Name: systems biology of metabolic regulation by estrogen receptor signaling in breast cancer
Uploaded: 2016-03-17T14:00:00.000+00:00
Duration: 10 min 36 s
Description: Watch this Scientific Journal Video about Systems Biology of Metabolic Regulation by Estrogen Receptor Signaling in Breast Cancer at JoVE.com

这项工作是由粮食和农业研究所，美国农业部，屡获ILLU-698-909（ZME）和辉瑞公司（ZME）资助。其内容完全是作者的责任，并不一定代表美国农业部的官方意见。

1. RNA测序样品的制备<ol><li>细胞培养和治疗<ol><li>培养的MCF7细胞中改进的MEM（IMEM）加了10％热非活性的FBS，1％青霉素/在湿润的5％CO 2的气氛链霉素，在37℃的酚红培养基。 注意:相同的细胞系和细胞培养条件在整个研究中使用。 </li><li>种子100000 MCF7细胞每孔在6孔板中。对每个治疗一式三份。在第3天，除去培养基，加2ml含或不含10 -8 M的E2向每个孔新鲜培养基中。孵育所述板在湿润的5％CO 2的气氛中在37℃24小时。 </li><li>收获细胞，加入1ml RNA分离试剂（酸性硫氰酸胍 - 苯酚 - 氯仿）到每个孔中，并孵育在室温（RT）下5分钟。然后收集吹打并沉积在细胞裂解液试剂进入1.5毫升管</li></ol></li><li> RNA分离<ol><li>加入200μlchlorofoRM到1ml细胞溶解物试剂（1.1.3），涡旋10秒。在室温下孵育5分钟。离心样品以16,000 xg离心在4℃下15分钟。离心后，将水相（上部）转移到新的管中，而不会干扰其它相。 </li><li>将500μl100％异丙醇添加到与所传送的水相的新管中，并通过颠倒混合。孵育在-20℃过夜。离心样品以16,000 xg离心在4℃下10分钟。在管的底部观察的颗粒。弃去上清液并通过简短涡旋洗净用750微升不含RNA酶的70％的乙醇沉淀。 </li><li>离心样品在4℃6000×g离心5分钟。除去上清液，并在4℃下以16,000 xg离心离心样品1分钟以收集所有剩余的乙醇。通过移液用凝胶装载尖除去剩余的乙醇，并设置在管上侧向下风干10分钟。溶解在依赖新生20-50微升DEPC处理水沉淀丁对丸粒的大小。 </li></ol></li><li>使用RNA清理试剂盒按照协议4纯化的RNA。使用基于荧光检测试剂盒按照协议9测量RNA浓度。 注意:OD 260nm处取来确定在260nm的OD的RNA浓度和比率和280nm被用来确定样品的纯度。建议检查使用Bio-分析器测定RNA的质量。 </li><li>取用于测序约0.5至1微克纯化的RNA。 注:生物技术中心准备测序文库，并执行使用以前2详细方法配对末端测序读。 </li></ol> 2.芯片起样品的制备<ol><li>细胞培养和治疗<ol><li>种子每10cm盘500000 MCF7细胞在生长培养基中。 注意:对于每个拉下使用了16片。在第3天，除去培养基并加入5毫升或不含10 -8 M的E2到每个新鲜培养基盘子。处理细胞45分钟（37℃，在潮湿的5％CO 2的气氛中）。 </li></ol></li><li>交联和细胞收获<ol><li>加入250微升37％甲醛入5ml培养基交联染色质，在室温下孵育15分钟。停止洗涤细胞用5ml冰冷的1×MEM交联。 </li><li>收获在每板250μl的细胞裂解缓冲液（10mM EDTA，50mM的三盐酸，1％SDS，0.5％N，N-二dimethyldodecan -1-胺氧化物）的细胞。 </li></ol></li><li>染色质的碎片<ol><li>超声处理的细胞30秒×8以30放大器来达到所需的染色质的大小。 30秒的超声处理后在冰上冷却各样品。 </li><li>离心机在16，000×g离心在4℃下10分钟，并小心地吸取上清液，而不在底部干扰颗粒。 </li><li>取上清的5微升等分试样，并通过1.5％琼脂糖凝胶检查如先前10描述的电泳的DNA大小。理想的大小的DNA应当是200-500碱基对之间。 </li></ol></li><li>染色质免疫沉淀<ol><li>保存25微升细胞裂解液作为输入。在50ml管中，将4- ml细胞裂解液用20毫升的IP缓冲液（2mM EDTA的，100毫摩尔NaCl，20毫摩尔Tris-HCl和0.5％的Triton X），ERα抗体（500微升F10和500μl的HC- 20），和500微升PROTA和ProtG蛋白涂覆的磁珠。孵育通过旋转该混合物在4℃下过夜，使染色质 - 抗体复合物的形成。 </li></ol></li><li>清洗和反向交联<ol><li>用磁力架收集关于磁化侧磁珠去除洗涤缓冲液。 </li><li>洗磁珠在25ml洗涤缓冲液I（2毫摩尔EDTA，20毫摩尔Tris盐酸，01％SDS，1％的Triton X，和150毫摩尔NaCl）。彻底清洗磁珠不干扰DNA的蛋白质复合物，加入25 1ml洗涤慢慢缓冲到沿着侧的管然后反转管，直到所有的磁珠混合以及无线TH洗涤缓冲液没有出现一个小球。作为替代，使用旋转器以反转管。不要涡旋样品。用同样的方法在下面的步骤洗涤磁珠。 </li><li>在25ml洗涤缓冲液II（2毫摩尔EDTA，20毫摩尔Tris盐酸，01％SDS，1％的Triton X，和500mM的氯化钠）洗涤磁珠。 </li><li>洗磁珠在25ml洗涤缓冲液III（1.6毫摩尔EDTA，10毫摩尔Tris盐酸，1％NP-40，1％Dexycholate和250mM的氯化锂）。 </li><li>在25ml 1×TE缓冲液（1mM EDTA和10毫摩尔Tris HCl）的两次洗涤磁珠。 </li><li>洗脱的DNA于500μl洗脱缓冲液（1％SDS，和10mM的NaHCO 3），然后分配500微升的DNA洗脱到4个试管。从投入的DNA，洗脱125微升洗脱缓冲液的DNA。 </li><li>在65℃孵育管过夜加热块上。用铝箔覆盖在加热块上的管，以保持热甚至在所有的管子。将一个砝码放在管，以防止他们打开。&lt;/ LI&gt; </ol></li><li> DNA提取<ol><li>冷却在RT管5分钟。隔离使用DNA芯片分离试剂盒下拉样本的DNA。请遵循制造商的指示。 <ol><li>预热在65℃的洗脱缓冲液。添加625微升结合缓冲液至每个管。如果形成沉淀，再添加入250μl结合缓冲液，直至溶液澄清。 </li><li>加载样品上柱和离心机以16,000×g离心30秒。洗净用250微升洗涤液样品。记得要加乙醇洗涤缓冲液，当第一次使用它。重复洗。 </li><li>洗脱在15微升洗脱缓冲液将DNA。联合收割机一样的DNA，从4管拔下来，实现约55微升的DNA。 </li></ol></li><li>隔离输入DNA用DNA纯化试剂盒。 <ol><li>预热在65℃的洗脱缓冲液。添加625微升结合缓冲液中的每个管中并孵育10分钟。加载样品到柱上在2 ml collecti上管。 </li><li>洗用750微升洗涤缓冲液，离心样品以16,000 xg离心1分钟。重复离心以除去任何剩余的缓冲区。洗脱DNA插入EB的30微升。 </li></ol></li><li>测量DNA浓度使用荧光染料的商业与试验修改11。 <ol><li>稀释20倍TE缓冲到1倍的TE，作为稀释试剂和DNA样品的测定缓冲液。取5微升提取的DNA，并将其稀释成50微升的TE 1X。 </li><li>从2微克/毫升DNA原液进行DNA标准从毛坯到1，10，100，1000微克/毫升。在一个96孔板，分配每个标准的25微升以及在重复DNA稀释。 （推荐标准一式三份）。 </li><li>通过在塑料制管，从双链DNA试剂的200倍稀释于1×TE缓冲液制备工作试剂。用铝箔覆盖工作试剂溶液，以避免光。加入200μl工作试剂到每个样本。在cubate在室温下5分钟。用铝箔覆盖该板，以避免光。 </li><li>测量使用荧光板读数器（激发在480nm，发射520纳米）的样品的荧光。生成花期与DNA浓度的DNA标准曲线。确定基于所产生的DNA的标准曲线对样品的浓度。 </li></ol></li></ol></li><li>沉淀实验验证使用qPCR <ol><li>取2微升提取的DNA和稀释成20微升水。通过混合2.5微升DNA稀释液，5微升绿我PCR预混，1微升底漆和1.5微升无核酸酶水设置一式三份的qPCR实验。 </li><li>使用以下程序的快速实时PCR系统上运行的qPCR:步骤1:95℃60秒，第2步:95℃，10秒，60℃60秒，重复执行步骤2 39次。 </li></ol></li><li>送10纳克DNA样本，以生物技术中心进行测序。 注:生物技术H中央根据指令准备DNA芯片进入图书馆，并执行使用以前2详细方法单读测序。 </li></ol> 3.代谢组学分析样品制备<ol><li>细胞培养和治疗<ol><li>种子每10cm平板400000 MCF7细胞在生长培养基中。每个处理制备三个样品。使用每个样品中两个板块获得足够的细胞用于检测。在第3天，除去培养基并加入5毫升或不含10 -8 M的E2到细胞的新鲜培养基。处理细胞24小时（37℃下在湿润的5％CO 2的气氛中）。 </li></ol></li><li>样品采集<ol><li>加入5毫升冰水预冷的1×PBS的板块，经过几次短暂倾斜板吸出PBS。重复两次，在最后一次洗涤后除去尽可能多的PBS越好。 </li><li>添加750微升预冷的丙酮至各板和刮细胞。结合细胞的丙酮从两个板在冰上2ml的管中。 </li><li>正常化细胞计数<ol><li>对于每个处理，使用细胞量化两个额外板。从板到解附着细胞，加入2毫升，HE缓冲液（1×HBSS，10mM的HEPES，0.075％ 碳酸氢钠，和1mM EDTA）插入每个板并孵育5分钟。 </li><li>收集并通过在HE缓冲吹打细胞拌匀细胞。使用总共20微升细胞溶液到计数使用血球细胞数。 </li></ol></li></ol></li><li>发送到代谢中心来识别和使用气相色谱质谱（GC-MS）分析量化代谢物之前储存于-80℃的样品。 </li></ol> 4.综合分析<ol><li> RNA测序数据分析: <ol><li>执行使用FastQC FASTQ文件质量检查:读QC（http://galaxy.illinois.edu） <ol><li>选择FastQC:NGS的工具下阅读:QC和操纵。载从当前histor原始数据文件年。 注:建议给一个标题为输出文件提醒你什么工作了，因为对于可能有多个输出。 </li></ol></li><li>执行和输出文件将历史记录下。重复相同的步骤，对每个序列文件。修剪适配器使用NGS下剪辑:FASTX工具包。 </li><li>对齐读取使用Tophat2与RefSeq的基因组参考注释12（默认值）hg19。 <ol><li>根据NGS的工具选择Tophat2:RNA分析。指示库配对类型（单端VS末端配对）。 </li><li>上传从目前的历史上输入FASTQ文件。选择参考基因组。选择默认设置。或使用完整的参数表进行修改。 </li><li>执行和它会产生两个输出文件:一个是路口UCSC床轨道;另一种是在BAM格式读出比对的列表。 </li></ol></li><li>上传BAM文件测序数据分析工具。计算使用定量表达值ification工具。 </li><li>确定使用默认值使用表达分析工具差异上调的基因和倍数变化&gt; 2且p值 &lt;0.05。同样地，确定使用默认值使用表达分析工具差异下调的基因和倍数变化&lt;0.5和p值&lt;0.05。 </li></ol></li><li>芯片起数据分析: <ol><li>对齐FASTQ读取到银河系中的使用Bowtie2 13（默认值）hg19。 <ol><li> NSC的工具下选择Bowtie2:映射。验证该库是队友配对（单端VS末端配对）。上传从目前的历史FASTQ文件。 </li><li>选择参考基因组。使用默认的参数设置。执行和它会产生输出文件，映射顺序读为BAM文件。重复该过程的每个序列文件。 </li></ol></li><li>识别使用MACS 14 6.0E-7的p值截止和0.01 FDR峰，如前所述2。 </li></OL&gt; </li><li>代谢组学数据分析: <ol><li>转换代谢产物的化学名称KEGG_IDs。 </li><li>通过比较在车辆检测VS。E2水平鉴定差异调节的代谢物处理的样品（2倍截止在本研究中使用）。 </li></ol></li><li>综合分析: <ol><li>识别已经使用的测序数据分析工具ERα结合位点的基因。翻译地区使用20 KB作为距离截止基因的功能。 </li><li>使用倍数变化&gt; 2确定从RNA测序数据分析差异上调和下调的基因和倍数变化&lt;0.5，分别与p值 &lt;0.05为切断。比较使用维恩图比较ERα结合位点含有基因的列表中。 </li><li>使用此列表中使用的Cytoscape的Metscape 15模块识别E2调制的代谢途径。 <ol><li>通过从应用Metscape模块中，选择建立网络，然后途径-BAS编辑选项。 </li><li>选择生物体（人类）。 E2无管制化合物列表中选择的数据文件（KEGG ID），以及E2上调基因列表。 </li><li>选择化合物-Reaction - 酶 - 基因作为网络类型。选择化合物/基因作为查询。 </li><li>构建网络。重复使用E2下调化合物和基因。 </li></ol></li></ol></li></ol>

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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Performance+of+genetic+programming+optimised+Bowtie2+on+genome+comparison+and+analytic+testing+(GCAT)+benchmarks.">Performance of genetic programming optimised Bowtie2 on genome comparison and analytic testing (GCAT) benchmarks.</a> BioData Min. 8, (2015).</li><li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Model-based+analysis+of+ChIP-Seq(MACS).">Model-based analysis of ChIP-Seq(MACS).</a> Genome Biol. 9 (9), R137 (2008).</li><li>Gao, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metscape:+a+Cytoscape+plug-in+for+visualizing+and+interpreting+metabolomic+data+in+the+context+of+human+metabolic+networks.">Metscape: a Cytoscape plug-in for visualizing and interpreting metabolomic data in the context of human metabolic networks.</a> Bioinformatics. 26, 971-973 (2010).</li><li>Schroeder, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+RIN:+an+RNA+integrity+number+for+assigning+integrity+values+to+RNA+measurements.">The RIN: an RNA integrity number for assigning integrity values to RNA measurements.</a> BMC Mol Biol. 7 (3), (2006).</li><li>Mokry, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+double+fragmentation+ChIP-seq+provides+nucleotide+resolution+protein-DNA+binding+profiles.">Efficient double fragmentation ChIP-seq provides nucleotide resolution protein-DNA binding profiles.</a> PLoS One. 5 (11), e15092 (2010).</li><li>Park, P. 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伊利诺伊大学从辉瑞公司ZME收到调查发起的赠款和YCZ从这笔款项收到薪水的支持。

在本文中，我们描述了从与E2处理MCF-7细胞生成和RNA测序，芯片起和代谢组学数据的综合分析。我们制定了一套运筹帷幄，利用最有效的方法和用户友好的软商品即产生生物相关的发现协议。据我们所知，我们是从三个不同的分析和ERα在乳腺癌细胞直接调节确定的新的代谢途径整合组学数据的第一项研究。 转录 关键是要开始与高品质的RNA的转录成功分析。好的做法将消除RNA降解的可能性，包括创建无RNase长椅，经常改变手套，在-80存储℃并在使用中保持在冰上。在-20ºC过夜培养的加入提高了RNA回收。为了避免冻融RNA反反复复，可以分配给下游再修改的的的RNA在分离和纯化上课。对测序错误的存在，定量RT-PCR，使用该cDNA从样品中被经常执行以验证RNA测序鉴定的差异表达的基因。因此，足够的RNA应保留用于此目的。我们进行配对末端测序，使我们能够分析剪接变体，非编码RNA和微RNA。为了避免高测序成本，单末端测序也可被执行。相比微阵列，RNA测序更灵敏，并不限定于设置在芯片上的探针。我们得到不仅对编码序列，而且还对非编码基因和差异剪接转录信息。目前，我们正在使用这种技术来分析从小鼠实验中获得的配体的治疗以及组织后，各种细胞系的转录。 Cistromics 超高通量测序方法需要图书馆建设最少5纳克DNA芯片。在标准通道IP-SEQ协议18，建议有至少10纳克。定量用荧光是至关重要的。在这个协议中，我们使用的双链DNA检测分析以确定DNA浓度。人们也可以使用荧光计测定精确DNA定量。对于设备和材料的不同，每个实验室建立自己的超声协议，产生理想的DNA片段的大小是非常重要的。过度剪切DNA芯片可能会导致目标DNA的富集的损失，同时大尺寸芯片DNA创造高效的排序问题。该DNA提交测序之前，它始终是重要通过qPCR来验证一些已知区域的富集。该技术仍然被用于免疫沉淀的抗体的效率和特异性的限制。为了获得能够在比较分析可靠地使用结合位点的信息，我们验证了我们以下ENCODE的ChIP-SEQ标准试剂，19和使用STANDARdized协议为我们提供统一的数据集。再加上后期转录因子结合位点富集分析，芯片起仍然是最好的方法之一，以确定直接结合的转录因子染色质。目前，我们正在使用该协议招聘激酶，共调节因子和其他转录因子的研究在其他细胞系以及雌激素反应小鼠组织染色质。 代谢组学 在人类乳腺癌细胞成功代谢轮廓，仔细的样品制备是至关重要的。在这个协议中使用和细胞培养条件的细胞的量是最佳的MCF7细胞。其它细胞系，可能需要检测的代谢物的一个显著数目或多或少的细胞。我们获得围绕在每个实验中100代谢物。该方法只限于检测那些以低浓度存在短暂的代谢物或代谢物。这种方法提供了方便的USI纳克单个样品制备，以识别多个代谢物。我们目前正在使用这种技术，以确定在血清和小鼠不同组织中的雌激素调节的代谢物。 数据集成 众多转录和cistromics研究，完成调查雌激素信号和基因调节在乳腺癌细胞，包括MCF7细胞2,5-7,20。在这项研究中，在第一次，我们增加代谢数据，并试图推断是直接由ERα在配体除了调节代谢网络。这种新方法使我们能够通过针的雌激素影响细胞代谢调控分子点设计电路。总的来说，我们的研究结果支持ERα的作用，乳腺癌生物学的主要调控。 总之，我们的雌激素治疗和INTEGR后提供协议的详细汇编用于产生RNA测序，从乳腺癌细胞芯片起和代谢样品从这些组学的方法所获得的数据的ative分析。这些协议将使研究人员能够对其他转录因子，核受体配体和营养条件做类似的分析。

雌激素是两个女性和男性包括生殖组织，组织代谢，脑和骨1许多生理过程的重要调节。除了在这些组织中的有益作用，雌激素也驱动，从乳腺和生殖组织出现的癌症。雌激素主要是合作，通过减排额来诱导细胞类型特异性作用。通过使用芯片起RNA测序和全基因组ERαDNA结合位点分析ERα调节成绩单深度测序被证明是有用的了解ERα是如何工作的，从他们的出现的不同的组织和癌症。我们和其他人发表不同的受体（ERαVSERβ）相关基因表达谱2,3，不同的配体3-5和不同的共调节因子-2,4,6,7-。 RNA测序的主要方法检测转录，提供更高的精度和效率相比，芯片巴SED基因表达分析8。从细胞系2-4,7，组织或肿瘤样品获得的RNA进行测序，映射到可用的基因组组件和差异调节的基因被鉴定出来。染色质免疫沉淀（ChIP）被用来解剖转录因子和coregulator染色质结合已知调节结合位点。芯片起（芯片后跟高通量测序）提供全球性的结合位点的检测偏见。代谢组学是另一个越来越多地采用系统生物学的方法，它定量措施，生命系统的各种刺激，包括化学和遗传扰动的动态响应多参数。 通过进行全局代谢轮廓，功能性读出可以从细胞，组织和血液中获得。此外，从转录实验信息并不总是反映在有助于生化途径的酶的水平的实际变化。综合转录和代谢分析数据，使我们能够识别并关联与实际代谢物的改变的基因表达的变化。利用从所有这些大型数据集提供的机械细节的信息，了解调控复杂生物系统，特别是那些涉及到人类发展和疾病，如癌症和糖尿病的转录因子的作用。 哺乳动物基因组的复杂性使得它具有挑战性的整合并充分解释从转录，顺反组和代谢组实验中获得的数据。识别功能的结合事件，这将导致的变化，因为一旦功能性结合位点识别的目标基因的表达是重要的;随后的分析，包括转录结合（TF）的基序分析，可以以更高的精度来执行。这导致生物学上有意义的TF级联和机制的鉴定。也可直接comparis上的RNA测序和芯片起实验并不总是可能的，因为从每个实验的数据具有不同的尺度和噪音，在某些情况下，有意义的信号是由人口噪声掩盖。我们不知道，集成这三个独立而又相关的方法信息，了解乳腺癌ERα由直接代谢调控任何研究的。因此，我们在本文的总体目标是生产关系的结合事件的基因表达和代谢物的变化。为了实现这一目标，我们从RNA测序，芯片起和代谢实验的综合数据，并确定那些诱导的ERα的雌激素结合，这将导致在代谢途径基因表达的变化的事件。这是第一次，我们提供了一套完整的生成芯片起，RNA测序和代谢组学分析和数据进行综合分析方案（ 图1）来发现新的基因调节电路乳房的新陈代谢癌细胞系。

<table><tbody><tr><td>Triglyceride Mix</td><td>Sigma</td><td>17810-1AMP-S</td><td></td></tr><tr><td>Oleic acid&amp;nbsp;</td><td>Sigma</td><td>O1257-10MG</td><td>Oleic Acid-Water Soluble powder&amp;nbsp;</td></tr><tr><td>TRIzol Reagent</td><td>Life technologies&amp;nbsp;</td><td>15596-026</td><td></td></tr><tr><td>Dynabeads Protein A</td><td>Life technologies&amp;nbsp;</td><td>10006D</td><td></td></tr><tr><td>Dynabeads Protein G</td><td>Life technologies&amp;nbsp;</td><td>10007D</td><td></td></tr><tr><td>QIAquick PCR Purification Kit</td><td>QIAGEN</td><td>28106</td><td>DNA isolation of input sample&amp;nbsp;</td></tr><tr><td>ZYMO ChIP-DNA Isolation kit&amp;nbsp;</td><td>Zymo Research&amp;nbsp;</td><td>D5205</td><td>ChIP DNA Clean &amp;amp; Concentrator</td></tr><tr><td>Quant-iT&amp;ordf; PicoGreen&amp;uml; dsDNA Assay Kit</td><td>Invitrogen&amp;nbsp;</td><td>P7589</td><td>DNA Quantitation</td></tr><tr><td>RNase-Free DNase Set</td><td>QIAGEN</td><td>79254</td><td>RNA purification</td></tr><tr><td>DEPC Treated Water&amp;nbsp;</td><td>LIFE TECH</td><td>462224</td><td>Dnase/Rnase Free</td></tr><tr><td>Model 120 Sonic Dismembrator</td><td>Fisher Scientific&amp;nbsp;</td><td>FB120110</td><td>With 1/8 probe&amp;nbsp;</td></tr><tr><td>&amp;nbsp;Fisher Scientific Sound Enclosure</td><td>Fisher Scientific&amp;nbsp;</td><td>FB-432-A</td><td>For sound reduction</td></tr><tr><td>&amp;nbsp;Eppendorf Tubes 5.0 ml</td><td>Eppendorf&amp;nbsp;</td><td>0030 119.401</td><td>200 tubes (2 bags of 100 ea.)</td></tr><tr><td>Random Primer Mix&amp;nbsp;</td><td>NEB</td><td>S1330S</td><td></td></tr><tr><td>DNTP MIX</td><td>NEB</td><td>N0447S</td><td></td></tr><tr><td>M-MuLV Reverse Transcriptase</td><td>NEB</td><td>M0253S</td><td></td></tr><tr><td>FastStart SYBR Green Master</td><td>ROCHE</td><td>4673484001</td><td></td></tr><tr><td>Agarose</td><td>Fisher&amp;nbsp;</td><td>BP160100</td><td>1.5% agarose gel&amp;nbsp;</td></tr><tr><td>Qubit RNA HS Assay Kit</td><td>Life technologies&amp;nbsp;</td><td>Q32852</td><td>RNA quantification (100 assays)&amp;nbsp;</td></tr><tr><td>Formaldehyde</td><td>Sigma</td><td>F8775</td><td></td></tr><tr><td>Protease Inhibitor tablets&amp;nbsp;</td><td>Roche&amp;nbsp;</td><td>4693116001</td><td>Each tablet is sufficient for 10 ml lysis buffer</td></tr><tr><td>PhosSTOP Phosphatase Inhibitor Cocktail Tablets</td><td>Roche&amp;nbsp;</td><td>4906845001</td><td>Each tablet is sufficient for 10 ml lysis buffer</td></tr><tr><td>Qubit Assay Kits</td><td>Life technologies&amp;nbsp;</td><td>Q32850</td><td>For 100 assays</td></tr><tr><td>Automated cell counter</td><td>ORFLO</td><td>MXZ000</td><td></td></tr><tr><td>tube Rotator&amp;nbsp;</td><td>VWR</td><td>10136-084</td><td></td></tr><tr><td>Victor X5 Multilple plate reader&amp;nbsp;</td><td>PerkinElmer</td><td>2030-0050</td><td></td></tr><tr><td>Microcentrifuge 5417R</td><td>Eppendorf</td><td>22621807</td><td></td></tr><tr><td>Magnetic Stand</td><td>Diagenode</td><td>B04000001&amp;nbsp;</td><td></td></tr><tr><td>Fast Real-time PCR system</td><td>Applied Science&amp;nbsp;</td><td>4351405</td><td></td></tr></tbody></table>

systems biology of metabolic regulation by estrogen receptor signaling in breast cancer

随着组学的出现接近我们的慢性疾病，如癌症和代谢综合征的认识有所提高。但是，这是由基因表达微阵列，深度测序实验或代谢分析获得大规模数据集的信息的有效开采是必不可少的发现，然后有效的病变靶细胞表型的关键调节因子。雌激素受体α（ERα）​​是调节这对于雌激素反应乳腺癌重要的基因程序的主转录因子中的一个。为了理解到的ERα在乳腺癌代谢信令作用我们从雌二醇处理的MCF-7细胞利用转录，cistromic和代谢数据。在这份报告中，我们描述了一代样品进行RNA测序，芯片起和代谢组学实验和获得的数据综合计算分析。这种方法是在描绘小说摩尔有用丘拉尔机制和基因调节电路是由一个特定的转录因子调节的正常或患病细胞的这影响代谢。

转录 分析差异表达的基因由E2治疗，我们选择了要执行的RNA测序实验。除了提供关于mRNA水平的信息，RNA测序数据也可被用来监测在非编码RNA（长的非编码RNA，微小RNA）和选择性剪接事件的变化。我们没有提供的非编码RNA或备选转录基因的分析信息，因为我们的研究范围是确定蛋白质编码基因是对于代谢调节很重要的。然而，RNA-Seq中获得的差异表达基因事件的信息的一个很好的方式。 为了获得高质量的读出，它获得高纯度的RNA是重要的。分离RNA后，我们使用纯化RNA一个清理工具包中的RNA。不幸的是近50％的RNA的在此过程中会丢失，起始RNA的量应采取到c中onsideration以获得由纯化步骤结束时所需的量（100-500纳克）。 我们量化使用荧光计的RNA HS测定，这是非常准确的，是低丰度RNA的定量优选的方法中的RNA产量。接着，将RNA样品的完整性和整体质量使用bioanlayzer，这是使用RNA最小量的凝胶电泳一个可行的替代进行了检查。分析表明所有样品都18S和28S个rRNA的锋利和清洁带指示样本（图2B）的完整性和纯度。 RNA完整性（RIN）用作RNA的质量控制标准。 RIN 10表示完整的RNA，RIN 6部分降解和RIN3强烈降低16。 它建议RIN为至少7用于测序实验。我们所有的RNA样品收到9.6-9.9之间RIN值&lt;STRONG&gt;（图2A）。测序文库从高质量的RNA为500ng采用超高通量测序系统构成从每个样品如Illumina公司2500平均1800万的高品质的测序读取每个样品（表1），这是准备用于下行得到分析。要检查高通量序列原始数据的整体素质，FastQC对每个样品的读取运行。一辆车RNA测序数据的代表性FastQC报告显示（ 图3）。最读取序列长度为约100bp的（ 图3A）。对于每个读，质量得分是32-34的前5 bp和36-38来自6-100bp（ 图3B）。其中17990863读取此样品产生，其中大部分存在质量得分高于34（ 图3C）。在序列中每读GC含量也跟着，平均48％GC的正态分布。整体质量的读取成绩是非常高的。 ERα的结合位点的Cistromic分析 为了实现高效的深度测序，在所希望的尺寸获得的DNA片段是其中的重要因素之一。当前测序方法可能对DNA片段有不同的要求，例如300-600碱基对用于超高通量测序系统，100-200碱基对AB /固17。在该实验的大部分DNA片段为300 bp和600 bp的作为凝胶所示（图4）之间。作为替代方案，读者可能会选择使用MNase消化编写自己的染色质，但是我们觉得我们既定的协议提供高品质要求的样品，并提供可重复的结果。从每个处理的DNA和其对应的输入样本的十纳克在EB缓冲液制备。该芯片是DNA单读取使用超高通量测序系统测序。 DNA芯片样品产生约20000000读取而输入的DNA产生多个35.000,000读取（表1）。 代谢组学 以用GCMS量化在哺乳动物细胞中的代谢物，细胞质量的至少10微克是必需的。我们开始40万MCF7细胞与实验。通过收获的时候，大约有500,000个细胞在每块板。由峰表示的代谢物的概述进行控制和雌二醇处理的样品（图5）之间进行比较。约100代谢物进行鉴定，其计数在哺乳动物已知总数的60％。这里，我们提出的顶部10的代谢物，其显示在E2治疗（表2）显著变化的列表。总体而言，E2上调途径，包括果糖和甘露糖代谢，组氨酸代谢，嘌呤代谢紊乱，胆固醇的生物合成和维生素B3的新陈代谢。该下调的途径是丁metabolis男，脂肪酸从头生物合成，戊糖磷酸途径，尿素循环以及精氨酸代谢（ 表3）。 数据分析和整合 从RNA测序，芯片起和GC-MS的数据进行处理和分析，通过一系列步骤（图6）。在RNA测序和芯片起鉴定的基因之间的比较之后，一组的上调和由雌二醇下调的基因的一组中提取。从代谢数据，我们通过雌二醇处理得到两套上调和下调的化合物。下一步，我们使用Metscape，对于Cytoscape的应用程式，以可视化和解释人体代谢15的上下文中的基因表达和代谢的数据。首先，我们选择了包括在网络中的化合物的元素，反应，酶和基因。我们选择的化合物/基因作为查询和E2上调（ 图7A）的两个网络和向下-regulation（ 图7B）被示出。该Metscape还提供了选择性途径构建网络的选项。例如，使用相同的一组数据，作为构建下调整体网络，通过从化合物/基因的具体途径切换查询生成雄激素的雌激素的生物合成和代谢途径的子网。有显著改变的基因或化合物在亮的颜色得到强调。除了选择从所描述的下拉窗口的特定途径，还可以通过施加"创建子网"功能所选区域创建子网络。如在图6A和B，ERα活化和直接结合到基因调控区示出影响在乳腺癌细胞中许多代谢途径。我们的综合分析表明，ERα直接结合并调节FASN基因，它是脂肪酸从头合成途径重要的表达（Figure 7C）。另外E2治疗通过这些途径抑制CYP4Z1，CYP2E1，CYP4B1，CYP2C8，TPO，GSTA4和GSTM2表达下调雄激素雌激素的生物合成途径。 <img alt="图1" src="/files/ftp_upload/53832/53832fig1.jpg" /> 图1:制剂的RNA，DNA样本和细胞裂解物的用于RNA测序，芯片起，和代谢组分析的工作流程。 <img alt="图2" src="/files/ftp_upload/53832/53832fig2.jpg" /> 图2:与安捷伦2100生物分析仪 RNA完整性（RIN），RNA的质量指标分析RNA的代表电泳 ，被分析为9.9给出。 （ 一 ）在RNA的一个虚拟的凝胶图片样张26S和18S的两个频段是雪亮的。 （B）的叔单个跟踪的荧光信号他的RNA显示，28S峰的强度大于18S高峰，无污染看到。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53832/53832fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53832/53832fig3.jpg" /> 图3:在RNA测序原始数据的代表性质量检查其中的车辆样本的序列数据，使用FastQC分析。 （A），它是100碱基对的文库的序列长度分布。 （ 二 ）质量分数沿读取的位置。 （ 三 ）每个序列质量评分显示所有序列的通用高品质。 （D）相匹配的理论分布每次读取GC含量。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53832/53832fig3large.jpg" target="_blank">请点击此处查看拉</a>rger版本这个数字。 <img alt="图4" src="/files/ftp_upload/53832/53832fig4.jpg" /> 图4:超声处理的染色质片段的凝胶电泳将5μl各样品的细胞裂解物在具有1000 bp的加梯在第一车道1.5％琼脂糖凝胶上运行。大多数碎片是300和BP之间600 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53832/53832fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/53832/53832fig5.jpg" /> 图5:表示从由GC-MS代谢物峰的概况的上面板显示，同时从控制下面板E2类处理的样品中确定的峰。封闭单间是在该地区以显示每个P代表的确切代谢产物<html> EAK。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53832/53832fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/53832/53832fig6.jpg" /> 图6:综合聚类方法的流程图。 <img alt="图7" src="/files/ftp_upload/53832/53832fig7.jpg" /> 图7:在Metscape综合基因-代谢物数据的可视化 （A）的途径上调E2的网络。 （B）的途径的网络由E2下调。 （℃）E2刺激ERα招募FASN附近的区域，该区域被发现，通过施加子网函数产生的脂肪酸从头合成网络中来调节。3832fig7large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="表格1" src="/files/ftp_upload/53832/53832table1.jpg" /> 表1:RNA 测序和芯片起的超高通量测序的总结。 <img alt="表2" src="/files/ftp_upload/53832/53832table2.jpg" /> 表2: 排名前10位的代谢产物与E2治疗显著的更改列表。 <img alt="表3" src="/files/ftp_upload/53832/53832table3.jpg" /> 表3:途径上调和下调由E2治疗。

Watch this Scientific Journal Video about Systems Biology of Metabolic Regulation by Estrogen Receptor Signaling in Breast Cancer at JoVE.com

Systems Biology of Metabolic Regulation by Estrogen Receptor Signaling in Breast Cancer

乳腺癌代谢调控的系统生物学雌激素受体信号

Integration of data from genome-wide sequencing experiments and metabolomics experiments is a challenge. In this paper we report, for the first time, generation, analysis and integration of transcriptome, cistrome and metabolome data from breast cancer cells treated with estradiol.

With the advent of the -omics approaches our understanding of the chronic diseases like cancer and metabolic syndrome has improved. However, effective ...

systems-biology-metabolic-regulation-estrogen-receptor-signaling

Nanyang Technological University

Research

JoVE Journal

Medicine

10.3K 次观看.  University of Illinois, Urbana-Champaign. Integration of data from genome-wide sequencing experiments and metabolomics experiments is a challenge. In this paper we report, for the first time, generation, analysis and integration of transcriptome, cistrome and metabolome data from breast cancer cells treated with estradiol. 

视频: Systems Biology of Metabolic Regulation by Estrogen Receptor Signaling in Breast Cancer

An In Vivo Estrogen Deficiency Mouse Model for Screening Exogenous Estrogen Treatments of Cardiovascular Dysfunction After Menopause

Postmenopausal women are at greater risk of developing cardiovascular diseases than premenopausal women. Female mice ovariectomized (OVX) at weaning display increased atherosclerotic lesions in the aorta compared with female mice with intact ovarian function. However, laboratory models involving estrogen-deficient mice with atherosclerosis-prone status are lacking. This deficit is crucial because clinical estrogen deficiency in menopausal women may aggravate the incidence of pre-existing or ongoing lipid disruption and atherosclerosis. In this study, we establish an in vivo estrogen-deficient mouse model by bilateral ovariectomy via a double dorsal-lateral incision in apolipoprotein E (apoE)-/- mice. We then compare the effects of 17&#946;-estradiol and pseudoprotodioscin (PPD) (a phytoestrogen) perorally administered via hazelnut spread. We find that although PPD exerts some effect on reducing final body weight and plasma TG in OVX apoE-/- mice, it has anti-atherosclerotic and cardiac-protective capacities comparable with its 17&#946;-estradiol counterpart. PPD is a phytoestrogen that has been reported to exert anti-tumor properties. Thus, the proposed method is applicable for screening phytoestrogens via peroral administration to substitute for traditional hormone replacement therapy in postmenopausal women, which has been reported to have potentially deleterious tumorigenetic capacity. Peroral administration via hazelnut spread is noninvasive, rendering it widely applicable to many patients. This article contains step-by-step demonstrations of bilateral ovariectomy via the double dorsal-lateral incision in apoE-/- mice and peroral 17&#946;-estradiol or phytoestrogen hormone replacement via hazelnut spread. Plasma lipid and cardiovascular function analyses using echocardiography follow.

Clinically, estrogen deficiency in menopausal women may aggravate the incidence of lipid disruption and atherosclerosis. We established an in vivo estrogen deficiency model by bilateral ovariectomy via a double dorsal-lateral incision in apoE-/- mice. The mouse model is applicable for screening exogenous estrogen treatments of cardiovascular dysfunction after menopause.

用于筛查绝经后心血管功能障碍的外源雌激素治疗的活体雌激素缺乏小鼠模型

Postmenopausal women are at greater risk of developing cardiovascular diseases than premenopausal women. Female mice ovariectomized (OVX) at weaning ...

科研

生物化学

Modeling Breast Cancer in Human Breast Tissue using a Microphysiological System

Breast cancer (BC) remains a leading cause of death for women. Despite more than $700 million invested in BC research annually, 97% of candidate BC drugs fail clinical trials. Therefore, new models are needed to improve our understanding of the disease. The NIH Microphysiological Systems (MPS) program was developed to improve the clinical translation of basic science discoveries and promising new therapeutic strategies. Here we present a method for generating MPS for breast cancers (BC-MPS). This model adapts a previously described approach of culturing primary human white adipose tissue (WAT) by sandwiching WAT between adipose-derived stem cell sheets (ASC)s. Novel aspects of our BC-MPS include seeding BC cells into non-diseased human breast tissue (HBT) containing native extracellular matrix, mature adipocytes, resident fibroblasts, and immune cells; and sandwiching the BC-HBT admixture between HBT-derived ASC sheets. The resulting BC-MPS is stable in culture ex vivo for at least 14 days. This model system contains multiple elements of the microenvironment that influence BC including adipocytes, stromal cells, immune cells, and the extracellular matrix. Thus BC-MPS can be used to study the interactions between BC and its microenvironment. 
We demonstrate the advantages of our BC-MPS by studying two BC behaviors known to influence cancer progression and metastasis: 1) BC motility and 2) BC-HBT metabolic crosstalk. While BC motility has previously been demonstrated using intravital imaging, BC-MPS allows for high-resolution time-lapse imaging using fluorescence microscopy over several days. Furthermore, while metabolic crosstalk was previously demonstrated using BC cells and murine pre-adipocytes differentiated into immature adipocytes, our BC-MPS model is the first system to demonstrate this crosstalk between primary human mammary adipocytes and BC cells in vitro.

This protocol describes the construction of an in vitro microphysiological system for studying breast cancer using primary human breast tissue with off the shelf materials.

使用微生理系统模拟人体乳房组织中的乳腺癌

Breast cancer (BC) remains a leading cause of death for women. Despite more than $700 million invested in BC research annually, 97% of candidate BC drugs ...

癌症研究

Screening for Phytoestrogens using a Cell-based Estrogen Receptor  &#946; Reporter Assay

Plants are a source of food for many animals, and&#160;they can produce thousands of chemicals. Some of these compounds affect physiological processes in the vertebrates that consume them, such as endocrine function. Phytoestrogens, the most well studied endocrine-active phytochemicals, directly interact with the hypothalamo-pituitary gonadal axis of the vertebrate endocrine system. Here we present the novel use of a cell-based assay to screen plant extracts for the presence of compounds that have estrogenic biological activity. This assay uses mammalian cells engineered to highly express estrogen receptor beta (ER&#946;) and that have been transfected with a luciferase gene. Exposure to compounds with estrogenic activity results in the cells producing light. This assay is a reliable and simple way to test for biological estrogenic activity. It has several improvements over transient transfection assays, most notably, ease of use, the stability of the cells, and the sensitivity of the assay.

We have optimized a commercially available estrogen receptor &#946; reporter assay for screening human and nonhuman primate foods for estrogenic activity. We validated this assay by showing that the known estrogenic human food soy registers high, while other foods show no activity.

使用基于细胞的雌激素受体对植物雌激素进行筛查β记者分析

Plants are a source of food for many animals, and&#160;they can produce thousands of chemicals. Some of these compounds affect physiological processes in ...

Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen receptors (ERs), ER&#945;, and ER&#946;. ER&#945; is frequently upregulated in breast cancer and drives the proliferation of breast cancer cells. The ERs function as transcription factors and regulate gene expression. Whereas ER&#945;&#39;s regulation of protein-coding genes is well established, its regulation of noncoding microRNA (miRNA) is less explored. miRNAs play a major role in the post-transcriptional regulation of genes, inhibiting their translation or degrading their mRNA. miRNAs can function as oncogenes or tumor suppressors&#160;and are also promising biomarkers. Among the miRNA assays available, microarray and quantitative real-time polymerase chain reaction (qPCR) have been extensively used to detect and quantify miRNA levels. To identify miRNAs regulated by estrogen signaling in breast cancer, their expression in ER&#945;-positive breast cancer cell lines were compared before and after estrogen-activation using both the &#181;Paraflo-microfluidic microarrays and Dual Labeled Probes-low density arrays. Results were validated using specific qPCR assays, applying both Cyanine dye-based and Dual Labeled Probes-based chemistry. Furthermore, a time-point assay was used to identify regulations over time. Advantages of the miRNA assay approach used in this study is that it enables a fast screening of mature miRNA regulations in numerous samples, even with limited sample amounts. The layout, including the specific conditions for cell culture and estrogen treatment, biological and technical replicates, and large-scale screening followed by in-depth confirmations using separate techniques, ensures a robust detection of miRNA regulations,&#160;and eliminates false positives and other artifacts. However, mutated or unknown miRNAs, or regulations at the primary and precursor transcript level, will not be detected. The method presented here represents a thorough investigation of estrogen-mediated miRNA regulation.

Molecular signaling through both estrogen and microRNAs are critical in breast cancer development and growth. Estrogen activates the estrogen receptors, which are transcription factors. Many transcription factors can regulate the expression of microRNAs, and estrogen-regulated microRNAs can be profiled using different large-scale techniques.

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 ...

医学

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the actual molecules towards which the cells migrate remain elusive. This modified invasion assay has been developed to identify targets which drive cell migration and invasion. This technique compares the invasion index under three conditions to determine whether a specific hormone, growth factor, or cytokine plays a role in mediating the invasive potential of a cancer cell. These conditions include i) normal fetal bovine serum (FBS), ii) charcoal-stripped FBS (CS-FBS), which removes hormones, growth factors, and cytokines and iii) CS-FBS + molecule (denoted &#8220;X&#8221;). A significant change in cell invasion with CS-FBS as compared to FBS, indicates the involvement of hormones, cytokines or growth factors in mediating the change. Individual molecules can then be added back to CS-FBS to assay their ability to reverse or rescue the invasion phenotype. Furthermore, two or more factors can be combined to evaluate the additive or synergistic effects of multiple molecules in driving or inhibiting invasion. Overall, this method enables the investigator to determine whether hormones, cytokines, and/or growth factors play a role in cell invasion by serving as chemoattractants or inhibitors of invasion for a particular type of cancer cell or a specific mutant. By identifying specific chemoattractants and inhibitors, this modified invasion assay may help to elucidate signaling pathways that direct cancer cell invasion.

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

This video article describes a modified in vitro method to examine the role of hormones, cytokines and/or growth factors in driving cancer cell invasion.

一种改进<em&gt;在体外</em&gt;入侵检测，以确定激素，在调节肿瘤细胞侵袭的细胞因子和/或生长因子的潜在作用

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the ...

An Ex vivo Model to Study Hormone Action in the Human Breast

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial cells tend to lose hormone receptor expression. Widely used hormone receptor positive breast cancer cell lines are of limited relevance to the in vivo situation. Here, we describe an ex vivo model to study hormone action in the human breast. Fresh human breast tissue specimens from surgical discard material such as reduction mammoplasties or mammectomies are mechanically and enzymatically digested to obtain tissue fragments containing ducts and lobules and multiple stromal cell types. These tissue microstructures kept in basal medium without growth factors preserve their intercellular contacts, the tissue architecture, and remain hormone responsive for several days. They are readily processed for RNA and protein extraction, histological analysis or stored in freezing medium. Fluorescence activated cell sorting (FACS) can be used to enrich for specific cell populations. This protocol provides a straightforward, standard approach for translational studies with highly complex, varied human specimens.

An Ex vivo Model to Study Hormone Action in the Human Breast

We have developed a novel ex vivo model to study hormone action in the human breast. It is based on tissue microstructures isolated from surgical breast tissue specimens which preserve tissue architecture, intercellular interactions, and paracrine signaling.

一个<em&gt;体外</em&gt;模型在乳腺癌研究激素的作用

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial ...

Using 16&#945;-[18F]-Fluoro-17&#946;-Estradiol PET to Visualize Estrogen Receptor &#945; Expression in Human Breast Cancer Xenografts in Female Ovariectomized Mice

To demonstrate how estrogen receptor alpha (ER&#945;) positive breast cancer xenografts may be visualized in BALB/c nude mice using 16&#945;-[18F]-fluoro-17&#946;-estradiol (18F-FES) positron emission tomography (PET), ovariectomized BALB/c nude mice were injected with ER&#945;-positive breast cancer cells (MCF-7, 3 &#215; 106 cells; shoulder [n = 10] or 4th inguinal mammary fat pad [n = 10]) or ER&#945;-negative breast cancer cells (MDA-MB-231, 1 &#215; 106 cells; mammary fat pad [n = 5]). Mice harboring MCF-7 cells received subcutaneous injections of 20 &#181;g of 17&#946;-estradiol (20 &#181;g/20 &#181;L; corn oil:ethanol, 9:1) in the nape of their necks 2 days prior to cell injection, followed by daily injections five times per week for 5 weeks. Tumor volumes were measured according to the formula: (L*W2)/2 (L; length, W; width). Once tumor volumes reached approximately 100 mm3, 17&#946;-estradiol injections were halted 2 days prior to mice receiving 18F-FES for PET imaging to avoid competitive binding with ER&#945;. Upon 18F-FES administration via the lateral tail vein, PET/MRI was performed for 15 min at 1 h to 1.5 h post-injection. 18F-FES uptake was not observed in ER&#945;-negative, MDA-MB-231 tumor-bearing mice. 18F-FES uptake was most pronounced in mice harboring MCF-7 tumors in the shoulder. In MCF-7 tumors grown in the inguinal mammary fat pad, 18F-FES uptake was less visible, as the intestinal excretion pattern of 18F-FES obscured the radioactivity detectable in these tumors. To use 18F-FES PET as a tool to visualize ER&#945; expression in ER&#945;-positive breast xenografts, we demonstrate that the visibility of 18F-FES uptake is clear in tumors located away from the abdominal region of mice, such as in the shoulder.

Here, we demonstrate a protocol to use 16&#945;-[18F]-fluoro-17&#946;-estradiol (18F-FES) positron emission tomography (PET) as a tool to visualize ER&#945; expression in ER&#945;-positive breast xenografts.

使用 16α-[18F]-氟-17β-雌二醇 PET 可视化雌性卵巢切除小鼠人乳腺癌异种移植物中雌激素受体α表达

To demonstrate how estrogen receptor alpha (ER&#945;) positive breast cancer xenografts may be visualized in BALB/c nude mice using ...

红景天苷抑制 MCF-7 细胞迁移和增殖的机制

Exploring the Pharmacological Action and Molecular Mechanism of Salidroside in Inhibiting MCF-7 Cell Proliferation and Migration

Salidroside (Sal) contains anti-carcinogenic, anti-hypoxic, and anti-inflammatory pharmacological activities. However, its underlying anti-breast cancer mechanisms have been only incompletely elucidated. Hence, this protocol intended to decode the potential of Sal in regulating the PI3K-AKT-HIF-1&#945;-FoxO1 pathway in the malignant proliferation of human breast cancer MCF-7 cells. First, the pharmacological activity of Sal against MCF-7 was evaluated by CCK-8 and cell scratch assays. Moreover, the resistance of MCF-7 cells was measured by migration and Matrigel invasion assays. For cell apoptosis and cycle assays, MCF-7 cells were processed in steps with annexin V-FITC/PI and cell cycle-staining detection kits for flow cytometry analyses, respectively. The levels of reactive oxygen species (ROS) and Ca2+ were examined by DCFH-DA and Fluo-4 AM immunofluorescence staining. The activities of Na+-K+-ATPase and Ca2+-ATPase were determined using the corresponding commercial kits. The protein and gene expression levels in apoptosis and the PI3K-AKT-HIF-1&#945;-FoxO1 pathway were further determined using western blot and qRT-PCR analyses, respectively. We found that Sal treatment significantly restricted the proliferation, migration, and invasion of MCF-7 cells with dose-dependent effects. Meanwhile, Sal administration also dramatically forced MCF-7 cells to undergo apoptosis and cell cycle arrest. The immunofluorescence tests showed that Sal observably stimulated ROS and Ca2+ production in MCF-7 cells. Further data confirmed that Sal promoted the expression levels of pro-apoptotic proteins, Bax, Bim, cleaved caspase-9/7/3, and their corresponding genes. Consistently, Sal intervention prominently reduced the expression of the Bcl-2, p-PI3K/PI3K, p-AKT/AKT, mTOR, HIF-1&#945;, and FoxO1 proteins and their corresponding genes. In conclusion, Sal can be used as a potential herb-derived compound for treating breast cancer, as it may reduce the malignant proliferation, migration, and invasion of MCF-7 cells by inhibiting the PI3K-AKT-HIF-1&#945;-FoxO1 pathway.

作者聚焦：探索红景天苷在乳腺癌治疗中的分子机制 

The present protocol describesa comprehensive strategy for evaluating the pharmacological action and mechanism of salidroside in inhibiting MCF-7 cell proliferation and migration.

探究红景天苷抑制MCF-7细胞增殖和迁移的药理作用及分子机制

Salidroside (Sal) contains anti-carcinogenic, anti-hypoxic, and anti-inflammatory pharmacological activities. However, its underlying anti-breast cancer ...

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探究红景天苷抑制MCF-7细胞增殖和迁移的药理作用及分子机制

探究红景天苷抑制MCF-7细胞增殖和迁移的药理作用及分子机制

探究红景天苷抑制MCF-7细胞增殖和迁移的药理作用及分子机制