Name: profiling of estrogen-regulated micrornas in breast cancer cells
Uploaded: 2014-02-21T14:30:00
Duration: 16 min 24 s
Description: Watch this Scientific Journal Video about Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells at JoVE.com

我们感谢孝廉高，休斯顿大学博士，为给LC科学miRNA芯片平台，提供咨询，卡琳爱德华松博士，卡罗林斯卡医学院，瑞典和Eylem Aydogdu博士，休斯顿大学，分享他们的专业知识的miRNA 。研究报告本出版物中得到了卫生部下奖号码R01CA172437国家研究院​​国立癌症研究所的支持。内容完全是作者的责任，并不一定代表美国国立卫生研究院的官方意见。这项工作也是由来自得克萨斯州的新兴科技基金，协议项下的任何资助。 300-9-1958。

1。细胞培养基的制备<ol><li>对于T47D细胞系培养基： <ol><li>混合500毫升Dulbecco改良的Eagle培养基（DMEM）中，用500ml F12 [DMEM/F12（1:1）]在一个高压灭菌1升的瓶子。 </li><li>加入50mL胎牛血清（这使得5％FBS）中，然后加入10 ml青霉素链霉素（这使得1％PEST）。完全混合内容。 </li><li>贮存于4℃。 </li></ol></li><li>对于MCF7细胞培养基： <ol><li>混合500毫升DMEM用25ml的胎牛血清（5％FBS）中，然后用5 ml青霉素链霉素（1％PEST）的。 </li><li>贮存于4℃。 </li><li>为了降低血清培养基： <ol><li>对T47D：混合500毫升无酚红的DMEM，用500ml F12（1:1）中的高压灭菌的1升瓶，并加50毫升的葡聚糖涂覆的木炭处理的（DCC）胎牛血清为5％DCC-FBS的。做一个独立的一瓶5毫升DCC-FBS的0.5％DCC-FBS。然后，加入10 ml PEST（1％PEST）给每个瓶子。完全混合内容，存储Ë在4°C。 </li><li>对MCF7：混合500毫升无酚红DMEM中，用5ml 100倍的L-谷氨酰胺（200 mM的终浓度）。取25 mL DCC-FBS（5％DCC-FBS）的。使用2.5ml DCC-FBS（0.5％DCC-FBS）的一个单独的瓶子。然后，加入5 ml PEST（1％PEST）给每个瓶子。完全混合内容，储存于4°C。 </li></ol></li><li>对磷酸盐缓冲盐水（PBS）溶液： <ol><li>填用超纯水或蒸馏水的1L高压灭菌玻璃瓶。 </li><li>新增一个平板电脑的PBS和动摇偶尔直到片剂溶解。 </li><li>高压灭菌的PBS液并储存于室温。 </li></ol></li><li>制备配的股票： <ol><li>制备的E2 100mM的储备液通过在无菌的1.5 ml离心管中溶解于100μl乙醇或DMSO 2.72毫克17β-雌二醇，并轻轻混合内容。简要地旋转，并连续稀释（1:10），得到10毫米，1毫米和0.1毫米的股票concentr使用溶剂ations。存储所有E2储备液在-20°C。 </li><li>准备ICI的100毫米原液在无菌1.5 ml离心管溶解在100μl乙醇或DMSO 6.07毫克ICI 182,780，轻轻混匀内容。简单地旋转，使连续稀释（1:10），得到10毫米，1毫米，使用溶剂0.1毫米的股票浓度。存储所有ICI股票的解决方案在-20°C </li><li>车辆在溶剂（乙醇或DMSO）中。放置0.5-1毫升100％乙醇或DMSO中在无菌的1.5 ml离心管，并储存在-20℃。 </li></ol></li></ol></li></ol> 2。细胞培养及处理执行每个治疗双重或三重板的技术重复。使用相同的细胞系，其将作为该细胞系的生物学重复的不同的通道重复的细胞培养过程和治疗。用不同的细胞系，以REPL重复该过程在一个以上的细胞系icate发现。所有的细胞培养技术，应在无菌条件下在层流罩中进行。 <ol><li>细胞培养启动<ol><li>暖媒体37℃，在无菌温水澡。 </li><li>解冻T47D和MCF7细胞的冷冻瓶。 </li><li>用70％乙醇清洁的小瓶和媒体瓶的外侧，然后将两个小瓶和瓶的无菌层流净化罩。 </li><li>标签无菌的T-75烧瓶相应（在通风橱）。 </li><li>加12-15的相应的媒体毫升到使用无菌的血清吸液管的烧瓶（确保表面被完全覆盖的介质）。 </li><li>从药瓶细胞转移到烧瓶中，并轻轻混匀。 </li><li>把烧瓶于37℃培养箱中，用5％的CO 2供给。 </li></ol></li><li>用于制备治疗细胞<ol><li>取出来的细胞培养箱中，并在显微镜下观察，以确保细胞至少80％的共nfluent。这是为了确保有足够的细胞用于实验。烧瓶转移到无菌罩。 </li><li>用无菌巴斯德移液管连接到真空从烧瓶中取出介质。用PBS轻轻洗涤两次附着的细胞。 </li><li>加入1毫升温胰蛋白酶-EDTA的向烧瓶中，并把烧瓶中孵化2分钟。这是为了使从烧瓶中的细胞分离。 </li><li>约3-4毫升的适当温暖媒体添加到烧瓶中，并用5毫升的血清吸液管磨碎的分离的细胞。磨碎由卷取细胞在血清吸液管和用吸管置于对烧瓶的底部，以增加压力的顶端释放的细胞。这是打破细胞分割成单个细胞。 </li><li>计数细胞， 例如用细胞计数器，根据制造商的方案。 </li><li>标签百毫米板（如需要），并加入约10ml介质板。板约2.0×10 6个细胞/皿。由GE细胞分布ntle旋流​​板。 </li><li>孵育细胞24-48小时，直至约80％汇合。 </li><li>在80％汇合，用PBS洗涤细胞两次，并添加相应的5％DCC-FBS的培养基。然后，孵育细胞24小时。 </li><li> 24小时后，用PBS洗涤细胞两次，并添加相应的0.5％DCC-FBS的培养基。然后，细胞孵育额外的48小时。 </li></ol></li><li>细胞治疗<ol><li> 48小时后，用PBS洗涤细胞两次。一定要去除尽可能多的PBS中成为可能。 </li><li>在一个15毫升的锥形管，加入10毫升相应的0.5％DCC-FBS的培养基中。那么，对于10纳米的终浓度添加1微升0.1 MM配股票（E2或ICI）的。此外，加入1μl车辆〜10毫升媒体的对照实验的单独管。 </li><li>在试管混匀内容物轻轻地，并加入到细胞板。这应该是每板1的配体。 </li><li>孵育细胞所选择的时间点（0-72小时）。 </li></ol></li></ol> &lt;P类=“j​​ove_title”&gt; 3。 RNA的提取及质量控制<ol><li> RNA提取<ol><li>后处理结束的时间所需的时间，用PBS洗涤细胞两次，然后加入约1-2毫升异硫氰酸胍 - 苯酚溶液中的细胞的板。注意：硫氰酸胍 - 酚的解决方案是通过与皮肤或眼睛接触有毒，吸入或误吞。穿戴适当的防护服，手套和眼睛/面保护，并使用通风柜。 </li><li>确保电池的体积不超过10％的硫氰酸胍 - 苯酚溶液的体积，并且该板覆盖该溶液，并允许静置1分钟。然后，用橡胶刮刀刮去的细胞并转移到一个离心管中。细胞可以被存储在该溶液中，在-80℃下进行周。 </li><li>提取和使用硫氰酸胍 - 苯酚 - 氯仿法随后离心柱纯化和DNA酶从每个处理纯化的总RNA余DNA降解法对动物细胞。 </li><li>萃取后，将细胞洗脱在60微升不含RNA酶的水。在-80°C等分1-2μL的RNA进行定量分析和存储的RNA </li><li>使用1微升RNA和aspectrophotometer的测量RNA浓度。确保有采取任何测量完成之前的空白。此外，彻底清除分光光度计到达时间测量。浓度以ng /μL通常显示。保存结果显示RNA浓度。 </li></ol></li><li> RNA的质量控制<ol><li>取每个样品并置于微量离心管中的约100ng的RNA。 </li><li>使用生物分析仪测量RNA的完整性。通常12个样本可以在同一时间在一个生物分析仪上运行。运行通常需要大约25分钟。 </li><li>比较与核糖核酸梯子的结果，保证了RNA是良好的实验。保存并打印结果。 </li></ol></li></ol> 4。对待确认换货：蛋白质编码基因的定量PCR <ol><li> cDNA合成<ol><li>取总RNA（每个样品）的500毫微克或1微克，放入1.5ml的微量离心管中。把每根管子的音量使用无RNase水10微升。 </li><li>加入2μl的50μM的随机六聚体引物和热管至70℃10分钟。 </li><li>传输管到冰上5分钟。 </li><li>对于每个样品，加入4微升5×第一链缓冲液，1微升的0.1M DTT，1微升的10mM的dNTP，0.5μl的上标III和1.5微升不含RNA酶的水。 </li><li>地方管在25℃的加热块或水浴中10分钟。 </li><li>换热管以1小时46℃加热块。然后，传输管，以15分钟，70℃加热块，使反应停止。 </li><li>用无RNA酶的水稀释，并储存在-20°C。稀释的cDNA的5纳克/微升股票（使用总RNA输入计算） </li></ol></li><li>定量PCR <ol><li>获得引物的兴趣和参照基因的基因。引物可以很容易地使用在NCBI的底漆-BLAST程序12的引物设计工具设计。这通常会产生一个正向和反向引物为每个感兴趣的基因。引物应为18-22碱基对的长度，具有52-58℃的熔化温度，有40-60％的GC含量，和100-180 bp的扩增子的长度。 </li><li>对于每个qPCR的反应均匀，加入10纳克的cDNA（2微升库存浓度从步骤4.1.7），1皮摩尔各正向和反向的感兴趣的基因的引物（来自步骤4.2.1），和5μl的2X菁染料的PCR母液。每个孔中的反应应该有一个10微升的最终反应体积。 </li><li>确保有一式三份孔的每个样品的每个基因（用于技术重复）。将96孔反应板都可以使用。 </li><li>对定量RT-PCR检测系统软件，指派记者和目标，输入反应体积中，选择比较循环阈值（ΔΔCT）方法，并定义样品孔。 </li><li>请一定要进行熔解曲线分析所有菁染料的运行，以确认一个特定片段的扩增。使用该运行的默认设置运行板块。 </li><li>保存结果运行后，和出口数据（尤其是CT值到MS Excel）。 </li></ol></li><li> 使用ΔΔCT公式定量PCR数据分析 相对mRNA表达的变化可以计算变化相对倍数控制在Excel中使用以下步骤每个样品： <ol><li>所有导出结果从上面的定量PCR应具备的Ct值包括在内。计算使用下面的公式ΔCT值： <ul><li> ΔCT= CT（目的基因） - CT（内参基因） </li></ul>这应该对每个样品进行。我们的目标是感兴趣的基因或miRNA。 </li><li>接着，计算出总的标准偏差（SD）为各样品。首先计算对SD的参考基因，然后计算在SD目标基因（这可以在Excel中使用的CT值的标准偏差函数来完成）。然后计算出总的SD使用这个公式每个样品： <ul><li>总标准差=（SD2（目标）+ SD2（参照））1/2 </li></ul></li><li>通过计算各样品的ΔΔCT的基因（目标）内： <ul><li> ΔΔCT=ΔΔCT（治疗/试验样品） - ΔΔCT（控制/校准样品） </li></ul>该校准样品是未经处理的样品。 </li><li>最后，计算使用公式各样品的倍数​​变化（FC）的值： <ul><li> FC = 2-ΔΔCT </li></ul>各样品的倍数​​变化值给出了基因/ miRNA的已归一化到参考基因和对照样品的相对表达。 </li><li>学生氏t-检验可以通过使用双尾分布和双样本方差不等用于统计分析参数：（p &lt;0.001（***）P &lt;0.01（**）和p &lt;0.05（*））。这可以在Excel中实现。确认治疗导致已知目标调控继续进行miRNA表达谱之前。 </li></ol></li></ol> 5。 miRNA表达谱分析<ol><li> miRNA芯片<ol><li>取5微克的分离的RNA与从载体或配体，并置于微量离心管中处理过的细胞。调整管体积至20μl。每个比较应一式两份或一式三份进行。 </li><li>使用上述RNA样品进行miRNA芯片。 miRNA芯片表达谱应该利用的μParaflo微流控生物芯片技术人类miRNA芯片双色样品阵列（桑格发布的miRBase 14.0）13来确定。 </li><li>结果应该显示差异表达的miRNA。考虑显著miRNA的表达当p &lt;0.05.p值&lt;0.10，也可以考虑进一步合作nfirmatory分析使用qPCR。保存此miRNA的名单作进一步的验证与定量PCR。 </li></ol></li><li> miRNA表达谱：DLP-LDA <ol><li>每个样品应一式两份或一式三份进行分析。取700纳克从与载体或配体处理的细胞的总RNA，并将其放置在一个离心管中。调整管卷3微升。 </li><li>使用双标记探针的miRNA检测方法及10倍的RT引物进行cDNA的合成。总体积为每个反应应该是7.5微升。 </li><li>将反应管中PCR系统热循环仪，并设置为推荐的参数作为双标记探针的miRNA检测方法表示。开始运行。这将产生的cDNA。注意，该cDNA可以储存在-20℃下至少1周。 </li><li>虽然RT反应是跑，冒了DLP-LDA板，并允许在室温下坐。每个阵列板包含384个孔，其中包含独特的miRNA引物和对照引物。 </li><li>取6μL的cDNA的（从5.2.3）并置于1.5 ml离心管中。加入450μL双标记探针2X通用PCR扩增预混试剂，并加入444μL无RNase水。 </li><li>逆转管约6次以混合内容物，并短暂离心​​。 </li><li>当DLP-LDA板已经达到室温，负载100微升到每个八景&#39;填写端口“的板块。然后，离心阵列板。 </li><li>打开定量RT-PCR检测系统，并检查该块是，对于384孔板。使用SDS软件设置运行。选择相对定量（CT），选择好数量和数组类型，并输入样本描述来定义井。 </li><li>使用默认的热循环条件下运行的数组。当运行结束后，将结果保存和导出到MS Excel和使用ΔΔCT公式（步骤4.3）除作进一步的分析。 </li></ol></li></ol> 6。使用单独的miRNA规定的确认定量PCR分析<ol><li>菁染料定量PCR分析<ol><li>设计引物所需的miRNA分析。成熟miRNA，它等于正向引物的序列，可以从mirbase.org 14来获得。将所有“U”到“T”。 </li><li>制备cDNA：取1μg总RNA，并装在1.5 ml离心管中。遵循多聚腺苷酸从miRNA的第一链cDNA合成和定量RT-PCR方法拖尾和第一链cDNA合成的miRNA的指示。稀释所得到的cDNA（1:10），并储存在-20℃。 </li><li>获得一个96孔反应板。 </li><li>对于各miRNA的qPCR反应良好，从步骤6.1.2添加16纳克的polyA的cDNA（来自步骤6.1.2），2皮摩尔各特异性正向引物（来自步骤6.1.1）和通用引物（由试剂盒）和5微升2倍的qPCR预混的。最终反应体积为10微升/孔。 </li><li>保证有个复孔每个样品各miRNA（技术重复）。还，确保参照基因（通常U6小核RNA）被包括在内。 </li><li>对定量RT-PCR检测系统软件，指派记者和目标，输入反应体积中，选择比较阈值循环（ΔΔCT）方法，并定义样品孔。 </li><li>使用该运行的默认设置运行板块。请一定要进行熔解曲线分析所有菁染料的运行。每个解链曲线应该只显示一个特定的峰来确认一个特定片段的扩增。如果一个以上的峰或无明显的峰被观测到，在引物不特定和数据不能被使用。 </li><li>保存结果运行后，和出口数据（尤其是CT值到MS Excel）。 </li></ol></li><li>双标记探针定量PCR分析<ol><li>取10ng的每个总RNA，每个在5微升体积和地点在0.2ml微量离心管中。 </li><li>遵循使用双标记探针微执行反转录（RT）的协议指令RNA反转录试剂盒15。每个反应总体积为15微升。请注意，对于双标记探针RT反应中，有用于各miRNA待研究的特异性引物。 </li><li>处的反应管中的PCR热循环仪系统，并根据该协议设定的参数表示在上述步骤6.2.2。开始运行。这大约需要70-80分钟。 </li><li> RT反应后，稀释样品的cDNA中以1:5的比例，并存储所有的cDNA在黑暗中在-20℃下</li><li>获得一个96孔反应板。 </li><li>对于每个miRNA的qPCR的反应很好，添加以下内容：10μL双标记探针的2个通用PCR扩增预混试剂（试剂相同的步骤5.2.5），7.67微升的无RNase水，1微升20×实时检测的引物（具体为各miRNA），和1.33微升cDNA的来自上述步骤6.2.4。这应该是一个20微升的最终反应体积。混合内容轻轻的，短暂离心。 </li><li>确保日ERE的是一式三份孔的每个采样的各miRNA（为技术重复）。此外，请确保参照基因（通常U6小核RNA）被包括在内。 </li><li>对定量RT-PCR检测系统软件，为每个感兴趣的miRNA的（目标）指派记者（FAM）和淬灭剂（NFQ-MGB），输入反应体积中，选择比较阈值循环（ΔΔCT）方法，并定义样品孔。 </li><li>按照有关设置从双标记探针的microRNA分析方案运行参数定量PCR的说明。然后，开始运行。保存结果运行后，和出口数据（尤其是CT值到MS Excel）。 </li></ol></li></ol>

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作者什么都没有透露。

确定机制miRNA在乳腺癌细胞激素调节可用于理解这种疾病提供了一个平台，并且可以提供用于乳腺癌的治疗潜力。培养这些细胞，以提供激素作用的最佳条件是非常重要的。这里，一个程序，即确保利益（雌激素）的激素被排除在治疗前和E2的最佳剂量是用于在治疗过程中的适当时间进行了说明。其它激素和生长因子的作用被逐渐降低的血清水平和使用DCC-FBS的，这是FBS已经剥离了其大部分激素，细胞因子和生长因子的保持在最低限度。无酚红培养基进一步用于减少其他非荷尔蒙的影响，因为酚红已被报道具有雌激素活性和细胞系20,21影响某些功能。将细胞暴露于激素的时间是非常重要的。对基因的雌激素相关的法规，它先前已经表明，24 hrexposure产生的乳腺癌细胞中1,18直接靶基因显著响应。由于miRNA是由RNA聚合酶II转录，mRNA的一样，可以设想，这是一个合适的时间点来检测直接调节也是的miRNA。它是尚未确定是否以及如何将这些miRNA的影响在乳腺癌细胞中这些公知的目标，ER的表达。 miRNA表达谱可以是由于miRNA的，但事实上，成熟miRNA序列可还发现，在初级miRNA和miRNA前体的相对小的尺寸具有挑战性的，因为异质其GC含量22。几种分析技术和化学已经被用来克服这一困难。其中有微阵列和qPCR分析的不同的方法（ 图2）。虽然芯片被广泛接受的全基因组肛门ysis，它可以提供假阴性，且存在可用的平台之间的差异，从化学的印刷技术23。还分析了相对较少的miRNA基因，其中被计数以千比数以万计的mRNA转录物时，归一化处理是一个挑战。定量PCR，在另一方面，是更为敏感，并更好地应用于当较少的基因是要考虑的。然而，在大的384孔板中进行时，只有一个每板技术复制，多个板需要对每个样品使得分析成本进行分析。此外，在我们的手中，越来越多的假阴性结果采用DLP-LDA分析相比，基因芯片获得的。鉴于成熟miRNA序列是存在于初级miRNA和miRNA前体序列，它是感兴趣的这些转录物使用PCR技术24之间进行区分。 DLP探头也可用于预miRNA的，和SPecific引物也可以被设计来排除不同的成熟或前体变体使用花菁染料的定量PCR技术。 定量PCR是一个黄金标准的差异表达验证的评测结果。在定量PCR的效率，然而，依赖于几个参数，包括RNA提取，RNA的完整性（质量），合成cDNA，引物的设计，检测方法（化学），和参考基因对数据归一化1,22,25。引物设计的miRNA分析的选项是非常有限的，因为短期的miRNA序列的长度不给余地很大另类引物设计，只有一个引物可以在这短短的顺序进行包庇。因此，需要替代的操作， 如图2。技术复制是重要的，以验证扩增方法的鲁棒性和采用的工艺。生物复制所需的一般生物variatio的表示N，包括可变的反应配体处理，并表明在可见单元格的效果是可重复的。根据我们的经验，不同的细胞培养物之间的miRNA表达可发生。通常这些差异都小于1.3倍，和值的平均值和相应的SD标识自然和技术的变化。这种变化可能导致由各种因素，包括，细胞密度和事实，即细胞的功能可以由通道切换到通道。识别从配体治疗导致统计学显著表达式，一个广泛接受的意义是，当p值小于0.05。这里，对使用双尾分布和双样本方差不等参数的生物学和技术重复学生的t-试验已被使用。其他t-检验的参数存在，而选择依赖于实验装置26。 在评估成熟miR激素法规的详细协议适用于乳腺癌细胞中已经提供。在分析和验证这些miRNA的表达重要技术和化学已经解释清楚。选择了miRNA在乳腺癌细胞研究的技术最终取决于究竟正在调查中。

雌激素是一种激素，乳腺发育过程中是非常重要的。雌激素也在开发，维护，风险和治疗乳腺癌1中起着重要的作用。雌激素通过结合到急诊室，这是转录因子，调节特定靶基因发挥其功能。两个受体变异体，ERα是乳腺癌细胞的雌激素依赖性增殖是必不可少的。大多数乳腺癌是雌激素受体α阳性和依赖于雌激素生长。这使得雌激素信号和ERα治疗激素受体阳性乳腺癌的目标。了解ERα的基本机制是很重要的，以提高治疗效果，克服阻力的治疗，并了解乳腺癌的发展。 微RNA是由于转录后基因调控的影响主要在细胞功能的重要作用。 miRNA是19-24个核苷酸短的单链，非编码是首先通过转录RNA聚合酶II进入初级miRNA转录（初级miRNA）的RNA。它们是由Drosha酶在核内加工成短发夹前体的微RNA（miRNA前体），然后被Dicer加工，分离成单链，以形成在细胞质中成熟的miRNA。单链成熟miRNA被转移到Argonaute蛋白形成RISC复合物。然后，miRNA能够杂交到靶mRNA的3&#39;-非翻译区（3&#39;-UTR），导致转录后调节通过阻断翻译或降解靶mRNA 2。 由于这两种雌激素和微RNA在肿瘤进展中起，确定正常或破坏雌激素信号相关的miRNA的重要作用，以提高我们对发展的理解，提高乳腺癌的治疗是很重要的。虽然有一个很好的理解ERα如何调节蛋白编码基因，扩展和非编码RNA法规的细节还有待调查清楚。最初的研究目的是澄清的miRNA的ERα调节在乳腺癌细胞系中已经产生了相互矛盾的结果，即使在相同的细胞系进行了分析3-6。这可能是不同的治疗，生物变型中，使用不同的技术，而事实上，miRNA的小尺寸使它们具有挑战性的分析结果。在这里，一个协议，对变化和方法控制工件描述。 以确定哪些miRNA是由雌激素调节，ERα活性的良好定义的乳腺癌模型的分析是第一步。几个细胞系已经产生来自人ERα阳性乳腺癌肿瘤，这是依赖于雌激素相似，大多数临床乳癌。两个这些细胞系，T47D和MCF7，包括ERα和其下游靶的表达的分子性质，在孕激素受体 ​​（PR），缺乏膜受体HER2表达的，随着雌激素反应和管腔上皮分化的基因表达，使它们适合作为乳腺肿瘤7-10的Luminal亚型车型。 17β-雌二醇（E2）是雌激素的主要形式，而浓度与时间点对ERα的最佳转录激活已经表征了在多个研究。在此协议为10nM E2处理24小时的使用和T47D和MCF7作为在乳腺癌细胞中雌激素受体​​α1活性的模型。另外，依赖于时间的miRNA的规定可以具体为例如 1-72小时的时间间隔进行分析。 其次，分析的miRNA具有独立的挑战，部分原因是他们的短尺寸。 miRNA是没有得到很好的保留在总RNA样品时经常胍thiocyanate-phenol/chloroform/isopropanol RNA沉淀执行，或当STandard柱纯化使用。特别的预防措施需要采取维持或富集RNA的小部分。通过增加异丙醇的体积，离心（-80℃），然后降低温度，并省略洗涤在70％乙醇中，miRNA的保持可以沉淀过程中得到增强。或者，特定列的缓冲器和一个高品质和鲁棒制备含有miRNA的总RNA，都可以使用。也为分析本身，他们的短尺寸来制作的挑战。对于全基因组miRNA的筛选，有三种常见的miRNA表达谱技术可以选择：芯片，定量PCR和新一代测序。每种技术可以使用多个不同的平台上进行，并且不同的样品准备工作是必需的，每个引入的工件不同的风险。在miRNA芯片，幻灯片有斑点或与成千上万的寡核苷酸的合成。这些寡核苷酸被用作探针，每个探针的s被设计用于杂交到一个特定的miRNA序列。对于芯片的样品制备，如本研究中进行，首先为丰富的miRNA，然后介绍的Cy5和Cy3标记标签上的miRNA。微阵列提供了机会，同时观察的大量基因的相对表达水平，速度快，适合于大量样品的筛选，但可以仅分析该序列存在于微阵列和将例如未发现变化，在未知的或突变的miRNA。如本方案进行微阵列分析也需要相对大量的每个样品的总RNA进行分析，大约5微克。低密度qPCR的miRNA表达谱，需要更少的材料（700毫微克/样品和复制），并且允许进行miRNA的检测和定量。转录水平可以被确定，并且数量可以是绝对量或相对量。定量PCR分析，首先需要转换的miRNA成cDNA，这里使用一个环形底漆为每个特定的miRNA确保只有成熟的miRNA分析。这会产生一个更长的模板，可以利用一个miRNA的特异性正向引物和一个通用反向引物互补的环状序列进行扩增，并能够携带进一个双标记探针的特异性。定量PCR可以在其中可以并行使用一种或以每孔11个别引物对几个384孔板进行检测数百miRNA的低密度的格式来使用。新一代测序，在另一方面，是唯一的这些技术，它允许新的，突变的或编辑的miRNA的发现，作为样品中所有的RNA可测序11。这种技术，但是，需要多个步骤来丰富的小RNA，并使用几个步骤接头连接反应和纯化的带有后续增强调制样品之间的相对表达水平的风险，产生小RNA文库。这还需要显著bioinformatiCS分析。给出的各种技术对于miRNA表达谱，最合适的技术取决于应用。芯片是最适合当材料是比较丰富的，而利息是确定的已知的miRNA表达差异。低密度的qPCR阵列是最适合当一个有限的样本量是可用和低表达的miRNA的高灵敏度是必需的。测序是当需要未知的miRNA，突变的或miRNA的不同同种型的分析最合适。 在乳腺癌中雌激素调节的miRNA研究中，2模型细胞系的使用，T47D和MCF7，其中是容易得到大量的RNA。每个细胞系中使用不同的通道，每个治疗技术重复复制的细胞培养物进行了分析。这使得强大的检测的可重复性，ERα调节的miRNA。同时使用miRNA芯片和相对miRNA的表达水平进行比较双标记探针 - 低密度阵列（DLP-LDA）和验证的结果与特定的qPCR同时使用花青染料和DLP化学品。 miRNA的法规，然后进一步在时间序列分析，以确定它们随时间的精确调节，能帮助雌激素诱导分化法规随机或昼夜变化，并表示从二次效应主要影响。与ERα的染色质结合生物信息学研究的比较可以在初级与次级效应的分化进一步帮助。该法导致了miRNA的法规在那里它可以建立，经过24小时的治疗E2蛋白编码转录物易于调节，但成熟的miRNA并没有受到影响1一个可靠的评估。然而，这是可能的，miRNA是调节在稍后的时间点，如在选定的miRNA 1的时间序列分析。细节需要进一步探讨和这里介绍的协议提供了一个ROBUS吨方式来研究成熟miRNA在乳腺癌细胞系中的激素调节。

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			<td height="21" style="height:21px;width:515px;">DMEM</td>
			<td style="width:364px;">Life Technologies</td>
			<td style="width:399px;">11885092</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">F12</td>
			<td>Life Technologies</td>
			<td>11765054</td>
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		<tr height="21">
			<td height="21" style="height:21px;">FBS</td>
			<td>Sigma-Aldrich</td>
			<td>F0926-500ML</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Penicillin Streptomycin</td>
			<td>Life Technologies</td>
			<td>15140122</td>
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		<tr height="21">
			<td height="21" style="height:21px;">phenol red-free DMEM&#160;</td>
			<td>Life Technologies</td>
			<td>11054020</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Ultrapure Water</td>
			<td>EMD Millipore</td>
			<td>Specific equipment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DCC-FBS</td>
			<td>Sigma-Aldrich</td>
			<td>F6765-500ML</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100X L-glutamine</td>
			<td>Life Technologies</td>
			<td>25030081</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS tablet</td>
			<td>Medicago, Accurate chemicals</td>
			<td>Accurate (BF092051-100), Medicago (09-9400-100)</td>
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		<tr height="21">
			<td height="21" style="height:21px;">17&#946;-estradiol</td>
			<td>Sigma-Aldrich</td>
			<td>E2758</td>
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		<tr height="21">
			<td height="21" style="height:21px;">ICI 182,780&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>I4409</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>E7023-600ML</td>
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		<tr height="21">
			<td height="21" style="height:21px;">T47D or MCF7 cells lines</td>
			<td>American Type Culture Collection (ATCC)</td>
			<td>T47D (ATCC HTB-133) and MCF7 (ATCC HTB-22)</td>
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		<tr height="21">
			<td height="21" style="height:21px;">T-75 flask&#160;</td>
			<td>VWR International LLC</td>
			<td>BD353136 (vented cap)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin-EDTA</td>
			<td>Life Technologies</td>
			<td>25300062</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Serological pipet</td>
			<td>VWR International LLC</td>
			<td>53300. For 5mL (53300-421)</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Countess Cell Counting chamber</td>
			<td>Life Technologies</td>
			<td>C10228</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Countess Automated Cell Counter</td>
			<td>Life Technologies</td>
			<td>C10227</td>
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		<tr height="21">
			<td height="21" style="height:21px;">100 mm plates&#160;</td>
			<td>VWR International LLC</td>
			<td>25382-166</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Guanidinium thiocyanate-Phenol solution (TRizol)</td>
			<td>Life Technologies</td>
			<td>15596026</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rubber cell scraper&#160;</td>
			<td>VWR International LLC</td>
			<td>82050-470</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Guanidinium thiocyanate-Phenol-Chloroform method (miRNeasy Mini Kit/Trizol)</td>
			<td>Qiagen</td>
			<td>217004</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rnase-Free DNase I&#160; kit (DNA degradation)</td>
			<td>Qiagen</td>
			<td>79254</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNase-free water (Nuclease-free water)</td>
			<td>Thermoscientific</td>
			<td>SH30538.02</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Nanodrop 1000 Spectrophotometer&#160;</td>
			<td>Thermoscientific</td>
			<td>ND-1000</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Agilent RNA 6000 Nano Assay kit</td>
			<td>Agilent</td>
			<td>5067-1512</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Agilent 2100 Bioanalyzer</td>
			<td>Agilent</td>
			<td>G2938C and for software (G2946)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">All primers (except TaqMan primers)</td>
			<td>Integrated DNA Technologies</td>
			<td>Specific per order for each gene/miRNA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">First strand buffer, DTT, dNTPs, superscript III (Superscript III cDNA synthesis kit)&#160;</td>
			<td>Life Technologies</td>
			<td>18080085</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MicroAmp Fast 96-Well reaction plate&#160;</td>
			<td>Life Technologies</td>
			<td>4346906</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fast Optical Adhesive Covers</td>
			<td>Life Technologies</td>
			<td>4311971</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2X SYBR green PCR master mix (Cyanine dye)</td>
			<td>Life Technologies</td>
			<td>4385612</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">7500 Fast Real-Time PCR System and software</td>
			<td>Life Technologies</td>
			<td>4351106</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#956;Paraflo Microfluidic Biochip Technology&#160;</td>
			<td>LCSciences</td>
			<td>MRA-1001 and AS-2001(for result analysis)</td>
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		<tr height="21">
			<td height="21" style="height:21px;">DLP-LDA (TLDA) Megaplex Pools Assay (Rt primers/Human pool set)</td>
			<td>Life Technologies</td>
			<td>4400928</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dual Labeled Probes (TaqMan) miRNA RT kit&#160;</td>
			<td>Life Technologies</td>
			<td>4366596</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GeneAmp PCR System 9700 thermocycler</td>
			<td>Life Technologies</td>
			<td>4310899</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dual Labeled Probes (TaqMan) 2X Universal PCR Master Mix-No AmpErase UNG</td>
			<td>Life Technologies</td>
			<td>4324018</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">7900HT Fast Real-Time PCR system</td>
			<td>Life Technologies</td>
			<td>4329001</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SDS and RQ manager softwares</td>
			<td>Life Technologies</td>
			<td>4444202</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NCode miRNA First-Strand cDNA Synthesis and qRT-PCR Kit</td>
			<td>Life Technologies</td>
			<td>MIRC-50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10X DPL (TaqMan) RT primers</td>
			<td>Life Technologies</td>
			<td>Specific for each miRNA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">20X DPL (TaqMan) real-time assay primer&#160;</td>
			<td>Life Technologies</td>
			<td>Specific for each miRNA</td>
		</tr>
	</tbody>
</table>

profiling of estrogen-regulated micrornas in breast cancer cells

雌激素在乳腺发育和乳腺癌​​发展中起着至关重要的作用。它介导通过结合并激活雌激素受体（ERS），ERα和ERβ其功能。 ERα经常上调在乳腺癌和驱动乳腺癌细胞的增殖。雇员再培训计划作为转录因子和调节基因的表达。而蛋白质编码基因的雌激素受体α的调控是公认的，非编码小分子RNA（miRNA）的，其监管较少游客。微RNA在基因的转录后调控中发挥了重要作用，抑制其翻译或降低其mRNA的表达。的miRNA可以作为癌基因或抑癌基因，并且也有为的生物标志物。其中可用的miRNA的测定法，微阵列和实时定量聚合酶链式反应（定量PCR）已被广泛用于检测和定量miRNA水平。以识别的miRNA通过雌激素信号在乳腺癌，日规EIR表达ERα阳性的乳腺癌细胞系进行了对比之前和之后同时使用的μParaflo微流控芯片和双标记探针低密度阵列雌激素激活。结果使用特定的qPCR实验进行了验证，同时应用花青染料型和双标记探针为基础的化学反应。此外，一个时间点测定法用于鉴定规定时间的推移。在这项研究中所使用的miRNA的检测方法的优点是，它使成熟的miRNA法规快速筛选大量样品中，即使在有限的样品量。的布局，包括用于细胞培养的特定条件和雌激素治疗，生物技术和重复，并且大规模筛选随后深入的确认使用单独的技术，确保了韧性检测的miRNA的规定，并消除误报和其他伪影。然而，突变或未知的miRNA，或在小学和前体的转录LEVE法规升，将不会被检测到。这里介绍的方法代表了雌激素介导的miRNA调控了深入调查。

该方法的概述示于图1和各样品的准备和需要为每个技术核酸修饰的比较被可视化在图2中 。 在用激素治疗前的细胞的条件和环境是决定激素16的实际效果非常重要。一些介质和血清含有可能影响结果的生长因子，使细胞血清饥饿，以确保所有激素效应已被降低到最小。因此，与E2治疗时，有更多的确定性所观察到的影响E2相关。一个重要因素是细胞的汇合，从而影响细胞 - 细胞接触和行为，例如细胞 - 细胞信号传导和细胞增殖。让细胞在80％汇合（ 图3A）和很好地附着，以允许生长和避免细胞在随后的洗涤和介质通道损失橘色。 RNA提取和储存过程中的条件是对于RNA的质量和后续的分析非常重要。它也被称为该细胞的数量，提取方法，和miRNA的GC含量会影响二者的mRNA和miRNA的17的产量和质量。因此，有必要在无RNA酶的条件下进行RNA提取和在进行实验之前，检查RNA的质量。萃取后，该RNA的定量提供了关于（含有两个基因和微RNA）的总RNA，从而使观察收率的浓度信息。它还提供看出，在图3B的结果作为一个图形表示，这允许在观察样品（通常由一个峰，顶板表示）的纯度。 RNA的产量不佳会产生不特定吸收在260 nm处（ 图3B，底部面板）。要确定RNA是否是高质量的，RNA完整性进行了测量。 9-10之间的RNA的完整性号（RIN）确保RNA不降解，是最佳的质量。此外，RNA的图形表示应观察和18S和28S rRNA基因应具有峰值， 如图4（中图）。用梯子（ 图4，上图）的比较识别的峰值的大小。一个降解的RNA会有那么明显的峰值和18S和28S rRNA基因的相对减少量（ 图4，底部面板）。小RNA的部分可以进一步使用安捷伦小RNA试剂盒保证。因此建议以验证雌激素反应处理后，在规定的试验条件下，进行到miRNA的筛选前。一个可以定量PCR已知的ER靶基因，如PS2或SPINK4 18上执行，利用cDNA模板。这些基因通常会在E2治疗后1-24小时上调。一旦RNA的质量和雌激素应答经评估，进一步的实验可以进行。 微阵列仍然是一个广泛使用的技术进行大规模筛选在细胞中miRNA的表达。例如，一旦使用了miRNA芯片包含894成熟的miRNA和50控制独特的探针四胞胎1。杂交是在染料交换过程重复进行。该miRNA芯片结果通常是通过图形化的热图来表示。 图5A显示热图，即从车辆和E2处理的样品之间的miRNA芯片比较数据。红色表明miRNA的上调E2的处理的样品（高表达）中，而绿色表示miRNA的（低表达）的下调。 miRNA的选择通常取决于它们在表达意义，而且通常的p值小于0.05被认为是显著。被指示进行差异表达的miRNA的应进一步验证瓦特第i个定量PCR，从误报区分它们。 该DLP-阵列LDA，另一方面，使用定量PCR为基础的方法来筛查在384 - 孔格式调节的miRNA。通常有两种384孔板（卡）提供，池​​A和B。A组通常包含更好的表征，更高效表达的miRNA比乙组各miRNA的相对水平是由定量PCR，其中一个miRNA是每个分析确定良好（ 图5B）。较高的Ct值表示较小的miRNA的表达。很像miRNA芯片，结果从DLP-LDA的实现还需要进一步验证，以确保准确性。 验证可以使用qPCR来执行。在这里，两个定量PCR检测方法进行了说明，以防止偏置方法和允许彻底确认和miRNA的规定调查。的花青染料为基础的检测化学工程比DLP不同的DLP-LDA谱是基于，并且是适合于确认振动性的目的。因为它检测到所有扩增的双链DNA，但样品的均匀性可以通过执行熔化曲线分析。 图6A（左图）进行评估显示了miRNA的熔化曲线分析也可以是不特定，和一个明确规定统一的峰值可以看出。如果该引物是引物二聚体的非特异性或显著量被形成（ 图6A，右图）多个峰值会被观察到。另一方面DLP miRNA的检测，更具体为miRNA的检测目标的唯一扩增。扩增曲线的每个靶miRNA的可观察到的，如例示于图6B中 。以控制对多个扩增产物用熔解曲线分析的出现的选项，但是，不能与这种化学和花菁染料的qPCR是较便宜的。菁染料和DLP qPCR实验有时会产生稍微不同的结果。对于这两种qPCR实验，比较的参考资料，希望能帮rence基因需要确定基因的两个样本之间的相对表现。参照基因，也称为看家基因，应该是一个基因，其不改变的表达和其表达水平相似的感兴趣的基因。一个合适的参考基因在这些乳腺癌细胞系的一个例子是ARHGDIA，作为Rho GDP解离抑制因子，其功能该R​​ho蛋白的GDP / GTP交换反应。如观察到在图6C（顶部），则不会在车辆和配体处理的样品之间变化ARHGDIA的mRNA水平。的18S rRNA基因，也可以使用，虽然它的高表达，需要一个单独的1:500稀释的cDNA的库存，因而是不适合的。对于miRNA的分析，还有一个明确的参照基因进行辩论。到目前为止，U6 snRNA的是，已被广泛接受为miRNA的分析的参照基因。 U6 snRNA的是〜110个核苷酸的非编码小核的功能在核mRNA前体RNA的SPL结冰19。如观察到在图6C（底部），U6的表达水平大约是相同在显示的所有样本，从而允许对miRNA的可变水平的计算。的与miRNA的qPCR结果为已被归一化至参考U6车辆和E2处理的细胞之间的比较的图形表示可以在图6D中被观察到。这说明被检测到后24小时E2治疗作为非管制，一个miRNA的，但怀有ER染色质结合位点接近其基因组的位置和时间序列分析处理后识别显著调节72小时。 <img alt="图1" fo:content-width="5in" fo:src="/files/ftp_upload/51285/51285fig1highres.jpg" src="/files/ftp_upload/51285/51285fig1.jpg" /> 图1。用于检测miRNA在乳腺癌细胞激素调节的方法的概述。 </str翁&gt;过程开始于细胞培养和处理，接着通过大规模miRNA表达谱来识别可能的差异表达的miRNA，并且与阵列结果的定量RT-PCR验证结束。 <img alt="图2" fo:content-width="5in" fo:src="/files/ftp_upload/51285/51285fig2highres.jpg" src="/files/ftp_upload/51285/51285fig2.jpg" /> 图2。不同的样品制备和核酸操作的每个miRNA检测技术的比较分析。 （一）使用的μParaflo技术方法的miRNA微阵列包括富集，多聚A尾和结扎标签（B）DLP技术的样品制备和检测方法，包括一个环形cDNA合成引物的变性步骤之后扩展，并提供较长的片段窝藏反向引物和探针序列（C）花青染料技术的样品制备和检测方法包括多聚A尾，和cDNA合成与寡-dT引物包括一个通用的5&#39;序列。 <img alt="图3" fo:content-width="5in" fo:src="/files/ftp_upload/51285/51285fig3highres.jpg" src="/files/ftp_upload/51285/51285fig3.jpg" /> 图3。细胞培养和RNA的提取。 （一）培养的细胞提取后，说明配体治疗前需要80％汇合。（B）RNA的浓度和纯度的图形表示。每个峰代表一个样本和一个成功的提取过程产生的纯RNA（顶板）。一个提取的RNA不佳显示了在260 nm处（下图）没有明显的吸收峰。 <img alt="图4" fo:content-width="5in" fo:src="/files/ftp_upload/51285/51285fig4highres.jpg" src="/files/ftp_upload/51285/51285fig4.jpg" /> 图4。＆＃160; RNA质量控制结果从RNA完整性分析表示为阶梯（顶部），良好的质量核糖核酸（中间）和降解的RNA样品（下部）的图形表示。 <img alt="图5" fo:content-width="5in" fo:src="/files/ftp_upload/51285/51285fig5highres.jpg" src="/files/ftp_upload/51285/51285fig5.jpg" /> 图5。 miRNA的分析结果的说明。 （一）miRNA芯片结果差异表达的miRNA热图表示。的miR-A，B，C的上调E2的处理，样品中的以红色表示，而miR-D，E，F，G被下调的E2处理的样品中为绿色表示（B）扩增曲线的每个的miRNA从DLP-LDA结果。检测miRNA的扩增通过增大RN的值所指示的。 <img alt="图6" fo:content-width="5in" fO型：SRC =“/ files/ftp_upload/51285/51285fig6highres.jpg”SRC =“/ files/ftp_upload/51285/51285fig6.jpg”/&gt; 图6。 miRNA的规定的qPCR分析。一个miRNA的（A）的熔点曲线分析使用花菁染料的检测，显示所有测试的样品（左）和多个片段（右）的扩增均匀性分析（B）的扩增曲线的每个样本的miRNA的。这可以是从花青染料或DLP检测方法（C）的合适的参考基因表达分析ARHGDIA（顶部）和miRNA的分析的snRNA U6（底部），显示出样品之间没有显著差异表达的条形图表示（D）的miRNA的qPCR结果，说明了miR-135a的相对表达水平的比较，在一个时间过程。 图6D是从Katchy 等 1重印与从爱思唯尔权限。值通常是单独的exper的平均iments（生物重复）±SD。学生氏t-检验用于演示的意义：* P &lt;0.05。

Watch this Scientific Journal Video about Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells at JoVE.com

Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells

分子通过雌激素和小分子RNA的信号是在乳腺癌的发展和增长的关键。雌激素激活的雌激素受体，它是转录因子。许多转录因子能调节微RNA的表达，而雌激素调节的微小RNA可以使用不同的大规模的技术来成型。

在乳腺癌细胞雌激素调节小分子RNA的剖析

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen ...

profiling-of-estrogen-regulated-micrornas-in-breast-cancer-cells

Research

JoVE Journal

Medicine

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There is little knowledge about in situ, in vivo, tumor hypoxia during intracranial development of malignant brain tumors because of lack of efficient means to monitor it in these deep-seated orthotopic tumors. Bioluminescence imaging (BLI), based on the detection of light emitted by living cells expressing a luciferase gene, has been rapidly adopted for cancer research, in particular, to evaluate tumor growth or tumor size changes in response to treatment in preclinical animal studies. Moreover, by expressing a reporter gene under the control of a promoter sequence, the specific gene expression can be monitored non-invasively by BLI. Under hypoxic stress, signaling responses are mediated mainly via the hypoxia inducible factor-1&alpha; (HIF-1&alpha;) to drive transcription of various genes. Therefore, we have used a HIF-1&alpha; reporter construct, 5HRE-ODD-luc, stably transfected into human breast cancer MDA-MB231 cells (MDA-MB231/5HRE-ODD-luc). In vitro HIF-1&alpha; bioluminescence assay is performed by incubating the transfected cells in a hypoxic chamber (0.1% O2) for 24 hr before BLI, while the cells in normoxia (21% O2) serve as a control. Significantly higher photon flux observed for the cells under hypoxia suggests an increased HIF-1&alpha; binding to its promoter (HRE elements), as compared to those in normoxia. Cells are injected directly into the mouse brain to establish a breast cancer brain metastasis model. In vivo bioluminescence imaging of tumor hypoxia dynamics is initiated 2 wks after implantation and repeated once a week. BLI reveals increasing light signals from the brain as the tumor progresses, indicating increased intracranial tumor hypoxia. Histological and immunohistochemical studies are used to confirm the in vivo imaging results. Here, we will introduce approaches of in vitro HIF-1&alpha; bioluminescence assay, surgical establishment of a breast cancer brain metastasis in a nude mouse and application of in vivo bioluminescence imaging to monitor intracranial tumor hypoxia.

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

Bioluminescence imaging of hypoxia inducible factor-1&alpha; activity is applied to monitor intracranial tumor hypoxia development in a breast cancer brain metastasis mouse model.

在小鼠模型体内生物发光成像乳腺癌脑转移的肿瘤乏氧动力学

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There ...

科研

医学

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques.
For this purpose, we injected 105 MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg1. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula1. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US).
MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified2-4. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions5,6. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles7.
In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.

In the pathogenesis of bone metastasis, angiogenesis is a crucial process and therefore represents a target for imaging and therapy. Here, we present a rat model of site-specific breast cancer bone metastasis and describe strategies to non-invasively image angiogenesis in vivo using magnetic resonance imaging, volumetric computed tomography and ultrasound.

利用磁共振成像，容积电脑断层扫描及超音波的裸鼠乳腺癌骨转移大鼠模型血管生成的多模态成像

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell ...

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

实时成像在乳腺癌小鼠模型中的肿瘤微环境对药物的反应

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional ...

Three Dimensional Cultures: A Tool To Study Normal Acinar Architecture vs. Malignant Transformation Of Breast Cells

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The interplay of signals between cancer cells and their microenvironment exerts a powerful influence on breast cancer growth and biological behavior1. However, most of these signals from the extracellular matrix are lost or their relevance is understudied when cells are grown in two dimensional culture (2D) as a monolayer. In recent years, three dimensional (3D) culture on a reconstituted basement membrane has emerged as a method of choice to recapitulate the tissue architecture of benign and malignant breast cells. Cells grown in 3D retain the important cues from the extracellular matrix and provide a physiologically relevant ex&#160;vivo system2,3. Of note, there is growing evidence suggesting that cells behave differently when grown in 3D as compared to 2D4. 3D culture can be effectively used as a means to differentiate the malignant phenotype from the benign breast phenotype and for underpinning the cellular and molecular signaling involved3. One of the distinguishing characteristics of benign epithelial cells is that they are polarized so that the apical cytoplasm is towards the lumen and the basal cytoplasm rests on the basement membrane. This apico-basal polarity is lost in invasive breast carcinomas, which are characterized by cellular disorganization and formation of anastomosing and branching tubules that haphazardly infiltrates the surrounding stroma. These histopathological differences between benign gland and invasive carcinoma can be reproduced in 3D6,7. Using the appropriate read-outs like the quantitation of single round acinar structures, or differential expression of validated molecular markers for cell proliferation, polarity and apoptosis in combination with other molecular and cell biology techniques, 3D culture can provide an important tool to better understand the cellular changes during malignant transformation and for delineating the responsible signaling.

Three dimensional culture of mammary epithelial cells on a reconstituted basement membrane is a useful method to recapitulate the in vivo architecture of the benign breast, and to differentiate the malignant phenotype from the benign breast phenotype. Importantly, this system can be applied to study invasive carcinomas in other tissues.

三维文化：一种工具来研究正常腺泡结构与恶性转化的乳腺细胞

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The ...

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the extracellular matrix (ECM) has been demonstrated to be a critical regulator of cell behavior in culture and homeostasis in vivo. The current approach of culturing cells on two-dimensional (2D), plastic surfaces results in the disturbance and loss of complex interactions between cells and their microenvironment. Through the use of three-dimensional (3D) culture assays, the conditions for cell-microenvironment interaction are established resembling the in vivo microenvironment. This article provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment. In addition, we discuss how these 3D assays have the potential to examine the loss of signaling molecules that regulate epithelial morphology by immunostaining procedures. These studies aid to identify important mechanistic details into the processes regulating invasion, required for the spread of breast cancer.

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

This article provides detailed methodologies for the use of three-dimensional (3D) assays to quantify breast cancer cell invasion. Specifically, we discuss the procedures required to set up such assays, quantification, and data analysis, as well as methods to examine the loss of membrane integrity that occurs when cells invade.

乳腺癌细胞侵袭定量使用三维（3D）模型

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the ...

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the extracellular matrix (ECM). Characterizing the biology of this phenotypically distinct group of cells could substantially improve our understanding of early events during the metastatic cascade.

Tumor invasion is a dynamic process facilitated by bidirectional interactions between malignant epithelium and the cancer associated stroma. In order to examine cell-specific responses at the tumor stroma-interface we have combined organotypic co-culture and laser micro-dissection techniques.
Organotypic models, in which key stromal constituents such as fibroblasts are 3-dimentioanally co-cultured with cancer epithelial cells, are highly manipulatable experimental tools which enable invasion and cancer-stroma interactions to be studied in near-physiological conditions.

Laser microdissection (LMD) is a technique which entails the surgical dissection and extraction of the various strata within tumor tissue, with micron level precision.
By combining these techniques with genomic, transcriptomic and epigenetic profiling we aim to develop a deeper understanding of the molecular characteristics of invading tumor cells and surrounding stromal tissue, and in doing so potentially reveal novel biomarkers and opportunities for drug development in CRC.&#160;&#160;&#160;

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Molecular profiling of laser microdissected cells and stroma from synthetic, tissue-engineered colorectal cancer models represents a novel and manipulatable approach to characterize and dissect the distinctive biology at the interface between tumor cells at the invasive front and cancer associated stromal cells. &#160;

在结直肠癌细胞的三维模型的入侵肿瘤微环境的分子剖析和<em&gt;体外</em&gt;成纤维细胞

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the ...

Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the functions of proteins involved in oncogenic progression or help to discover new tumor suppressors. In addition, injecting cancer cells into mice with different genotypes might provide a better understanding of the importance of the stromal compartment. Many models may be useful to investigate certain aspects of disease progression but do not recapitulate the entire cancerous process. In contrast, breast cancer cells engraftment to the mammary fat pad of mice better recapitulates the location of the disease and presence of the proper stromal compartment and therefore better mimics human cancerous disease. In this article, we describe how to implant breast cancer cells into mice orthotopically and explain how to collect tissues to analyse the tumor milieu and metastasis to distant organs. Using this model, many aspects (growth, angiogenesis, and metastasis) of cancer can be investigated simply by providing a proper environment for tumor cells to grow.

Cancer is a complex disease that is influenced by the tissue surrounding the tumor as well as local pro- and anti-inflammatory mediators. Therefore, orthotropic injection models, rather than subcutaneous models may be useful to study cancer progression in a manner that better mimics human pathology.

注射原位乳腺癌细胞到小鼠乳腺脂肪垫来研究肿瘤的生长。

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the ...

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of women are alive 5 years after diagnosis. The omentum is a preferred site of HGSC metastasis formation. Despite the clinical importance of this microenvironment, the contribution of omental adipose tissue to ovarian cancer progression remains understudied. Omental adipose is unusual in that it contains structures known as milky spots, which are comprised of B, T, and NK cells, macrophages, and progenitor cells surrounding dense nests of vasculature. Milky spots play a key role in the physiologic functions of the omentum, which are required for peritoneal homeostasis. We have shown that milky spots also promote ovarian cancer metastatic colonization of peritoneal adipose, a key step in the development of peritoneal metastases. Here we describe the approaches we developed to evaluate and quantify milky spots in peritoneal adipose and study their functional contribution to ovarian cancer cell metastatic colonization of omental tissues both in vivo and ex vivo. These approaches are generalizable to additional mouse models and cell lines, thus enabling the study of ovarian cancer metastasis formation from initial localization of cells to milky spot structures to the development of widespread peritoneal metastases.

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

We outline a protocol that implements both in vivo and ex vivo approaches to study ovarian cancer colonization of peritoneal adipose tissues, particularly the omentum. Furthermore, we present a protocol to quantitate and analyze immune cell-structures in the omentum known as milky spots, which promote metastases of peritoneal adipose.

在体内和体外途径研究银河系结构现货卵巢癌转移定植腹膜脂肪

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of ...

Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation assay (hereafter called soft agar assay), which measures the ability of cells to proliferate in semi-solid matrices, remain a hallmark of cancer research. A key advantage of this technique over conventional 2D monolayer or 3D spheroid cell culture assays is the close mimicry of the 3D cellular environment to that seen in vivo. Importantly, the soft agar assay also provides an ideal tool to rigorously test the effects of novel compounds or treatment conditions on cell proliferation and migration. Additionally, this assay enables the quantitative assessment of cell transformation potential within the context of genetic perturbations. We recently identified peptidylarginine deiminase 2 (PADI2) as a potential breast cancer biomarker and therapeutic target. Here we highlight the utility of the soft agar assay for preclinical anti-cancer studies by testing the effects of the PADI inhibitor, BB-Cl-amidine (BB-CLA), on the tumorigenicity of human ductal carcinoma in situ (MCF10DCIS) cells.

Here, we document the use of the soft agar colony formation assay to test the effects of a peptidylarginine deiminase (PADI) enzyme inhibitor, BB-Cl-amidine, on breast cancer tumorigenicity in vitro.

软琼脂集落形成实验利用识别的致瘤性乳腺癌细胞抑制剂

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.