNA has been verified. The bioinformatics tool and molecular cloning biology were employed to investigate whether miRNAs regulate the posttranscriptional regulation of genes that play important role in cardiac remodeling. TIMP3 is expressed in the heart and downregulated in the heart failure. hsa-miR-1 was shown to be overexpressed in individuals with coronary artery disease, and that when overexpressed in normal or infarcted rat hearts, it exacerbates arrhythmogenesis. Elimination of hsa-miR-1 by an antisense inhibitor in infarcted rat hearts relieved arrhythmogenesis. Downregulation of hsa-miR-1 can induce cardiac hypertrophy dysregulation of miRNA expression contributes to heart disease. An important aim for the future will be to identify the mRNA targets of the miRNAs responsible for adverse cardiac remodeling. As miRNAs often have numerous target mRNAs, the effects of individual miRNAs on cardiac growth and function may reflect altered expression of the products of multiple mRNAs. Further analysis of the functions of the regulatory miRNAs described in this study during adverse cardiac remodeling, and during heart development, promises to provide insights into heart disease and potential therapeutic targets. The expression of TIMP3 and miR-1 are abbrerantly expressed in cancer, arthritis and heart disease. Transgenic TIMP3 has been found to induce the apoptosis of cancer cells. We hope we could provide a new tool for gene therapy by finding the naturally occurring miRNAs which regulate genes that play important role in cardiac remodeling.
Methods
1、bioinformatics prediction:
The analysis was done using the three algorithms, TargetScan, PicTar, and microinspector. commonly used to predict human miRNA gene targets.
2、cell culture
3、reverse transfection with Ambion transfection reagent siPort NeoFx which can save one day
4、Westernblot:
5、RNA isolation
Total RNA was isolated using the SV Total RNA isolation system(Promega,UK), followed manufacturer's instruction.
6、Reverse Transcription
7、Real time PCR
8、Polymerase Chain reaction (PCR)
9、double digestion(Amersham) of pGL3 control vector
10、Rapid ligation
11、Transformate into competent E-coli cells
12、Separation and extraction of DNA fragments by agarose gel electrophoresis
13、DNA sequencing
14、QIAGEN Endotoxin-free Plasmid Maxiprep
15、Dual Luciferase assay
Remove the growth medium from the cultured cells, and gently apply lml PBS to wash the surface of the 24-well plate. Swirl the vessel briefly to remove detached cells and residual growth medium. Completely remove the rinse solution before applying PLB reagent. Dispense into each culture well 100μl 1X Passive lysis buffer (Promega, UK) which coverd the cell monolayer. Place the culture plates on a rocking platform with gentle rocking to ensure complete and even coverage of the cell monolayer with 1X PLB. Rock the culture plates at room temperature for 15 minutes. Transfer the lysate to a 1.5ml eppendorf tube for further handling and storage. Clear the lysate samples for 30 seconds by centrifugation at top speed in a refrigerated microcentrifuge. Transfer cleared lysates to a new tube prior to reporter enzyme analyses. Prepare Luciferase Assay Reagent II (LARⅡ) by resuspending the provided lyophilized Luciferase Assay Substrate in 10ml of the supplied Luciferase AssayBufferⅡ. Prepare 1X Stop & Glo Substrate by adding 1 volume of 50X Stop & Glo Substrate to 50 volumes of Stop & Glo Buffer in a glass tube. The assays for firefly luciferase activity and Renilla luciferase activity are performed sequentially in one well Predispense 100μl of LAR II into the appropriate number of luminometer tubes to complete the desired number of DLR. Assays. Carefully transfer up to 20μl of cell lysate into the luminometer tube containing LARⅡ; mix by pipetting 2 or 3 times. Do not vortex. Place the tube in the luminometer and initiate reading. add 100μl of Stop & Glo(?) Reagent and vortex briefly to mix. Replace the sample in the luminometer, and initiate reading. Firefly and Renilla luciferase activities were measured consecutively by using dual-luciferase assays (Promega) 36 h after transfection.
16、Generating two inserts with mutations respectively by using the QuikChange Multi Site-Directed Mutagenesis Kit.
17、Immunoprecipatition
(一)Preparation of mRNP lysate from culture cells
1、Grow and harvest tissue culture cells by washing two times with ice-cold PBS and pellet by centrifugation at 4℃/2000rpm/5'. We need 2 big dishes of cells per sample. Loosen the final cell pellet by gently flicking the bottom of the tube and add an approximately equal pellet volume of ice-cold PLB buffer supplemented with RNAse inhibitors and protease inhibitors.
2、Mix cells by pumping several times with a hand pipettor (no vortex!) and place on ice for 10 min.
3、Spin 30 min at 14,000 rpm (20,000 x g)/4℃. Transfer supernatant to the fresh Ependorf tubes。Freeze and store at-80℃. (Its better to use lysate directly for IP).
4、Preclear the supernatant with 15μg (30μl from stock 0.5μg/μl) of IgG1 control, for 30 min/4℃. Add 50μl PAS non-coated with Ab, incubate 30 min/4℃with rotation. (Note that preclearing is not required for IP followed by RT-PCR. Its required only for IP followed by microarray) 5、Spin down 14,000 rpm/4℃. Save supernatant. This is your pre-cleared lysate. Do Bradford to measure protein concentration (measure 2μl of a 1:100 dilution). We routinely get 15-30μg/μl concentration of lysates and you will need anywhere from 50-100μl per IP of lysate.
(二)Antibody coating of protein A beads
1、Use PAS beads from Sigma (P-3391) (or preswollen beads from Sigma). In a 50 ml Falcon, swell beads overnight in 5% BSA solution at 4℃. Add extra solution until it covers beads 3-4 volumes (until 15 ml mark). In the morning, pour off excess so that the beads are 50% (vol/vol) slurry. May do this in advance but add 0.1% Na azide and store at 4℃.
2. Add 30μg (150μl from stock 200μg/ml) of antibody to 100μl volume of PAS beads using RNAse free Eppendorf tubes. Add about 100-200μl of NT-2 buffer. Bind overnight on rotator at 4℃. Note that the amount of Ab required for IP will depend on the protein. The optimal amount of Ab needed should be determined by doing IP with this protocol with 1,5,10 and 30 ug Ab.
3、Next morning, wash (14K rpm/5') the beads with 1 ml aliquots of ice-cold of NT25 times. After last spin take out the NT2. The beads are now ready to be used. May also do this in advance but add 0.1% Na azide and store at 4℃.
(三)Immunoprecipitation of mRNPs
1、Use 1.5 ml Eppendorf tubes. To precoated PAS/Ab (around 50μl), first add about 700μl NT-2 buffer. Then add all the additives 10μl 0.1 M DTT (do not add the DTT to the pellet directly, as this will reduce you antibody and the IP will not work!), 10μl RNAout,33μl 0.5 M EDTA. Add 100μl lysate (even if concentration of protein is lower than 30ug/ul) and fill-up with NT-2 to 1 ml mark on tube. Incubate 1-2 hrs at 4℃, end-over-end. Spin down (5000 g,5 mins). Wash pellet 5 times with 1 ml aliquots of ice-cold NT-2 buffer (5000 g,5 mins)
2、After last wash, add 100ul NT2 buffer having 5ul DNase I (2U/ul). Keep at 37 C (or 30 C) for 5-10 mins. Add 1 ml NT2 buffer, spin 5000 g,5 mins, discard supernatant. Then, add the following to the PAS pellet:5μl of Proteinase K (10mg/ml), 1μl 10%SDS and 100μl NT-2. If you have several samples, its good to make a mastermix of NT2 buffer, Proteinase K and SDS). Incubate at 55℃for 15-30 min, with mixing.
3、Spin 5000 g,5 mins, collect supernatant (～100ul)
4、To beads add 200ul NT2 buffer, spin 5000 g/2 mins, collect supernatant (-200ul), discard beads.
5、Combine supernatants (100ul and 200ul) and add 300ul lower layer of acid phenol-CHCl3 (Ambion)
6、Vortex,1'RT (or 37C in shaker), short spin at RT (imp)/1'/max speed.
7、Collect 250ul of upper layer, add 25ul sodium acetate pH 5.2,625ul 100% ETOH and 5ul glycoblue, mix well, keep O/N-20C.
8、Next day, mix the tubes by inversion 3-5 times, spin 14.000rpm/4C/30 mins and discard supernatant
9、To the blue pellet add lml of 70% ETOH and mix by inversion or vortexing, spin 14.000rpm/4C/2'
10、Discard supernatant, spin pellet 1'/14.000rpm/4C, pipette any 70% ETOH, air dry pellet at RT for 5', resuspend in 20-40 ul of water.
Results
1、Hsa-miR-1 downregulates the expression of TIMP3 potein in HEK 293 cells.
2、Hsa-miR-1 downregulates the expression of TIMP3 mRNA compared with the negative control groups in HEK 293 cells.
3、It has been validated that hsa-miR-1 has target site in TIMP3 3'UTR by cloning in HEK 293 cells, mutagenesis and dual luciferase assay. The luciferase activity of the mutant constructs are significantly higher that those of wild type constructs for the first potential target site, wheareas the negative control group did not show the same effect. For the second potential target site, the difference between the mutant constructs and wild type constructs are not significant.
4、It has been validated that pre-miR-21 significantly downregulated TIMP3 expression and upregulated MMP9 expression. And the antogomiR-21 significantly upregulated the TIMP3 expression and downregulated MMP9 expression human cardiac fibroblast cells. This suggested that miR-21 regulated the cardiac remodeling pathway by targeting the important regulators in the pathway. Since cadiac remodeling leads to heart failure. This suggests that knockdown miR-21 by antagomiR-21 might have therapeutical effect on heart failure. It has been verified that miR-21 is another trans-acting factor of TIMP3 gene.
5、It has been confirmed that ARE binding protein HuR contributes the TIMP3 mRNA stability in human cardiac fibroblast cells.
Conclusion
The experiment verified hsa-miR-1 directly regulates the posttranscriptional of TIMP3 gene in 293 cells. hsa-miR-1 downregulates mRNA and protein expression of TIMP3 gene. The target site in TIMP3 was confirmed. In addition to hsa-miR-1, this studay also confirmed another two trans-acting factors. miR-21 and ARE binding protein HuR. TIMP3 and miR-1 were aberrantly expressed in cancer, arthritis and heart diseases. It has been validated that pre-miR-21 significantly downregulated TIMP3 expression and upregulated MMP9 expression. And antogomiR-21 significantly upregulated the TIMP3 expression and downregulated MMP9 expression in the cadiac fibroblast. This suggested that miR-21 regulated the cardiac remodeling pathway by targeting the important regulators in the pathway. This suggests that knockdown miR-21 by antagomiR-21 might have therapeutical effect on heart failure since cadiac remodeling leads to heart failure. The experiment provides the clue for investigating the cause of the diseases and how to cure the diseases.
由于miRNA天然存在于动植物体内,探索miRNA对肿瘤抑制基因和疾病相关基因的作用无疑将会揭开这些疾病治疗的新篇章。目前只有少数miRNA的部分功能和靶点被鉴定。组织金属蛋白酶抑制因子3基因和miR-1,miR-2-(microRNA-21)均在心血管紊乱疾病和癌症中异常表达。miR-1广泛分布于心肌,骨骼肌。TIMP3(组织金属蛋白酶抑制因子3基因)在心脏中大量存在,在缺陷的心脏中表达下调。冠状动脉硬化的个体内miR-1过度表达。在正常或心肌梗塞的大鼠心脏里过表达miR-1会加速心律不齐。用反义的抑制剂除去心肌梗塞大鼠心脏内过表达的miR-1会减轻这种症状。miR-1表达低于正常水平可引起心肌肥大。miRNAs的失调成为心脏病的起因之一。未来的任务是确认这些失调miRNA的靶mRNA,因为miRNA通常有许多个靶mRNAs,每个miRNA对心肌生长和功能的作用反映了许多mRNA表达的改变。进一步分析这些miRNA的靶mRNA会为心血管疾病提供新的诊断,预后和治疗靶标。
心力衰竭高发病率、患病率和死亡率已经成为21世纪发达和发展中国家的重要公共卫生问题。目前的心衰治疗方案均有一定的副作用,因此继续研究疾病的基本病因以便选择特异性的对因治疗方案和靶点具有特殊的重要性和迫切性。几个不同的研究小组发现miR-21在心肌肥厚模型中表达持续增高。而且用反义核苷酸敲减miR-21的表达可以抑制心肌细胞肥大的表型。miRNA-21在心力衰竭模型鼠的成纤维细胞内大大增加。很重要的是,心力衰竭患者心肌亦有同样结果。心肌重构在心衰进展中起重要作用,目前尚未有人证明miR-21在心肌细胞内调节参与心肌重构通路基因的表达。正常情况下,MMPs(基质金属蛋白酶)和TIMPs(组织金属蛋白酶抑制剂)之间处于平衡状态。MMPs和TIMPs的失衡与心室扩张和重构有关,并将最终导致心力衰竭。终末期心衰患者心室肌中TIMP-3减少程度与心室基质重构程度平行。据文献报道MMPs和ADAMs(分离整和素金属蛋白酶)的有力抑制剂TIMP-3将会是一个很有希望的治疗靶点。近年来的研究已经明确导致心衰发生发展的基本机制是心肌重构,慢性心力衰竭治疗的关键还是逆转或者延缓心肌重构。心肌成纤维细胞分泌的MMPs／TIMPs(组织基质金属蛋白酶／组织基质金属蛋白酶抑制剂)的平衡在心室重构中起着十分重要的作用,因而成为心力衰竭治疗的一个新靶标。临床上已经开始应用多种药物增加组织会属蛋白酶抑制因子3基因表达减少MMPs水平来改善心室重构。基于这些研究基础,本课题以在心力衰竭中异常表达的mi R-21为靶点的反义核苷酸(antagomiR-21)对心肌成纤维细胞表达的TIMP3／MMPs进行调控,从而达到改善心肌重构,避免心脏纤维化,治疗心力衰竭的目的。本项目证明miR-21参与调控心肌重构重要基因,miR-21调控组织金属蛋白酶抑制因子3基因,MMP9在心肌细胞内未见相关报道。从而根据调控通路推断miR-21反义核苷酸同时可以抑制MMPs等参与心肌重构的重要基因在心力衰竭中的异常表达。进而可能抑制心力衰竭。
更好地理解转录后调节不仅需要研究mRNA中的顺式作用元件,而且需要确定反式作用因子(以mRNA中特殊基序为靶标的RNA结合蛋白)。
真核生物mRNA 3’非翻译区可以调节转录本的稳定性、亚细胞定位和翻译水平,决定某一特定mRNA的命运,是许多基因表达所必需的一个调节区。3’非翻译区介导的功能的修饰可影响一个或多个基因的表达,从而导致疾病的发生。对相关mRNA 3’非翻译区的调节序列和与这些序列特异结合的蛋白质等具体信息的认识,将成为药物设计的新的分子靶。
本实验用生物信息学工具,分子克隆等生物学手段探讨了是否micoRNA以及其反义核苷酸调节心肌重构重要基因表达水平,明确了调控其表达的顺式作用元件和反式作用因子。为心力衰竭和肿瘤治疗的临床前期研究打下了基础。
实验方法
一、生物信息学预测
用targetscan, pictar,microinspector等程序预测最可能以组织金属蛋白酶抑制因子3基因为靶基因的miRNAs.
二、细胞培养
三、转染
转染293细胞用siportneofx,根据Ambion的操作规程。
HuR siRNA转染试验
HuR siRNA终浓度为20nM,同时设置正常对照组和20nM终浓度的Ctrl.siRNA阴性转染对照组。oligoFectamine转染原代培养的美国ATCC人心脏成纤维细胞株HCF。
四、western blot
1、弃细胞培养液,冷PBS洗两次,细胞裂解液中加入蛋白酶抑制剂,每孔加100μl细胞裂解液。在4℃冷室内摇床上摇15分钟。细胞擦子刮细胞及细胞外基质,收集。15000 rpm,10分钟,4℃冷室内离心收集上清.
2、定量蛋白：吸取上清后,以BCA试剂做蛋白定量。
3、制胶：分离胶浓度为12%,浓缩胶浓度为4%。浓缩胶内加8M尿素。
4、SDS-PAGE凝胶电泳：用微量加样器分别吸取待分析样品,根据蛋白质浓度和加样孔体积决定加样量,BIORAD电泳板。浓缩胶80V，分离胶100V
5、转印
6、封闭
7、一抗孵育
8、二抗孵育
9、ECL显色
10、扫描胶片
五、RNA提取
按照操作说明用SV40 total RNA分离试剂盒提取转染后293细胞和美国ATCC人心脏成纤维细胞株HCF的mRNA。用nanodrop分光光度计进行定量确定mRNA质量和浓度后进行反转录。
六、反转录
向总量为～200 ng RNA加入随机引物(Invitrogen) 2μl,去离子水定容至11μl进行反转录。将样品在70℃恒温10分钟后,取出冰块骤冷5分钟,加入4μl10×第一链缓冲液和酶(250mM Tris-Hcl, PH8.3室温下375mM KCL;15mM MgCl2),1μl 10mM dNTP混合物(Roche),1μl RNAse抑制剂(40单位／μlPromega), lμl superscriptⅡ反转录酶(20u/μl Invitrogen),2μl 0.1M DTT(Invitrogen),42℃恒温1小时得产物。第一链产物cDNA将通过PCR扩增。
七、实时荧光定量PCR
用已知浓度的样品作标准曲线,将已知浓度的样品2μl分别加入8μl水中。其它样品为等体积含5ng cDNA的溶液。空白组则加入去离子水至10μl,加入151μl通用Taqman mastermix(1×Taqman buffer A,5.5mM Mgcl2,8%甘油,dATP,dCTP,dGTP各200μM, dUTP 400μM,0.01 units/μl of Amperase Uracil N Glycosylase (UNG), AmpliTaq GoldTM DNA多聚酶0.01 units/μl) 8.33μl,探针100nM及正向引物200nM,反向引物200nM,加水使总体积达到251μl。将反应混合液放入实时荧光定量PCR仪进行循环反应,起始温度设为50℃,保持2分钟。每个循环控制条件为95℃10分钟(cDNA链变性),95℃15秒,60℃60秒。此循环进行40次,约2小时完成全部循环。RNA／cDNA相对拷贝数用标准曲线表示出来并且除以各样品的18 SrRNA得到mRNA的相对数量
八、PCR扩增
以反转录的cDNA为模板,围绕miR-1在组织金属蛋白酶抑制因子3基因内的靶点用Pfx高保真聚合酶扩增。
九、双酶切
十、快速连接
用Promega快速连接系统将纯化的PCR片段与pGL3载体或pGEMT载体连接。0.8μl pGL3载体与3'UTR片段混合：3.2 or 3μl 5μl 2×加入酶的快速连接缓冲液,1μl快速连接酶.。将3'UTR片段与pGL3载体以1：3(or>3：1)的比例混合,在连接反应之前可以用电泳法大致测量。或者用分光光度计准确测量。在与pGEMT载体连接之前用Taq-多聚酶(Roche)为PCR片段加尾。设立连接反应的阴性对照实验。将连接反应置于室温下一小时。
十一、转化
建立阳性对照来检测转入活性细胞的效率。每份连接反应都取2μl加入50μl的Topo 10细胞(invitrogen)或JM109细胞.将转化物置冰上30分钟,然后在42℃下进行30秒钟(JM109细胞45秒)的热休克,接着再放入冰中冷却2分钟。在每份转化物种加入250μl的SOC medium,然后在震荡保温箱培养一个小时。每份转化物取100μl涂平板。
十二、琼脂糖凝胶电泳分离提纯DNA片段
1g琼脂糖溶于100 ml的1×Tris-Acetate/EDTA缓冲液,然后在微波炉中加热大约1分钟(至澄清),冷却之后加入5μl EB溶液(10mg／ml)并充分混合。取20μl PCR产物用1%琼脂糖凝胶电泳分析以确定PCR是否成功,分离切割正确的电泳带。凝胶电泳也可用来检测是否片断成功插入pGL3载体上。每20μl样品加入2μ1 10×上样液,同时加1kb plus ladder (invitrogen)或lkb ladder (invitrogen and promega)。将凝胶倒入封好的槽中,插入梳子并且放置40分钟凝固。连上电极后DNA会向阳极泳动。在凝胶电泳系统下100U电泳1小时后,在微弱的紫外光下显影,以防DNA发生突变。
十三、DNA测序分析
测序引物被设计成正好从pGL3载体上的插入片断之前开始测序。M13forward被用于pGEMT克隆测序。每个插入片断的测序反应都在正向引物或者反向引物的引导下完成。
DNA的痕迹文件可以用软件程序Chromos打开。最后用clustal和GeneDoc把这些序列与NCBI中的mRNA序列比对。
十四、质粒大提
按照质粒纯化作用手册的详细说明进行质粒DNA的大提纯化。在4℃15min6000×g离心后收集细菌细胞,沉淀物在缓冲溶液P1中重新悬浮,加入缓冲液P2,然后倒置3-4次使其混合,室温下放置5分钟。取10 ml冷冻了的缓冲液buffer P3加入溶菌产物,迅速倒置4-6次将其混合。将溶菌产物置于Qiafilter管中在室温(25℃)下放置10分钟。之后将其从QIAfilter滤过,用50ml falcon tube收集,加入2.5ml缓冲液ER倒置10次使其混合,在冰中放置30分钟。QIAGEN-tip 500用缓冲液QBT平衡后,将溶菌产物滤过。用缓冲液QC清洗QIAGEN-tip 500。用QIAGEN-tip 500和缓冲液QN洗脱DNA,用10.5 ml异丙醇沉淀并以15000×g离心30分钟。用70%的消毒酒精清洗片状沉淀物,以15000×g离心10分钟。风干片状沉淀物并在300-500μl TE中重新悬浮。用分光光度计测出DNA样品的浓度和纯度。
十五、用stratagene的Quick change变变试剂盒定点突变插入片断
stratagene的Quick change变变试剂盒能够一次性在多位点引发突变,首先用PCR扩增合成大量突变载体。热循环反应成份包括一个超螺旋的双链DNA模板,两个或更多含有所需突变的合成寡核苷酸引物,还有试剂盒提供的具有PfuTurbo(?)DNA多聚酶混合物。首先突变的引物结合到载体上(注意所有的寡核苷酸都设计成与模板DNA的同一条链结合)然后PfuTurbo DNA多聚酶高保真扩增,产生双链DNA分子,其中一条链包含多个突变位点并包含缺刻。缺口被酶混合物封住。突变引物包含突变的hsa-miR-1靶序列。按照产品说明进行聚合酶链式反应。在操作过程的第二步,用限制性内切酶Dpn I处理热循环反应产物。Dpn I对甲基化和半甲基化的DNA具有特异性,用于消化亲代DNA的模板。几乎从所有的大肠杆菌株中分离出的DNA都是甲基化的,因此对消化具有敏感性。在第三步,含有大量复制的突变单链DNA的反应混合物被转入XL10-Gold(?)超感受态细胞,在细胞内形成突变的闭合环状双链DNA。然后可以从转化细胞中制备出双链质粒DNA并用适当的方法分析鉴定包含所需突变的克隆。从大多数普遍使用的大肠杆菌株(dam+)中分离的质粒DNA都是甲基化的,但是从dam大肠杆菌株中分离的质粒DNA,包括JM110和SCS110,不适合这个试剂盒。
十六、双亮荧光酶测定法
将生长液从培养细胞中移除,轻轻地将lml PBS加至24孔板的表面。移除漂浮的细胞和残留的生长液。在用细胞裂解剂之前完全移除PBS。在每个培养孔中加入100μlX被动细胞裂解液(Promega)盖住单层细胞。将培养皿放在摇床上轻轻晃动以确保单层细胞完全被等量的1X被动细胞裂解液覆盖。在室温下摇培养皿15分钟。将细胞裂解液移入1.5ml eppendorf管。在冷冻微量离心机中以最高速度离心溶菌产物30秒。去上清液。在一个孔中连续测定荧火虫荧光素酶和海肾荧光素酶的活性。预先配制LARⅡ和Stop & Glo溶液。小心地将20μl细胞裂解液转移至盛有100ul LARⅡ测定板中,通过2或3次吸液将其混合。将测定板放入荧光分光光度计开始读数。然后加入100μl Stop&Glo(?)反应物并迅速晃动混合,迅速开始读数。在转染36小时后用双重荧光素酶测定法(Promega)连续测量荧火虫荧光素酶和Renilla荧光素酶的活力。
十七、免疫沉淀实验
(一)制备培养细胞1nRNP (mRNA蛋白质复合体)裂解物
1、培养原代培养的美国ATCC人心脏成纤维细胞株HCF细胞于10cm培养皿中,收集细胞前用冰PBS洗细胞两次。
2、4℃／2000rpm／5min离心得到细胞沉淀物。
3、轻弹离心管的底部加入大约和细胞沉淀物相等体积的含RNA酶和蛋白酶抑制剂的冰PLB缓冲液。
4、用移液器吹打混匀细胞,切勿用漩涡震荡器。置于冰上10分钟。
5、30 min,14,000 rpm(20,000 x g)／4℃离心,转移上清到新的Ependorf管中。
6、可以置于-80℃冰箱中(最好直接用于下面的IP实验中)
7、14,000 rpm／4℃离心,保留上清,做BCA实验测蛋白质浓度。我们通常得到蛋白质浓度为15-30μg/μl的裂解物。对于IP实验我们需要50-100μl的细胞裂解物。
(二)抗体包被ProteinA-Sepharose微球
1、在50ml的Falcon管内用5%BSA 4℃溶涨ProteinA-Sepharose微球(sigma),加3-4倍多余的溶液直到15ml.早上倒掉多余的溶液,加0.1%叠氮化钠储存于4℃。
2、加30μg的抗HuR抗体(Santa Cruz Biotech.)或IgG1 (BD Pharmingen)于100μl的ProteinA-Sepharose微球内。用免RNA酶的Eppendorf管,加大约100-200μl的NT-2缓冲液,4℃温和混合过夜。
3、第二天清晨,用1ml冰NT2洗五次,在最后一次离心后弃NT2,微球可以用了。
(三)免疫沉淀mRNP(mRNA蛋白质复合体)
1、用1.5ml Eppendorf管预包被ProteinA-Sepharose抗体微球,首先加入700μl NT-2缓冲液。然后加入10μl 0.1 M DTT(不要直接加到细胞沉淀上,这会让IP不工作)。10μl RNA酶抑制剂,33μl 0.5 M EDTA.加100μl细胞沉淀物并且加NT-2到1ml.4℃孵育1-2小时。5000 g,5 mins离心。用1ml冰NT-2缓冲液洗细胞沉淀物5次。
2、最后一次清洗后,加含5ul DNase I (2U/ul)的100μlNT2 buffer,37℃孵育5-10 mins.加1 ml NT2缓冲液,5000 g,5 mins离心,弃上清。然后加5μl蛋白激酶K,1μl 10% SDS and 100μl NT-2.55℃孵育15-30 min,混匀。
3、5000 g,5 mins离心,收集上清100μl。
4、加200ul NT2缓冲液于微球,5000g/2 mins离心收集上清,弃微球。
5、结合两次收集的上清(100μl和200μl),加300μl底层的苯磺酰氯酸。
6、于室温震荡混悬1分钟,最大速度离心一分钟。
7、收集上层250ul,加25ul pH 5.2的叠氮化钠,625 ul 100%的乙醇和5ulglycoblue,混匀,置于-20℃。
8、第二天,上下颠倒3-5次混匀试管,14.000rpm/4C/30 mins离心,弃上清。
9、加70%乙醇于蓝色的细胞沉淀,混匀,14.000rpm/4℃/2min离心。
10、弃上清,1min/14.000rpm/4℃离心沉淀物,加70%乙醇,室温空气风干5分钟,重悬于20-40 ul水中。
实验结果
1、Targetscan, pictar等生物信息学工具预测表明,hsa-miR-1,hsa-miR-20a, hsa-miR-106b, hsa-miR-30e-5p, hsa-miR-206, hsa-miR-144, hsa-miR-181, hsa-miR-30d, hsa-miR-221, hsa-miR-21, hsa-miR-30b, hsa-miR-101, hsa-miR-22等均可能以组织金属蛋白酶抑制因子3基因为靶基因,其中miR-1最为可能,因为它被许多miRNA预测程序列为前三个最有可能以组织金属蛋白酶抑制因子3基因为靶基因的miRNA。
2、Western blot证实has-miR-1(人microRNA-1)下调组织金属蛋白酶抑制因子3基因蛋白表达水平。
3、实时荧光定量PCR证明相对于阴性转染对照组,hsa-miR-1下调组织金属蛋白酶抑制因子3基因mRNA水平。
4、应用生物信息学技术预测hsa-miR-1在组织金属蛋白酶抑制因子3基因基因中的靶点,PCR技术扩增niR-1预测靶点附近组织金属蛋白酶抑制因子3基因,克隆入荧光素酶表达载体pGL3构建重组质粒pGL3-TIMP3,并用定点突变技术成功构建突变载体,电泳鉴定重组质粒表达及测序验证；
5、hsa-miR-1野生型与突变型载体与renilla和hsa-miR-1共转染粒转染293细胞,双荧光素酶实验证实了hsa-miR-1在组织金属蛋白酶抑制因子3基因内的第一个预测靶点为ACATTCCA。hsa-miR-1对组织金属蛋白酶抑制因子3基因的调控是直接的。从而确定了参与组织金属蛋白酶抑制因子3基因的顺式调控序列。用克隆,定点突变和双亮荧光酶测定技术证实了hsa-miR-1直接打靶组织金属蛋白酶抑制因子3基因基因,在组织金属蛋白酶抑制因子3基因3’非翻译区内存在靶点。对于hsa-miR-1在组织金属蛋白酶抑制因子3基因内的第一个预测靶点,突变型载体的荧光强度以海肾荧光素酶的荧光强度作内参优化后,明显高于野生型。T-test统计分析具有显著性p<0.001。而miRNA前体阴性对照转染组野生型和突变型的荧光素酶的活力以海参荧光素酶作内参优化后则呈不规则变化。对于miR-1在组织金属蛋白酶抑制因子3基因内的第二个预测靶点,在突变四个碱基的情况下,这种变化不明显,无统计学意义。
6、通过转染和实时荧光定量PCR实验证实了miR-21可以下调组织金属蛋白酶抑制因子3基因,增加MMP9的表达,同时证明了antagomiR-21可以增加TIMP3降低MMP9的表达。TIMP3在心肌肥大和心力衰竭中表达降低,而miR-21在这些心血管疾病中异常升高。TIMP3和MMP9是参与心肌重构的重要分子。而心力衰竭的基础就是心肌重构。实验结果提示了通过抑制miR-21在心肌肥大和心力衰竭中的异常表达,有望调控心肌重构途径。通过此试验证明了TIMP3受另外一个反式作用因子miR-21的调节。
7、通过IP和-RT-PCR实验证实HuR蛋白质结合组织金属蛋白酶抑制因子3基因3’未翻译区,通过siRNA knock down HuR从而下调组织金属蛋白酶抑制因子3基因mRNA水平证实HuR参与组织金属蛋白酶抑制因子3基因的转录后调节。其可能结合的区域由生物信息学方法预测。通过此试验证明了组织金属蛋白酶抑制因子3基因的另外一个反式作用因子：ARE结合蛋白HuR。
结论
实验证明了在HEK293细胞内hsa-miR-1直接调控肿瘤抑制基因组织金属蛋白酶抑制因子3基因的转录后调节。下调其mRNA和蛋白质表达水平。在组织金属蛋白酶抑制因子3基因3’未翻译区内存在组织金属蛋白酶抑制因子3基因的靶点。hsa-miR-1通过组织金属蛋白酶抑制因子3基因3'UTR调节的顺式作用元件得以确定。通过IP和RT-PCR实验证实ARE结合蛋白HuR结合组织金属蛋白酶抑制因子3基因3’未翻译区,通过siRNA敲减HuR基因表达从而下调组织金属蛋白酶抑制因子3基因mRNA水平证实HuR参与组织金属蛋白酶抑制因子3基因的转录后调节。从而确定了HuR为参与组织金属蛋白酶抑制因子3基因转录后调节的反式作用元件。通过转染和实时荧光定量PCR实验证实了miR-21可以下调组织金属蛋白酶抑制因子3基因,增加MMP9的表达,同时证明了antagomiR-21可以增加组织金属蛋白酶抑制因子3基因降低MMP9的表达。通过此试验证明了组织金属蛋白酶抑制因子3基因受另外一个反式作用元件miR-21的调节。
组织金属蛋白酶抑制因子3基因和hsa-miR-1在关节炎,癌症,以及心脏病中均异常表达,这为探索其致病机理,以及基因治疗这些疾病提供了线索。组织金属蛋白酶抑制因子3基因在心肌肥大和心力衰竭中表达降低,而miR-21在这些心血管疾病中异常升高。TIMP3和MMP9是参与心肌重构的重要分子。而心力衰竭的基础就是心肌重构。实验结果提示了通过抑制miR-21在心肌肥大和心力衰竭中的异常表达,有望调控心肌重构途径。由于miR-21在多种癌症中表达上调,而组织金属蛋白酶抑制因子3基因在这些癌症中表达沉默。以往的观点认为这可能是由于组织金属蛋白酶抑制因子3基因启动子甲基化引起,但是并非所有癌症都发现了组织金属蛋白酶抑制因子3基因启动子的甲基化,我们的研究为合理解释这一现象提供了开创性的提议和验证,就是miRNAs有可能是组织金属蛋白酶抑制因子3基因在癌症和心血管疾病中经常沉默的原因。
Objective
Naturally occurring microRNAs (miRNAs) are small posttranscriptional regulatory noncoding RNAs that regulate gene expression by affecting the translation or stability of target mRNAs.
Tissue inhibitors of matrix metalloproteinases (TIMPs) maintain a balance in the metabolism of the extracellular matrix.Disruption of this balance may result in diseases associated with uncontrolled turnover of matrix, such as arthritis, cancer, cardiovascular diseases, nephritis, and acute lung injury.
Research in the past five years has put miRNA into a prominent role in development and disease. MiRNAs have been discovered to be involved in tumorigenesis, development, control of cell proliferation, cell death,,fat metabolism,neuronal patterning in nematode, control of leaf and flower development in plants, insulin secretion, B-cell development, and neural stem cell fate, and they may be important for proper immune function.
As miRNAs naturally exist in plants and animals to investigate whether miRNA target the tumor suppressor gene and disease related gene will provide clue to cure these diseases. Currently only the function and target site of a few miRNA has been verified. The bioinformatics tool and molecular cloning biology were employed to investigate whether miRNAs regulate the posttranscriptional regulation of genes that play important role in cardiac remodeling. TIMP3 is expressed in the heart and downregulated in the heart failure. hsa-miR-1 was shown to be overexpressed in individuals with coronary artery disease, and that when overexpressed in normal or infarcted rat hearts, it exacerbates arrhythmogenesis. Elimination of hsa-miR-1 by an antisense inhibitor in infarcted rat hearts relieved arrhythmogenesis. Downregulation of hsa-miR-1 can induce cardiac hypertrophy dysregulation of miRNA expression contributes to heart disease. An important aim for the future will be to identify the mRNA targets of the miRNAs responsible for adverse cardiac remodeling. As miRNAs often have numerous target mRNAs, the effects of individual miRNAs on cardiac growth and function may reflect altered expression of the products of multiple mRNAs. Further analysis of the functions of the regulatory miRNAs described in this study during adverse cardiac remodeling, and during heart development, promises to provide insights into heart disease and potential therapeutic targets. The expression of TIMP3 and miR-1 are abbrerantly expressed in cancer, arthritis and heart disease. Transgenic TIMP3 has been found to induce the apoptosis of cancer cells. We hope we could provide a new tool for gene therapy by finding the naturally occurring miRNAs which regulate genes that play important role in cardiac remodeling.
Methods
1、bioinformatics prediction:
The analysis was done using the three algorithms, TargetScan, PicTar, and microinspector. commonly used to predict human miRNA gene targets.
2、cell culture
3、reverse transfection with Ambion transfection reagent siPort NeoFx which can save one day
4、Westernblot:
5、RNA isolation
Total RNA was isolated using the SV Total RNA isolation system(Promega,UK), followed manufacturer's instruction.
6、Reverse Transcription
7、Real time PCR
8、Polymerase Chain reaction (PCR)
9、double digestion(Amersham) of pGL3 control vector
10、Rapid ligation
11、Transformate into competent E-coli cells
12、Separation and extraction of DNA fragments by agarose gel electrophoresis
13、DNA sequencing
14、QIAGEN Endotoxin-free Plasmid Maxiprep
15、Dual Luciferase assay
Remove the growth medium from the cultured cells, and gently apply lml PBS to wash the surface of the 24-well plate. Swirl the vessel briefly to remove detached cells and residual growth medium. Completely remove the rinse solution before applying PLB reagent. Dispense into each culture well 100μl 1X Passive lysis buffer (Promega, UK) which coverd the cell monolayer. Place the culture plates on a rocking platform with gentle rocking to ensure complete and even coverage of the cell monolayer with 1X PLB. Rock the culture plates at room temperature for 15 minutes. Transfer the lysate to a 1.5ml eppendorf tube for further handling and storage. Clear the lysate samples for 30 seconds by centrifugation at top speed in a refrigerated microcentrifuge. Transfer cleared lysates to a new tube prior to reporter enzyme analyses. Prepare Luciferase Assay Reagent II (LARⅡ) by resuspending the provided lyophilized Luciferase Assay Substrate in 10ml of the supplied Luciferase AssayBufferⅡ. Prepare 1X Stop & Glo Substrate by adding 1 volume of 50X Stop & Glo Substrate to 50 volumes of Stop & Glo Buffer in a glass tube. The assays for firefly luciferase activity and Renilla luciferase activity are performed sequentially in one well Predispense 100μl of LAR II into the appropriate number of luminometer tubes to complete the desired number of DLR. Assays. Carefully transfer up to 20μl of cell lysate into the luminometer tube containing LARⅡ; mix by pipetting 2 or 3 times. Do not vortex. Place the tube in the luminometer and initiate reading. add 100μl of Stop & Glo(?) Reagent and vortex briefly to mix. Replace the sample in the luminometer, and initiate reading. Firefly and Renilla luciferase activities were measured consecutively by using dual-luciferase assays (Promega) 36 h after transfection.
16、Generating two inserts with mutations respectively by using the QuikChange Multi Site-Directed Mutagenesis Kit.
17、Immunoprecipatition
(一)Preparation of mRNP lysate from culture cells
1、Grow and harvest tissue culture cells by washing two times with ice-cold PBS and pellet by centrifugation at 4℃/2000rpm/5'. We need 2 big dishes of cells per sample. Loosen the final cell pellet by gently flicking the bottom of the tube and add an approximately equal pellet volume of ice-cold PLB buffer supplemented with RNAse inhibitors and protease inhibitors.
2、Mix cells by pumping several times with a hand pipettor (no vortex!) and place on ice for 10 min.
3、Spin 30 min at 14,000 rpm (20,000 x g)/4℃. Transfer supernatant to the fresh Ependorf tubes。Freeze and store at-80℃. (Its better to use lysate directly for IP).
4、Preclear the supernatant with 15μg (30μl from stock 0.5μg/μl) of IgG1 control, for 30 min/4℃. Add 50μl PAS non-coated with Ab, incubate 30 min/4℃with rotation. (Note that preclearing is not required for IP followed by RT-PCR. Its required only for IP followed by microarray) 5、Spin down 14,000 rpm/4℃. Save supernatant. This is your pre-cleared lysate. Do Bradford to measure protein concentration (measure 2μl of a 1:100 dilution). We routinely get 15-30μg/μl concentration of lysates and you will need anywhere from 50-100μl per IP of lysate.
(二)Antibody coating of protein A beads
1、Use PAS beads from Sigma (P-3391) (or preswollen beads from Sigma). In a 50 ml Falcon, swell beads overnight in 5% BSA solution at 4℃. Add extra solution until it covers beads 3-4 volumes (until 15 ml mark). In the morning, pour off excess so that the beads are 50% (vol/vol) slurry. May do this in advance but add 0.1% Na azide and store at 4℃.
2. Add 30μg (150μl from stock 200μg/ml) of antibody to 100μl volume of PAS beads using RNAse free Eppendorf tubes. Add about 100-200μl of NT-2 buffer. Bind overnight on rotator at 4℃. Note that the amount of Ab required for IP will depend on the protein. The optimal amount of Ab needed should be determined by doing IP with this protocol with 1,5,10 and 30 ug Ab.
3、Next morning, wash (14K rpm/5') the beads with 1 ml aliquots of ice-cold of NT25 times. After last spin take out the NT2. The beads are now ready to be used. May also do this in advance but add 0.1% Na azide and store at 4℃.
(三)Immunoprecipitation of mRNPs
1、Use 1.5 ml Eppendorf tubes. To precoated PAS/Ab (around 50μl), first add about 700μl NT-2 buffer. Then add all the additives 10μl 0.1 M DTT (do not add the DTT to the pellet directly, as this will reduce you antibody and the IP will not work!), 10μl RNAout,33μl 0.5 M EDTA. Add 100μl lysate (even if concentration of protein is lower than 30ug/ul) and fill-up with NT-2 to 1 ml mark on tube. Incubate 1-2 hrs at 4℃, end-over-end. Spin down (5000 g,5 mins). Wash pellet 5 times with 1 ml aliquots of ice-cold NT-2 buffer (5000 g,5 mins)
2、After last wash, add 100ul NT2 buffer having 5ul DNase I (2U/ul). Keep at 37 C (or 30 C) for 5-10 mins. Add 1 ml NT2 buffer, spin 5000 g,5 mins, discard supernatant. Then, add the following to the PAS pellet:5μl of Proteinase K (10mg/ml), 1μl 10%SDS and 100μl NT-2. If you have several samples, its good to make a mastermix of NT2 buffer, Proteinase K and SDS). Incubate at 55℃for 15-30 min, with mixing.
3、Spin 5000 g,5 mins, collect supernatant (～100ul)
4、To beads add 200ul NT2 buffer, spin 5000 g/2 mins, collect supernatant (-200ul), discard beads.
5、Combine supernatants (100ul and 200ul) and add 300ul lower layer of acid phenol-CHCl3 (Ambion)
6、Vortex,1'RT (or 37C in shaker), short spin at RT (imp)/1'/max speed.
7、Collect 250ul of upper layer, add 25ul sodium acetate pH 5.2,625ul 100% ETOH and 5ul glycoblue, mix well, keep O/N-20C.
8、Next day, mix the tubes by inversion 3-5 times, spin 14.000rpm/4C/30 mins and discard supernatant
9、To the blue pellet add lml of 70% ETOH and mix by inversion or vortexing, spin 14.000rpm/4C/2'
10、Discard supernatant, spin pellet 1'/14.000rpm/4C, pipette any 70% ETOH, air dry pellet at RT for 5', resuspend in 20-40 ul of water.
Results
1、Hsa-miR-1 downregulates the expression of TIMP3 potein in HEK 293 cells.
2、Hsa-miR-1 downregulates the expression of TIMP3 mRNA compared with the negative control groups in HEK 293 cells.
3、It has been validated that hsa-miR-1 has target site in TIMP3 3'UTR by cloning in HEK 293 cells, mutagenesis and dual luciferase assay. The luciferase activity of the mutant constructs are significantly higher that those of wild type constructs for the first potential target site, wheareas the negative control group did not show the same effect. For the second potential target site, the difference between the mutant constructs and wild type constructs are not significant.
4、It has been validated that pre-miR-21 significantly downregulated TIMP3 expression and upregulated MMP9 expression. And the antogomiR-21 significantly upregulated the TIMP3 expression and downregulated MMP9 expression human cardiac fibroblast cells. This suggested that miR-21 regulated the cardiac remodeling pathway by targeting the important regulators in the pathway. Since cadiac remodeling leads to heart failure. This suggests that knockdown miR-21 by antagomiR-21 might have therapeutical effect on heart failure. It has been verified that miR-21 is another trans-acting factor of TIMP3 gene.
5、It has been confirmed that ARE binding protein HuR contributes the TIMP3 mRNA stability in human cardiac fibroblast cells.
Conclusion
The experiment verified hsa-miR-1 directly regulates the posttranscriptional of TIMP3 gene in 293 cells. hsa-miR-1 downregulates mRNA and protein expression of TIMP3 gene. The target site in TIMP3 was confirmed. In addition to hsa-miR-1, this studay also confirmed another two trans-acting factors. miR-21 and ARE binding protein HuR. TIMP3 and miR-1 were aberrantly expressed in cancer, arthritis and heart diseases. It has been validated that pre-miR-21 significantly downregulated TIMP3 expression and upregulated MMP9 expression. And antogomiR-21 significantly upregulated the TIMP3 expression and downregulated MMP9 expression in the cadiac fibroblast. This suggested that miR-21 regulated the cardiac remodeling pathway by targeting the important regulators in the pathway. This suggests that knockdown miR-21 by antagomiR-21 might have therapeutical effect on heart failure since cadiac remodeling leads to heart failure. The experiment provides the clue for investigating the cause of the diseases and how to cure the diseases.
引文
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1 Lee RC, Feinbaum RL, Ambros V. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell,1993,75(5):843.
2 Mannello F, Gazzanelli G. Tissue inhibitors of metalloproteinases and programmed cell death: Conundrums, controversies and potential implications. Apoptosis,2001,6(6):479.
3 Calin GA, Sevignani C, Dumitru CD, et al. Human microRNA genes are frequently located at fragile sites and genomic regions involved in cancers. PNAS,2004,101(9):2999-3004.
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12 Lewis BP, Shih IH, Jones-Rhoades MW, et al. Prediction of mammalian microRNA targets. Cell,2003,115(7):787-798.
13 Federici M, Hribal ML, Menghini R, et al. Timp3 deficiency in insulin receptor-haploinsufficient mice promotes diabetes and vascular inflammation via increased TNF-{alpha}. J. Clin. Invest.,2005,115(12):3494-3505.
14 Leco KJ, Khokha R, Pavloff N, et al. Tissue inhibitor of metalloproteinases-3 (TIMP-3) is an extracellular matrix-associated protein with a distinctive pattern of expression in mouse cells and tissues. J. Biol. Chem.,1994,269(12):9352-9360.
15 Lui ELH, Loo WTY, Zhu L, et al. DNA hypermethylation of TIMP3 gene in invasive breast ductal carcinoma. Biomedecine & Pharmacotherapy,2005,59(Supplement 2):S363.
16 Bond M, Murphy G, Bennett MR, et al. Tissue Inhibitor of Metalloproteinase-3 Induces a Fas-associated Death Domain-dependent Type II Apoptotic Pathway. J. Biol. Chem.,2002,277(16): 13787-13795.
17 Drynda A, Quax PHA, Neumann M, et al. Gene Transfer of Tissue Inhibitor of Metalloproteinases-3 Reverses the Inhibitory Effects of TNF-{alpha} on Fas-Induced Apoptosis in Rheumatoid Arthritis Synovial Fibroblasts. J Immunol,2005,174(10):6524-6531.
18 Qi JH, Ebrahem Q, Yeow K, et al. Expression of Sorsby's Fundus Dystrophy Mutations in Human Retinal Pigment Epithelial Cells Reduces Matrix Metalloproteinase Inhibition and May Promote Angiogenesis. J. Biol. Chem.,2002,277(16):13394-13400.
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22 Cimmino A, Calin GA, Fabbri M, et al. miR-15 and miR-16 induce apoptosis by targeting BCL2. Proc Natl Acad Sci USA,2005,102(39):13944-13949.
23 He L, Thomson JM, Hemann MT, et al. A microRNA polycistron as a potential human oncogene. Nature,2005,435(7043):828-833.
24 Li Z, Zhan W, Wang Z, et al. Inhibition of PRL-3 gene expression in gastric cancer cell line SGC7901 via microRNA suppressed reduces peritoneal metastasis. Biochem Biophys Res Commun,2006,348(1):229-237.
25 Enright A, John B, Gaul U, et al. MicroRNA targets in Drosophila. Genome Biology,2003, 5(1):R1.
26 Stark A, Brennecke J, Russell RB, et al. Identification of Drosophila MicroRNA targets. PLoS Biology,2003,1(3):E60.
27 Lee Y, Ahn C, Han J, et al. The nuclear RNase III Drosha initiates microRNA processing. Nature,2003,425(6956):415.
28 Lim LP, Glasner ME, Yekta S, et al. Vertebrate MicroRNA Genes. Science,2003,299(5612): 1540-.
29 Lund E, Guttinger S, Calado A, et al. Nuclear Export of MicroRNA Precursors. Science,2004, 303(5654):95-98.
30 Yi R, Qin Y, Macara IG, et al. Exportin-5 mediates the nuclear export of pre-microRNAs and short hairpin RNAs. Genes Dev.,2003,17(24):3011-3016.
31 Hutvagner G, Zamore PD. A microRNA in a Multiple-Turnover RNAi Enzyme Complex. Science,2002,297(5589):2056-2060.
32 Naguibneva I, Ameyar-Zazoua M, Polesskaya A, et al. The microRNA miR-181 targets the homeobox protein Hox-All during mammalian myoblast differentiation. Nat Cell Biol,2006, 8(3):278-284.
33 Sewer A, Paul N, Landgraf P, et al. Identification of clustered microRNAs using an ab initio prediction method. BMC Bioinformatics,2005,6(1):267.
34 Valencia-Sanchez MA, Liu J, Hannon GJ, et al. Control of translation and mRNA degradation by miRNAs and siRNAs. Genes Dev.,2006,20(5):515-524.
35 Lagos-Quintana M, Rauhut R, Yalcin A, et al. Identification of Tissue-Specific MicroRNAs from Mouse. Current Biology,2002,12(9):735.
36 Brennecke J, Stark A, Russell RB, et al. Principles of microRNA-target recognition. PLoS biology.,2005,3(3).
37 Wightman B, Burglin TR, Gatto J, et al. Negative regulatory sequences in the lin-143'-untranslated region are necessary to generate a temporal switch during Caenorhabditis elegans development. Genes and Development,1991,5(10):1813.
38 Doench JG, Sharp PA. Specificity of microRNA target selection in translational repression. Genes Dev.,2004,18(5):504-511.
39 Saxena S, Jonsson ZO, Dutta A. Small RNAs with Imperfect Match to Endogenous mRNA Repress Translation:IMPLICATIONS FOR OFF-TARGET ACTIVITY OF SMALL INHIBITORY RNA IN MAMMALIAN CELLS. J. Biol. Chem.,2003,278(45):44312-44319.
40 Yekta S, Shih Ih, Bartel DP. MicroRNA-Directed Cleavage of HOXB8 mRNA. Science,2004, 304(5670):594-596.
41 Bagga S, Bracht J, Hunter S, et al. Regulation by let-7 and lin-4 miRNAs Results in Target mRNA Degradation. Cell,2005,122(4):553.
42 ing Q, Huang S, Guth S, et al. Involvement of MicroRNA in AU-Rich Element-Mediated mRNA Instability. Cell,2005,120(5):623.
43 Cimmino A, Calin GA, Fabbri M, et al. miR-15 and miR-16 induce apoptosis by targeting BCL2. PNAS,2005,102(39):13944-13949.
44 Fata JE, Leco KJ, Voura EB, et al. Accelerated apoptosis in the Timp-3-deficient mammary gland. J. Clin. Invest.,2001,108(6):831-841.
45 Brown BD, Venneri MA, Zingale A, et al. Endogenous microRNA regulation suppresses transgene expression in hematopoietic lineages and enables stable gene transfer. Nat Med, 2006, advanced online publication.
46 Takamizawa J, Konishi H, Yanagisawa K, et al. Reduced Expression of the let-7 MicroRNAs in Human Lung Cancers in Association with Shortened Postoperative Survival. Cancer Res, 2004,64(11):3753-3756.
47 Lu J, Getz G, Miska EA, et al. MicroRNA expression profiles classify human cancers. Nature, 2005,435(7043):834.
48 O'Donnell KA, Wentzel EA, Zeller KI, et al. c-Myc-regulated microRNAs modulate E2F1 expression. Nature,2005,435(7043):839.
49 Michael MZ, O'Connor SM, van Holst Pellekaan NG, et al. Reduced Accumulation of Specific MicroRNAs in Colorectal Neoplasia. Mol Cancer Res,2003,1(12):882-891.
50 Johnson SM, Grosshans H, Shingara J, et al. RAS Is Regulated by the let-7 MicroRNA Family. Cell,2005,120(5):635.
51 Akao Y, Nakagawa Y, Naoe T. let-7 MicroRNA Functions as a Potential Growth Suppressor in Human Colon Cancer Cells. Biol Pharm Bull,2006,29(5):903-906.
52 He L, Thomson JM, Hemann MT, et al. A microRNA polycistron as a potential human oncogene. Nature,2005,435(7043):828.
53 Fernandez PC, Frank SR, Wang L, et al. Genomic targets of the human c-Myc protein. Genes Dev.,2003,17(9):1115-1129.
54 Aravin A, Tuschl T, Harborth J, et al. Identification and characterization of small RNAs involved in RNA silencing Identification of essential genes in cultured mammalian cells using small interfering RNAs. FEBS Lett,2005,579(26):5830-5840.
55 Vatolin S, Navaratne K, Weil RJ. A Novel Method to Detect Functional MicroRNA Targets. Journal of Molecular Biology,2006,358(4):983.
56 Gao Z, Yang Z. Detection of microRNAs using electrocatalytic nanoparticle tags. Anal Chem, 2006,78(5):1470-1477.
57 Ramkissoon SH, Mainwaring LA, Sloand EM, et al. Nonisotopic detection of microRNA using digoxigenin labeled RNA probes. Molecular and Cellular Probes,2006,20(1):1.
2 Lee RC, Feinbaum RL, Ambros V. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell,1993,75(5):843.
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17 Lui ELH, Loo WTY, Zhu L, et al. DNA hypermethylation of TIMP3 gene in invasive breast ductal carcinoma. Biomedecine & Pharmacotherapy,2005,59(Supplement 2):S363.
18 Bond M, Murphy G, Bennett MR, et al. Tissue Inhibitor of Metalloproteinase-3 Induces a Fas-associated Death Domain-dependent Type II Apoptotic Pathway. J. Biol. Chem.,2002,277(16): 13787-13795.
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24 Li Z, Zhan W, Wang Z, et al. Inhibition of PRL-3 gene expression in gastric cancer cell line SGC7901 via microRNA suppressed reduces peritoneal metastasis. Biochem Biophys Res Commun,2006,348(1):229-237.
25 Enright A, John B, Gaul U, et al. MicroRNA targets in Drosophila. Genome Biology,2003,5(1): R1.
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27 Lee Y, Ahn C, Han J, et al. The nuclear RNase Ⅲ Drosha initiates microRNA processing. Nature, 2003,425(6956):415.
28 Lim LP, Glasner ME, Yekta S, et al. Vertebrate MicroRNA Genes. Science,2003,299(5612): 1540-.
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30 Yi R, Qin Y, Macara IG, et al. Exportin-5 mediates the nuclear export of pre-microRNAs and short hairpin RNAs. Genes Dev.,2003,17(24):3011-3016.
31 Hutvagner G, Zamore PD. A microRNA in a Multiple-Turnover RNAi Enzyme Complex. Science, 2002,297(5589):2056-2060.
32 Lewis BP, Shih IH, Jones-Rhoades MW, et al. Prediction of mammalian microRNA targets. Cell, 2003,115(7):787-798.
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37 Bagga S, Bracht J, Hunter S, et al. Regulation by let-7 and lin-4 miRNAs Results in Target mRNA Degradation. Cell,2005,122(4):553.
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1 Lee RC, Feinbaum RL, Ambros V. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell,1993,75(5):843.
2 Mannello F, Gazzanelli G. Tissue inhibitors of metalloproteinases and programmed cell death: Conundrums, controversies and potential implications. Apoptosis,2001,6(6):479.
3 Calin GA, Sevignani C, Dumitru CD, et al. Human microRNA genes are frequently located at fragile sites and genomic regions involved in cancers. PNAS,2004,101(9):2999-3004.
4 Markus Metzler MWKBSVAB. High expression of precursor microRNA-155/BIC RNA in children with Burkitt lymphoma. Genes, Chromosomes and Cancer,2004,39(2):167-169.
5 Esau C, Kang X, Peralta E, et al. MicroRNA-143 Regulates Adipocyte Differentiation. J. Biol. Chem.,2004,279(50):52361-52365.
6 McManus MT. Small RNAs and Immunity. Immunity,2004,21(6):747.
7 Poy MN, Eliasson L, Krutzfeldt J, et al. A pancreatic islet-specific microRNA regulates insulin secretion. Nature,2004,432(7014):226.
8 Chen C-Z, Lodish HF. MicroRNAs as regulators of mammalian hematopoiesis. Seminars in Immunology,2005,17(2):155.
9 Smirnova L, Grafe A, Seiler A, et al. Regulation of miRNA expression during neural cell specification. European Journal of Neuroscience,2005,21(6):1469-1477.
10 Krek A, Grun D, Poy MN, et al. Combinatorial microRNA target predictions. Nat Genet,2005, 37(5):495-500.
11 Lewis BP, Burge CB, Bartel DP. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell,2005,120(1):15-20.
12 Lewis BP, Shih IH, Jones-Rhoades MW, et al. Prediction of mammalian microRNA targets. Cell,2003,115(7):787-798.
13 Federici M, Hribal ML, Menghini R, et al. Timp3 deficiency in insulin receptor-haploinsufficient mice promotes diabetes and vascular inflammation via increased TNF-{alpha}. J. Clin. Invest.,2005,115(12):3494-3505.
14 Leco KJ, Khokha R, Pavloff N, et al. Tissue inhibitor of metalloproteinases-3 (TIMP-3) is an extracellular matrix-associated protein with a distinctive pattern of expression in mouse cells and tissues. J. Biol. Chem.,1994,269(12):9352-9360.
15 Lui ELH, Loo WTY, Zhu L, et al. DNA hypermethylation of TIMP3 gene in invasive breast ductal carcinoma. Biomedecine & Pharmacotherapy,2005,59(Supplement 2):S363.
16 Bond M, Murphy G, Bennett MR, et al. Tissue Inhibitor of Metalloproteinase-3 Induces a Fas-associated Death Domain-dependent Type II Apoptotic Pathway. J. Biol. Chem.,2002,277(16): 13787-13795.
17 Drynda A, Quax PHA, Neumann M, et al. Gene Transfer of Tissue Inhibitor of Metalloproteinases-3 Reverses the Inhibitory Effects of TNF-{alpha} on Fas-Induced Apoptosis in Rheumatoid Arthritis Synovial Fibroblasts. J Immunol,2005,174(10):6524-6531.
18 Qi JH, Ebrahem Q, Yeow K, et al. Expression of Sorsby's Fundus Dystrophy Mutations in Human Retinal Pigment Epithelial Cells Reduces Matrix Metalloproteinase Inhibition and May Promote Angiogenesis. J. Biol. Chem.,2002,277(16):13394-13400.
19 Baker AH, George SJ, Zaltsman AB, et al. Inhibition of invasion and induction of apoptotic cell death of cancer cell lines by overexpression of TIMP-3. Br J Cancer,1999,79(9-10): 1347-1355.
20 McCarthy JJ, Esser KA. MicroRNA-1 and microRNA-133a expression are decreased during skeletal muscle hypertrophy. J Appl Physiol,2007,102(1):306-313.
21 Hutvagner G, McLachlan J, Pasquinelli AE, et al. A cellular function for the RNA-interference enzyme Dicer in the maturation of the let-7 small temporal RNA. Science,2001,293(5531): 834-838.
22 Cimmino A, Calin GA, Fabbri M, et al. miR-15 and miR-16 induce apoptosis by targeting BCL2. Proc Natl Acad Sci USA,2005,102(39):13944-13949.
23 He L, Thomson JM, Hemann MT, et al. A microRNA polycistron as a potential human oncogene. Nature,2005,435(7043):828-833.
24 Li Z, Zhan W, Wang Z, et al. Inhibition of PRL-3 gene expression in gastric cancer cell line SGC7901 via microRNA suppressed reduces peritoneal metastasis. Biochem Biophys Res Commun,2006,348(1):229-237.
25 Enright A, John B, Gaul U, et al. MicroRNA targets in Drosophila. Genome Biology,2003, 5(1):R1.
26 Stark A, Brennecke J, Russell RB, et al. Identification of Drosophila MicroRNA targets. PLoS Biology,2003,1(3):E60.
27 Lee Y, Ahn C, Han J, et al. The nuclear RNase III Drosha initiates microRNA processing. Nature,2003,425(6956):415.
28 Lim LP, Glasner ME, Yekta S, et al. Vertebrate MicroRNA Genes. Science,2003,299(5612): 1540-.
29 Lund E, Guttinger S, Calado A, et al. Nuclear Export of MicroRNA Precursors. Science,2004, 303(5654):95-98.
30 Yi R, Qin Y, Macara IG, et al. Exportin-5 mediates the nuclear export of pre-microRNAs and short hairpin RNAs. Genes Dev.,2003,17(24):3011-3016.
31 Hutvagner G, Zamore PD. A microRNA in a Multiple-Turnover RNAi Enzyme Complex. Science,2002,297(5589):2056-2060.
32 Naguibneva I, Ameyar-Zazoua M, Polesskaya A, et al. The microRNA miR-181 targets the homeobox protein Hox-All during mammalian myoblast differentiation. Nat Cell Biol,2006, 8(3):278-284.
33 Sewer A, Paul N, Landgraf P, et al. Identification of clustered microRNAs using an ab initio prediction method. BMC Bioinformatics,2005,6(1):267.
34 Valencia-Sanchez MA, Liu J, Hannon GJ, et al. Control of translation and mRNA degradation by miRNAs and siRNAs. Genes Dev.,2006,20(5):515-524.
35 Lagos-Quintana M, Rauhut R, Yalcin A, et al. Identification of Tissue-Specific MicroRNAs from Mouse. Current Biology,2002,12(9):735.
36 Brennecke J, Stark A, Russell RB, et al. Principles of microRNA-target recognition. PLoS biology.,2005,3(3).
37 Wightman B, Burglin TR, Gatto J, et al. Negative regulatory sequences in the lin-143'-untranslated region are necessary to generate a temporal switch during Caenorhabditis elegans development. Genes and Development,1991,5(10):1813.
38 Doench JG, Sharp PA. Specificity of microRNA target selection in translational repression. Genes Dev.,2004,18(5):504-511.
39 Saxena S, Jonsson ZO, Dutta A. Small RNAs with Imperfect Match to Endogenous mRNA Repress Translation:IMPLICATIONS FOR OFF-TARGET ACTIVITY OF SMALL INHIBITORY RNA IN MAMMALIAN CELLS. J. Biol. Chem.,2003,278(45):44312-44319.
40 Yekta S, Shih Ih, Bartel DP. MicroRNA-Directed Cleavage of HOXB8 mRNA. Science,2004, 304(5670):594-596.
41 Bagga S, Bracht J, Hunter S, et al. Regulation by let-7 and lin-4 miRNAs Results in Target mRNA Degradation. Cell,2005,122(4):553.
42 ing Q, Huang S, Guth S, et al. Involvement of MicroRNA in AU-Rich Element-Mediated mRNA Instability. Cell,2005,120(5):623.
43 Cimmino A, Calin GA, Fabbri M, et al. miR-15 and miR-16 induce apoptosis by targeting BCL2. PNAS,2005,102(39):13944-13949.
44 Fata JE, Leco KJ, Voura EB, et al. Accelerated apoptosis in the Timp-3-deficient mammary gland. J. Clin. Invest.,2001,108(6):831-841.
45 Brown BD, Venneri MA, Zingale A, et al. Endogenous microRNA regulation suppresses transgene expression in hematopoietic lineages and enables stable gene transfer. Nat Med, 2006, advanced online publication.
46 Takamizawa J, Konishi H, Yanagisawa K, et al. Reduced Expression of the let-7 MicroRNAs in Human Lung Cancers in Association with Shortened Postoperative Survival. Cancer Res, 2004,64(11):3753-3756.
47 Lu J, Getz G, Miska EA, et al. MicroRNA expression profiles classify human cancers. Nature, 2005,435(7043):834.
48 O'Donnell KA, Wentzel EA, Zeller KI, et al. c-Myc-regulated microRNAs modulate E2F1 expression. Nature,2005,435(7043):839.
49 Michael MZ, O'Connor SM, van Holst Pellekaan NG, et al. Reduced Accumulation of Specific MicroRNAs in Colorectal Neoplasia. Mol Cancer Res,2003,1(12):882-891.
50 Johnson SM, Grosshans H, Shingara J, et al. RAS Is Regulated by the let-7 MicroRNA Family. Cell,2005,120(5):635.
51 Akao Y, Nakagawa Y, Naoe T. let-7 MicroRNA Functions as a Potential Growth Suppressor in Human Colon Cancer Cells. Biol Pharm Bull,2006,29(5):903-906.
52 He L, Thomson JM, Hemann MT, et al. A microRNA polycistron as a potential human oncogene. Nature,2005,435(7043):828.
53 Fernandez PC, Frank SR, Wang L, et al. Genomic targets of the human c-Myc protein. Genes Dev.,2003,17(9):1115-1129.
54 Aravin A, Tuschl T, Harborth J, et al. Identification and characterization of small RNAs involved in RNA silencing Identification of essential genes in cultured mammalian cells using small interfering RNAs. FEBS Lett,2005,579(26):5830-5840.
55 Vatolin S, Navaratne K, Weil RJ. A Novel Method to Detect Functional MicroRNA Targets. Journal of Molecular Biology,2006,358(4):983.
56 Gao Z, Yang Z. Detection of microRNAs using electrocatalytic nanoparticle tags. Anal Chem, 2006,78(5):1470-1477.
57 Ramkissoon SH, Mainwaring LA, Sloand EM, et al. Nonisotopic detection of microRNA using digoxigenin labeled RNA probes. Molecular and Cellular Probes,2006,20(1):1.