The authors would like to acknowledge Wensheng Liu for invaluable help with cell imaging. This work was supported by grants from the&#160;US National Institutes of Health (R01 CA182467), American Cancer Society (RSG-09-252-01-RMC), Lynn Sage Foundation, and A Sister&#8217;s Hope Foundation.

EMT诱导1细胞培养注:EMT可以通过治疗TGFβ或转录因子扭，蜗牛，或ZEB1 / 2在上皮细胞异常表达所诱导。另外，在本协议是通过扭ER融合蛋白在人永生化乳腺上皮细胞（HMLE /扭ER，J杨博士的礼物，加州大学圣地亚哥分校）9,11,29表达可诱导的EMT系统。当4-羟基他莫昔（TAM）的治疗中，融合蛋白的扭ER转位到细胞核中以驱动转录并产生一个完整的EMT的过渡在12-14天9,11,29。额外的EMT诱导系统，如HMLE /蜗牛-ER，HMLE / TGF-β，和MCF10A / TGF-β，可在我们以前的出版物11,30中找到。 <ol><li>在37℃的培养箱中，用5％的CO 2保持HMLE /扭ER的细胞在无血清乳腺上皮细胞生长培养基（MEGM）。通过细胞每2-3天，当他们达到80％汇合。孵育的细胞用0.15％胰蛋白酶在37℃下进行5-10分钟，然后失活胰蛋白酶，用5％小牛血清/ DMEM中。降速细胞和悬浮他们MEGM。板的细胞在新的组织培养皿中，在1〜4的比例。 </li><li>谭溶解在乙醇，使200微米的解决方案。从光存储保护TAM在-20℃。使用前，刚加谭成MEGM媒体在1:10,000稀释，使20纳米谭终浓度。 </li><li>对于诱导EMT的，板材1.6×10 6 HMLE /扭ER细胞变成10厘米的组织培养皿，一式两份。培养细胞与20纳米含谭MEGM媒体。 </li><li>板1.6×10 6个HMLE /扭ER细胞分化成一式两份的10cm平皿和治疗的细胞用0.01％的乙醇作为非TAM处理的控制。 </li><li>孵化两天后，拿在光学显微镜下的照片在放大10倍来记录细胞的形态变化。 </li><li>从控制或TAM治疗的一盘吸出培养基细胞。由报废一半的板块中的RNA裂解缓冲液中分离RNA并在RIPA缓冲液，另一半用于蛋白质分析洗涤细胞用冷的PBS中，并收获细胞。 </li><li>传代细胞，从在一式两份的10cm平皿的控制或TAM处理的菜肴由再镀1.6×10 6个细胞。连续处理的细胞用20nM的TAM或车辆。 </li><li>重复步骤5到7隔日一次，直到第14天，在此期间，TAM处理的扭ER的细胞表现出纺锤形的间充质的形态，而载体处理的对照细胞维持鹅卵石状上皮形态。 </li></ol> EMT的表征2。 注:完成EMT是通过表明:（1）细胞从鹅卵石样上皮细胞形态为梭形纤维母细胞样外观形态变化; （2）E-cadherin的定位在细胞与细胞连接处的损失;的EMT标志物和（3）转换表达式，由损耗定义上皮标记和间质标记物的增益。 细胞的形态变化是通过光学显微镜在EMT诱导的时间过程中放大10倍拍摄的，在本节中描述了第一节免疫荧光检测E-钙粘着蛋白在细胞结的说明。的EMT标志物的表达通过免疫印迹法检测。常见的上皮标记为E-钙粘蛋白，γ-catenin及occludin的，和间质标记物包括纤维连接蛋白，N-钙粘蛋白和波形。免疫印迹法的一般过程在第4节中描述。 <ol><li>谭处理，种子1×10 5细胞在12毫米的玻璃圆形盖玻片置于24孔板的孔的第12天。 </li><li>播种，吸中，洗涤细胞，预热的PBS后48小时。 </li><li>固定的细胞用预热的4％多聚甲醛固定15分钟。 </li><li>用PBS洗涤细胞三次。 </li><li>孵育的细胞用0.2％的Triton X-100的PBS中的15分在室温。 </li><li>用PBS洗涤细胞三次。 </li><li>孵育细胞在5％BSA / PBS进行1小时，在室温到阻断非特异性结合。 </li><li>孵育细胞与第一抗体的E-钙粘蛋白在1:50稀释1％BSA / PBS在湿盒在o / n 4℃。注:范围为初级抗体的稀释通常是1:50〜1:200，并为每个抗体的最佳稀释度应通过试验确定。 </li><li>滗出第一抗体并用PBS洗涤细胞三次。 </li><li>孵育在1细胞用荧光标记的二抗:500稀释为1小时，在室温。从在此步骤中，保护细胞免受光。 </li><li>滗二次抗体和洗三次，用PBS。 </li><li>染色的细胞用0.1〜1克/毫升的DAPI或Hoechst的10分钟。 </li><li>漂洗细胞，用PBS三次。 </li><li>摩盖玻片用一滴安装介质，并密封盖玻片指甲油。 </li><li>观察细胞，并采取à63X荧光显微镜下的图像。 </li></ol>用另外的抗体染色，可以执行确认EMT表型。例如，N-cadherin和α-平滑肌肌动蛋白可染的骨髓间充质表型29,31,32。细胞骨架结构的重组可以通过与鬼笔环肽的F-肌动蛋白11染色细胞进行监控。此外，测定对细胞运动性和细胞死亡的电阻可以被执行以检查EMT 11的属性。 利用定量RT-PCR检测3剪接异构体的检测<ol><li>引物设计 注:引物对，应精心设计，放大不同的剪接异构体。外显子跳跃，最常观察到的选择性剪接事件，在此示为一个例子来说明引物设计的策略。 图1A示出了四个外显子的mRNA前体，与所述第三外显子的可变外显子。纳入外显子3的结果生产更长的剪接异构体，而跳过外显子3的产生较短的剪接异构体。引物需要被设计以区分外显子3，包括同种型和外显子3跳过的同种型，并检测该mRNA的总转录物。 <ol><li>用于检测外显子包含的，精心设计的可变外显子3，并在附近的组成型外显子4的内侧的反向引物内的正向引物，允许可变外显子包含同种型的特异性扩增。确保不设计正向和反向的相同变量的外显子中的引物，以避免从基因组DNA或mRNA前体的PCR扩增产物。 </li><li>特异性扩增外显子跳跃事件，设计的横跨构外显子2和外显子4，当可变外显子3被包括，否则将被破坏的交界处的引物之一。设计构成的外显子4，这样里面的另一个引物，PCR产物生成，只有当可变外显子3 exclud埃德和本构外显子2和外显子4随后结合在一起。 </li><li>以检测该基因的总转录物，设计中的两个构个外显子1和2的引物，因此，PCR产物是从所有亚型扩增。 </li><li>确保熔化温度（Tm）为所有的引物是约55℃时，各引物的长度是约18-22个核苷酸，PCR产物的大小为基础对介于80和150。 </li></ol></li><li> RNA提取:提取的RNA使用总RNA提取试剂盒（见表材料）。 <ol><li>收集细胞350μL的RNA裂解液。 </li><li>加入350微升70％乙醇的裂解物并充分混合。 </li><li>将样品转移到RNA的纯化塔。 </li><li>离心机以10,000 xg离心1分钟，丢弃流过。 </li><li>用RNA洗涤缓冲液洗二柱曾用500微升的RNA洗涤缓冲液I和的两倍。 </li><li>去除残留的RNA洗涤缓冲液II通过离心从在最大速度下的柱2分钟。 </li><li>柱转移至新的1.5毫升试管中，加入50μl的无核酸酶的水在柱基质的中心，并通过离心分离以最大速度洗脱RNA。 </li></ol></li><li>第一链cDNA的合成<ol><li>确定浓度和RNA的质量用紫外吸收测量。注:质量好的RNA应该有大约2.0 260纳米/ 280纳米的吸收率。 </li><li>设置逆转录酶（RT）反应包含250ng的总RNA，0.05微克随机六聚体引物，50皮摩尔的MgCl 2，10皮摩尔的dNTP，2微升5×GoScript缓冲液中，加入1μl逆转录酶在20微升的最终体积。 </li><li>进行RT反应在42℃进行1小时，接着温育在70℃下5分钟，以灭活逆转录酶。 </li></ol></li><li>实时荧光定量PCR <ol><li>成立20微升反应，由10微升的SYBR Green预混液，0.1-1.0微升cDNA模板，和5皮摩尔的正向和反向引物。 </li><li>运行实时PCR用下列步骤进行40个循环:95℃变性10秒，58℃退火10秒，和60℃延伸30秒。一式三份进行PCR反应。 </li><li>检查解离曲线，并确保具有单峰被观测到的每个引物组。 </li><li>通过计算扩增曲线的二阶导数的值获得的量化周期（Cq的）值。 </li><li>相比，使用"增量增量Cq的（ΔΔCq）&#39;所示的由下述式方法的未诱导的样品计算诱导样品中的靶mRNA的相对量（RQ）。使用管家基因，如GAPDH或TBP，作为参考。 </li></ol></li></ol><img alt="式（1）" fo:content-width="3in" src="/files/ftp_upload/51845/51845eq1.jpg" /> 为了更准确地计算剪接异构体的相对值，叔他使​​用多个参考基因应该被考虑。结果，能使用由Pfaffl 等人 33,34所述的改性绝对值量化方法进行分析。 在蛋白质水平考试4剪接异构体的<ol><li>收集细胞在100μl冰冷的RIPA缓冲液。 </li><li>设置在冰上裂解物为约10分钟。 </li><li>离心机以14,000 xg离心10分钟，在4℃，并收集上清液。 </li><li>测定蛋白浓度用Bradford检测。 </li><li>稀释在SDS上样缓冲液的蛋白质样品并装载20-40微克蛋白质在10％SDS-PAGE凝胶。 </li><li>跑SDS-PAGE凝胶在20mA 1.5小时。 </li><li>根据靶蛋白的分子量转移蛋白至PVDF膜在湿的传送系统，在4℃下进行3小时，在85 V.调整传输时间。 </li><li>块膜在5％脱脂乳进行30分钟至1小时，在室温下。 </li><li>孵育膜无线TH一抗，4℃CO / N。的范围为初级抗体的稀释通常是在1:200〜1:5000，确定每个抗体实验的最佳稀释度。在这个协议中使用的抗体的稀释可以在"表特定的试剂和设备"中找到。 <ol><li>为了检测剪接异构体，使用识别特定亚型的特异性抗体。另外，使用该识别出组成型外显子编码区的抗原决定簇的抗体，同时检测剪接异构体根据其不同的蛋白质的大小。 </li><li>在同一时间，通过免疫印迹对EMT标志物监测EMT。使用E-钙粘蛋白，γ-catenin及occludin的上皮标记和纤维连接蛋白，N-钙粘蛋白和波形间充质标记。 </li></ol></li><li>洗膜三次，用TBS-T（50毫摩尔Tris，150 mM氯化钠，0.05％吐温20，pH 7.4）中，在5分钟的时间间隔。 </li><li>孵育膜与HRP偶联的二级蚂蚁ibody以1:10,000稀释为1小时，在室温。 </li><li>洗膜3次用TBS-T以5分钟的间隔。 </li><li>可视化蛋白用化学发光检测系统和所述膜暴露于放射自显影胶片。 </li></ol>

<ol>
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The authors declare that they have no competing financial interests.

这里描述的方法使得能够选择性剪接的检测中可诱导的EMT模型。如剪接同种型的表达，例如，动态改变可以在整个EMT的时间过程被捕获。这种方法比使用不同的上皮 - 间质或，代表细胞系的选择性剪接的比较优势，因为从联合国有关的细胞不同的遗传背景，可能不适当地影响选择性剪接。然而，成功诱导的EMT必须谨慎使用上的变化剪接任何结论之前，各种EMT的标志，可以得出证实。此外，除了此处所描述的扭ER-诱导的EMT的系统，其它的EMT系统，如那些诱导蜗牛-ER或TGFβ被推荐用于实验发现11,30的验证。 可在RNA和蛋白水平进行检测剪接异构体的EMT过程中的切换。拼接异形式特异性抗体优选用于蛋白质分析。当同种型特异性抗体是不可用时，可以根据它们在SDS-PAGE分子量通过免疫印迹来确定剪接同种型的蛋白质水平。每个异构体的RNA水平可以通过同种型特异性引物进行定量。使用定量RT-PCR测定，每个异构体的变化倍数可以是灵敏和精确地确定。特别是，当实验材料是有限的，如分析在临床标本中的特定剪接同种型的表达，实时定量RT-PCR分析提供了将弥补缺乏亚型特异性的抗体进行免疫组织化学的定量量度。 这里所提到的方法是适合的已知的剪接同种型的变化EMT过程中的检测。此外，这种方法可以扩展为全基因组选择性剪接的研究和对新的剪接同工型开关EMT过程中的识别。为了实现这一点，使用大规模的技术，如RNA深测序或拼接敏感的微阵列平台的，可以使用的RNA从上述诱导EMT模型分离进行。然而，对同种型变化的qRT-PCR验证需要以下任何大规模分析。 总之，可变剪接是动态的EMT，这一过程是在胚胎发育和肿瘤转移的关键过程中调节。考虑到在人类基因组中的选择性剪接的频率高，则很可能的可变剪接的调节起着许多其他生物和病理过程，包括细胞分化，组织发育和程序性细胞死亡35-41中起重要作用。本文所描述的剪接异构体的检测的协议将适用于这些其他系统也是如此。 附加的实验方式，诸如剪接记者小基因测定法，可以直接使用Ë用于进一步研究的顺式作用元件和控制剪接30,42,43反式作用因子的调控机制。此外，EMT过程中的特定剪接同种型的功能性作用可通过沉默或异位表达的特定同种型和监测的EMT诱导中所描述的EMT诱导系统11进行调查。

上皮 - 间质转化（EMT）是推动胚胎发育过程中的器官形态和组织重塑一个发展计划。当异常激活，EMT促进肿瘤转移和器官纤维化1,2。 EMT过程中引人注目的研究已经描述转录调控的重要性，通过几个转录因子，如扭曲，蜗牛和ZEB，这抑制了粘着连接蛋白的E-钙粘蛋白的表达，从而导致cobble-的损失定义石样上皮形态学和纺锤状的间充质表型3-8的增益。通过RNA的全基因组分析，最近的研究表明，存在一组基因的剪接方式有两种上皮或间质表型9,10相关联的。从我们的实验室工作功能连接选择性剪接和EMT。通过研究细胞表面粘附分子CD44，我们证明了CD44 ALTERNative剪接EMT过程中紧密调节，并且更重要的是，CD44剪接同种型转换因果有助于EMT 11。 选择性剪接表示基因调控的一种普遍的和保守的模型，如高达95％的人的多外显子基因可变剪接12-14。通过从单个基因产生多个蛋白的产品，选择性剪接构成蛋白多样性的重要机制，增加了另一层复杂性，以人类基因组。因此，选择性剪接的失调可能会导致引起人类疾病的深刻的生物学效应。事实上，在疾病的异常剪接都记载了十多年15-25，其中包括在基因编码的剪接突变机械骨髓增生异常综合征26-28通常发现最近的调查结果。因此，开发可靠的方法的选择性剪接í检测soforms是在不同的生物过程，包括EMT的研究非常重要的。 在这里，我们提供了一个协议来检测使用可诱导的EMT模型的变化剪接。该方法用于设计PCR引物和检测剪接异构体是合适的，不仅用于选择性剪接的EMT过程中的研究，而且对选择性剪接在其他生物过程的研究。 EMT过程中调查剪接是必须为了更好地理解EMT和肿瘤转移的机制，从而促进有效的策略的开发来治疗癌症转移。

<table><tbody><tr><td>4-hydroxytamoxifen</td><td>Sigma</td><td>H7904</td><td>Make stock solution by dissolving 4-hydroxytamoxifen in ethanol to 200 &amp;#956;M. Keep the solution at -20 &amp;#176;C protected from light.</td></tr><tr><td>E.Z.N.A. Total RNA Isolation kit</td><td>Omega Bio-Tek</td><td>R6731</td><td>Total RNA isolation kit</td></tr><tr><td>GoScript Reverse Transcription System</td><td>Promega</td><td>A5001</td><td>Reagent for qRT-PCR assay</td></tr><tr><td>GoTaq qPCR Master Mix</td><td>Promega</td><td>A6002</td><td>Reagent for qRT-PCR assay</td></tr><tr><td>LightCycler 480 Real-Time PCR System</td><td>Roche</td><td></td><td>Equipment for qRT-PCR assay</td></tr><tr><td>CD44 antibody</td><td>R&amp;amp;D Systems</td><td>BBA10</td><td>1:1,000 dilution</td></tr><tr><td>E-cadherin antibody</td><td>Cell Signaling Technology</td><td>4065</td><td>1:2,500 dilution for immunoblotting; 1:50 dilution for immunofluorescence</td></tr><tr><td>&amp;gamma;-Catenin antibody</td><td>Cell Signaling Technology</td><td>2309</td><td>1:1,000 dilution</td></tr><tr><td>Occludin antibody</td><td>Santa Cruz Biotechnology Inc.</td><td>sc-5562</td><td>1:500 dilution</td></tr><tr><td>Fibronectin antibody</td><td>BD Transduction Laboratories</td><td>610077</td><td>1:5,000 dilution</td></tr><tr><td>N-cadherin antibody</td><td>BD Transduction Laboratories</td><td>610920</td><td>1:2,000 dilution</td></tr><tr><td>Vimentin antibody</td><td>NeoMarkers</td><td>MS-129-p1</td><td>1:500 dilution</td></tr><tr><td>GAPDH antibody</td><td>Millipore Corporation</td><td>MAB374</td><td>1:10,000 dilution</td></tr><tr><td>Amasham ECL Western blotting detection reagent</td><td>GE Health Life Science</td><td>RPN2209</td><td>Chemiluminescence system</td></tr></tbody></table>

detection of alternative splicing during epithelial-mesenchymal transition

Alternative splicing plays a critical role in the epithelial-mesenchymal transition (EMT), an essential cellular program that occurs in various physiological and pathological processes. Here we describe a strategy to detect alternative splicing during EMT using an inducible EMT model by expressing the transcription repressor Twist. EMT is monitored by changes in cell morphology, loss of E-cadherin localization at cell-cell junctions, and the switched expression of EMT markers, such as loss of epithelial markers E-cadherin and &#947;-catenin and gain of mesenchymal markers N-cadherin and vimentin. Using isoform-specific primer sets, the alternative splicing of interested mRNAs are analyzed by quantitative RT-PCR. The production of corresponding protein isoforms is validated by immunoblotting assays. The method of detecting splice isoforms described here is also suitable for the study of alternative splicing in other biological processes.

上述过程提供了一个可靠的方法EMT过程中检测可变剪接。的CD44剪接同种型转换时扭诱导的EMT代表性的结果在下面给出的一个例子。 在HMLE /扭ER细胞扭诱导的EMT的特点是从鹅卵石状上皮细胞表型的细长成纤维细胞表型的转变（ 图2A），无上皮标志物E-钙粘蛋白，γ-catenin和occludin和中上调的间质标记物纤连蛋白，N-钙粘蛋白，波形蛋白（ 图2B）。此外，EMT通过E-钙粘蛋白定位在细胞-细胞连接（ 图2C）的损失评估。 的CD44剪接异构体的EMT过程中的切换表达进行了检查，定量RT-PCR和免疫印迹。人CD44基因包含9个变量外显子。 CD44选择性剪接使得细胞亲达斯CD44的变体（CD44V），包括至少一个可变的外显子，和CD44的标准（CD44s的）即不含所有可变外显子的图3A中示出的引物设计的各种CD44剪接异构体的检测的战略。对于外显子跳跃的产物CD44s的检测中，正向引物位于组成型外显子5，而反向引物跨越构外显子5和6之间的交界处对于CD44V剪接异构体含有V5和V6变量的检测外显子，正向和反向引物的V5和V6内的设计，分别。引物进行检测的其他CD44可变含有外显子 - 异构体可以使用相同的策略进行设计。此外，总的CD44转录物使用正向和反向位于组成型外显子2和外显子3，分别为（ 图3A）的引物进行检测。如图3B所示，定量RT-PCR分析利用这些引物组，表示在光盘显著减少44V mRNA和谭后处理中的扭ER表达HMLE细胞14天增加CD44s的表达。相反，CD44的总转录物的EMT（ 图3C）中保持不变。一致以定量RT-PCR的结果，CD44V的蛋白水平显着下降，而CD44s的蛋白质的表达被大大上调（ 图3D）。因此，CD44的主要同种型，从CD44V在EMT过程转移到CD44s的。 <img alt="图1" fo:content-width="3.5in" src="/files/ftp_upload/51845/51845fig1highres.jpg" width="600" /> 图1引物设计。示意图示出用于检测剪接异构体中的外显子跳跃模式引物的位置。灰色方块代表组成的外显子，橙色框表示可变外显子和细线连接框表示内含子。引物的位置是indicat编按箭头:黑色箭头表示的引物组，用于检测总mRNA，橙色箭头表示引物用于扩增外显子，包括同种型，和蓝色箭头表示引物，用于检测外显子跳跃的异构体。 <img alt="图2" fo:content-width="6in" src="/files/ftp_upload/51845/51845fig2highres.jpg" width="600" /> 图2诱导的EMT。 （一）相位对比图像（10X）表示HMLE /扭ER细胞形态学变化之前（未处理）和谭后治疗14天。（二）在HMLE /扭质细胞前EMT标志物免疫印迹分析（未处理）后谭治疗14天。当谭治疗，上皮标记E-钙粘蛋白，γ-catenin及occludin的表达下调，而间质纤维连接蛋白标志物，N-钙粘蛋白和波形蛋白表达上调。（三）免疫荧光图像（63X），表明E-cadherin的损失定位在细胞 - 细胞连接后TAM治疗14天。绿染色表明E-cadherin的，和DAPI染色（蓝色）表示核。比例尺= 10微米。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/51845/51845fig2highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图3" fo:content-width="5in" src="/files/ftp_upload/51845/51845fig3highres.jpg" width="500" /> 图3的EMT过程中切换的CD44剪接异构体。 （一）引物设计的CD44剪接异构体的检测。灰色方块代表组成的外显子，橙色框表示可变外显子和细线连接框表示内含子。引物位置是箭头所示:黑色箭头表示引物，用于检测CD44的总mRNA，橙色箭头表示引物用于扩增CD44v5-6和蓝色箭头表示的引物组对检测CD44s的（B，C）的 CD44同种型的使用引物EMT过程中的水平特异性地检测CD44V含有可变外显子V5和V6（V5 / 6）和CD44s的（B），和CD44的总mRNA的定量RT-PCR分析（C）的 。 mRNA在日间的相对表达水平14细胞进行归一化到对应的第0天的细胞中TAM治疗和非治疗组。误差棒表示SEM; N = 3（四）免疫印迹CD44亚型的过程中HMLE /扭ER细胞谭诱导的EMT分析。 E-钙粘蛋白和N-cadherin蛋白水平进行了监测，以确定EMT的地位。

Watch this Scientific Journal Video about Detection of Alternative Splicing During Epithelial-Mesenchymal Transition at JoVE.com

Detection of Alternative Splicing During Epithelial-Mesenchymal Transition

Alternative splicing regulation has been shown to contribute to the epithelial-mesenchymal transition (EMT), an essential cellular program in various physiological and pathological processes. Here we describe a method utilizing an inducible EMT model for the detection of alternative splicing during EMT.

检测选择性剪接的过程中上皮 - 间质转化

Alternative splicing plays a critical role in the epithelial-mesenchymal transition (EMT), an essential cellular program that occurs in various ...

detection-alternative-splicing-during-epithelial-mesenchymal

Universidad San Sebastian

Research

JoVE Journal

Biology

12.9K 次观看.  Northwestern University Feinberg School of Medicine. Alternative splicing regulation has been shown to contribute to the epithelial-mesenchymal transition (EMT), an essential cellular program in various physiological and pathological processes. Here we describe a method utilizing an inducible EMT model for the detection of alternative splicing during EMT.

视频: Detection of Alternative Splicing During Epithelial-Mesenchymal Transition

Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in In Vitro Cancer Models

Drug resistance remains a major problem in the treatment of cancer for both hematological malignancies and solid tumors. Intrinsic or acquired resistance can be caused by a range of mechanisms, including increased drug elimination, decreased drug uptake, drug inactivation and alterations of drug targets. Recent data showed that other than by well-known genetic (mutation, amplification) and epigenetic (DNA hypermethylation, histone post-translational modification) modifications, drug resistance mechanisms might also be regulated by splicing aberrations. This is a rapidly growing field of investigation that deserves future attention in order to plan more effective therapeutic approaches. The protocol described in this paper is aimed at investigating the impact of aberrant splicing on drug resistance in solid tumors and hematological malignancies. To this goal, we analyzed the transcriptomic profiles of several in vitro models through RNA-seq and established a qRT-PCR based method to validate candidate genes. In particular, we evaluated the differential splicing of DDX5 and PKM transcripts. The aberrant splicing detected by the computational tool MATS was validated in leukemic cells, showing that different DDX5 splice variants are expressed in the parental vs. resistant cells. In these cells, we also observed a higher PKM2/PKM1 ratio, which was not detected in the Panc-1 gemcitabine-resistant counterpart compared to parental Panc-1 cells, suggesting a different mechanism of drug-resistance induced by gemcitabine exposure.

Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in In Vitro Cancer Models

Here we describe a protocol aimed at investigating the impact of aberrant splicing on drug resistance in solid tumors and hematological malignancies. To this goal, we analyzed the transcriptomic profiles of parental and resistant in vitro models through RNA-seq and established a qRT-PCR based method to validate candidate genes.

利用RNA测序来检测新剪接变异体在相关耐药<em&gt;体外</em&gt;癌症模型

Drug resistance remains a major problem in the treatment of cancer for both hematological malignancies and solid tumors. Intrinsic or acquired resistance ...

科研

癌症研究

Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells

The processing of most eukaryotic RNAs is mediated by RNA Binding Proteins (RBPs) with modular configurations, including an RNA recognition module, which specifically binds the pre-mRNA target and an effector domain. Previously, we have taken advantage of the unique RNA binding mode of the PUF domain in human Pumilio 1 to generate a programmable RNA binding scaffold, which was used to engineer various artificial RBPs to manipulate RNA metabolism. Here, a detailed protocol is described to construct Engineered Splicing Factors (ESFs) that are specifically designed to modulate the alternative splicing of target genes. The protocol includes how to design and construct a customized PUF scaffold for a specific RNA target, how to construct an ESF expression plasmid by fusing a designer PUF domain and an effector domain, and how to use ESFs to manipulate the splicing of target genes. In the representative results of this method, we have also described the common assays of ESF activities using splicing reporters, the application of ESF in cultured human cells, and the subsequent effect of splicing changes. By following the detailed protocols in this report, it is possible to design and generate ESFs for the regulation of different types of Alternative Splicing (AS), providing a new strategy to study splicing regulation and the function of different splicing isoforms. Moreover, by fusing different functional domains with a designed PUF domain, researchers can engineer artificial factors that target specific RNAs to manipulate various steps of RNA processing.

This report describes a bioengineering method to design and construct novel Artificial Splicing Factors (ASFs) that specifically modulate the splicing of target genes in mammalian cells. This method can be further expanded to engineer various artificial factors to manipulate other aspects of RNA metabolism.

工程人为因素来特异性地操纵人细胞中的选择性剪接

The processing of most eukaryotic RNAs is mediated by RNA Binding Proteins (RBPs) with modular configurations, including an RNA recognition module, which ...

遗传学

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

Alternative splicing (AS) occurs in more than 90% of human genes. The expression pattern of an alternatively spliced exon is often regulated in a cell type-specific fashion. AS expression patterns are typically analyzed by RT-PCR and RNA-seq using RNA samples isolated from a population of cells. In situ examination of AS expression patterns for a particular biological structure can be carried out by RNA in situ hybridization (ISH) using exon-specific probes. However, this particular use of ISH has been limited because alternative exons are generally too short to design exon-specific probes. In this report, the use of BaseScope, a recently developed technology that employs short antisense oligonucleotides in RNA ISH, is described to analyze AS expression patterns in mouse brain sections. Exon 23a of neurofibromatosis type 1 (Nf1) is used as an example to illustrate that short exon-exon junction probes exhibit robust hybridization signals with high specificity in RNA ISH analysis on mouse brain sections. More importantly, signals detected with exon inclusion- and skipping-specific probes can be used to reliably calculate the percent spliced in values of Nf1 exon 23a expression in different anatomical areas of a mouse brain. The experimental protocol and calculation method for AS analysis are presented. The results indicate that BaseScope provides a powerful new tool to assess AS expression patterns in situ.

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

An in situ hybridization (ISH) protocol that uses short antisense oligonucleotides to detect alternative pre-mRNA splicing patterns in mouse brain sections is described.

RNA原位杂交法定量分析小鼠脑切片中的替代前 mRNA 拼接方法

Alternative splicing (AS) occurs in more than 90% of human genes. The expression pattern of an alternatively spliced exon is often regulated in a cell ...

Induction of Mesenchymal-Epithelial Transitions in Sarcoma Cells

Phenotypic plasticity refers to a phenomenon in which cells transiently gain traits of another lineage. During carcinoma progression, phenotypic plasticity drives invasion, dissemination and metastasis. Indeed, while most of the studies of phenotypic plasticity have been in the context of epithelial-derived carcinomas, it turns out sarcomas, which are mesenchymal in origin, also exhibit phenotypic plasticity, with a subset of sarcomas undergoing a phenomenon that resembles a mesenchymal-epithelial transition (MET). Here, we developed a method comprising the miR-200 family and grainyhead-like 2 (GRHL2) to mimic this MET-like phenomenon observed in sarcoma patient samples.We sequentially express GRHL2 and the miR-200 family using cell transduction and transfection, respectively, to better understand the molecular underpinnings of these phenotypic transitions in sarcoma cells. Sarcoma cells expressing miR-200s and GRHL2 demonstrated enhanced epithelial characteristics in cell morphology and alteration of epithelial and mesenchymal biomarkers. Future studies using these methods can be used to better understand the phenotypic consequences of MET-like processes on sarcoma cells, such as migration, invasion, metastatic propensity, and therapy resistance.

We present here a cell culture method for inducing mesenchymal-epithelial transitions (MET) in sarcoma cells based on combined ectopic expression of microRNA-200 family members and grainyhead-like 2 (GRHL2). This method is suitable for better understanding the biological impact of phenotypic plasticity on cancer aggressiveness and treatments.

在肉瘤细胞间质上皮转变的诱导

Phenotypic plasticity refers to a phenomenon in which cells transiently gain traits of another lineage. During carcinoma progression, phenotypic ...

发育生物学

Studying TGF-&#946; Signaling and TGF-&#946;-induced Epithelial-to-mesenchymal Transition in Breast Cancer and Normal Cells

Transforming growth factor-&#946; (TGF-&#946;) is a secreted multifunctional factor that plays a key role in intercellular communication. Perturbations of TGF-&#946; signaling can lead to breast cancer. TGF-&#946; elicits its effects on proliferation and differentiation via specific cell surface TGF-&#946; type I and type II receptors (i.e., T&#946;RI and T&#946;RII) that contain an intrinsic serine/threonine kinase domain. Upon TGF-&#946;-induced heteromeric complex formation, activated T&#946;RI elicits intracellular signaling by phosphorylating SMAD2 and SMAD3. These activated SMADs form heteromeric complexes with SMAD4 to regulate specific target genes, including plasminogen activation inhibitor 1 (PAI-1, encoded by the SERPINE1 gene). The induction of epithelial-to-mesenchymal transition (EMT) allows epithelial cancer cells at the primary site or during colonization at distant sites to gain an invasive phenotype and drive tumor progression. TGF-&#946; acts as a potent inducer of breast cancer invasion by driving EMT. Here, we describe systematic methods to investigate TGF-&#946; signaling and EMT responses using premalignant human MCF10A-RAS (M2) cells and mouse NMuMG epithelial cells as examples. We describe methods to determine TGF-&#946;-induced SMAD2 phosphorylation by Western blotting, SMAD3/SMAD4-dependent transcriptional activity using luciferase reporter activity and SERPINE1 target gene expression by quantitative real-time-polymerase chain reaction (qRT-PCR). In addition, methods are described to examine TGF-&#946;-induced EMT by measuring changes in morphology, epithelial and mesenchymal marker expression, filamentous actin staining and immunofluorescence staining of E-cadherin. Two selective small molecule TGF-&#946; receptor kinase inhibitors, GW788388 and SB431542, were used to block TGF-&#946;-induced SMAD2 phosphorylation, target genes and changes in EMT marker expression. Moreover, we describe the transdifferentiation of mesenchymal breast Py2T murine epithelial tumor cells into adipocytes. Methods to examine TGF-&#946;-induced signaling and EMT in breast cancer may contribute to new therapeutic approaches for breast cancer.

We describe a systematic workflow to investigate TGF-&#946; signaling and TGF-&#946;-induced EMT by studying the protein and gene expression involved in this signaling pathway. The methods include Western blotting, a luciferase reporter assay, qPCR, and immunofluorescence staining.

研究TGF-β信号和TGF-β引起的乳腺癌和正常细胞的上皮到中位过渡

Transforming growth factor-&#946; (TGF-&#946;) is a secreted multifunctional factor that plays a key role in intercellular communication. Perturbations of ...

Using the E1A Minigene Tool to Study mRNA Splicing Changes

mRNA processing involves multiple simultaneous steps to prepare mRNA for translation, such as 5&#180;capping, poly-A addition and splicing. Besides constitutive splicing, alternative mRNA splicing allows the expression of multifunctional proteins from one gene. As interactome studies are generally the first analysis for new or unknown proteins, the association of the bait protein with splicing factors is an indication that it can participate in mRNA splicing process, but to determine in what context or what genes are regulated is an empirical process. A good starting point to evaluate this function is using the classical minigene tool. Here we present the adenoviral E1A minigene usage for evaluating the alternative splicing changes after different cellular stress stimuli. We evaluated the splicing of E1A minigene in HEK293 stably overexpressing Nek4 protein after different stressing treatments. The protocol includes E1A minigene transfection, cell treatment, RNA extraction and cDNA synthesis, followed by PCR and gel analysis and quantification of the E1A spliced variants. The use of this simple and well-established method combined with specific treatments is a reliable starting point to shed light on cellular processes or what genes can be regulated by mRNA splicing.

This protocol presents a rapid and useful tool for evaluating the role of a protein with uncharacterized function in alternative splicing regulation after chemotherapeutic treatment.

使用 E1A 微型工具研究 mRNA 拼接更改

mRNA processing involves multiple simultaneous steps to prepare mRNA for translation, such as 5&#180;capping, poly-A addition and splicing. Besides ...

A Reporter Based Cellular Assay for Monitoring Splicing Efficiency

During gene expression, the vital step of pre-mRNA splicing involves accurate recognition of splice sites and efficient assembly of spliceosomal complexes to join exons and remove introns prior to cytoplasmic export of the mature mRNA. Splicing efficiency can be altered by the presence of mutations at splice sites, the influence of trans-acting splicing factors, or the activity of therapeutics. Here, we describe the protocol for a cellular assay that can be applied for monitoring the splicing efficiency of any given exon. The assay uses an adaptable plasmid encoded 3-exon/2-intron minigene reporter, which can be expressed in mammalian cells by transient transfection. Post-transfection, total cellular RNA is isolated, and the efficiency of exon splicing in the reporter mRNA is determined by either primer extension or semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). We describe how the impact of disease associated 5&#8242; splice-site mutations can be determined by introducing them in the reporter; and how the suppression of these mutations can be achieved by co-transfection with U1 small nuclear RNA (snRNA) construct carrying compensatory mutations in its 5&#8242; region that basepairs with the 5&#8242;-splice sites at exon-intron junctions in pre-mRNAs. Thus, the reporter can be used for the design of therapeutic U1 particles to improve recognition of mutant 5&#8242; splice-sites. Insertion of cis-acting&#160;regulatory sites, such as splicing enhancer or silencer sequences, into the reporter can also be used to examine the role of U1 snRNP in regulation mediated by a specific alternative splicing factor. Finally, reporter expressing cells can be incubated with small molecules to determine the effect of potential therapeutics on constitutive pre-mRNA splicing or on exons carrying mutant 5&#8242; splice sites. Overall, the reporter assay can be applied to monitor splicing efficiency in a variety of conditions to study fundamental splicing mechanisms and splicing-associated diseases.

This protocol describes a minigene reporter assay to monitor the impact of 5&#180;-splice site mutations on splicing and develops suppressor U1 snRNA for the rescue of mutation-induced splicing inhibition. The reporter and suppressor U1 snRNA constructs are expressed in HeLa cells, and splicing is analyzed by primer extension or RT-PCR.

一种基于报告基因的细胞检测，用于监测剪接效率

During gene expression, the vital step of pre-mRNA splicing involves accurate recognition of splice sites and efficient assembly of spliceosomal complexes ...

生物化学

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

诱导上皮和分析间质转化

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

生物学

Identification of Alternative Splicing and Polyadenylation in RNA-seq Data

As well as the typical analysis of RNA-Seq to measure differential gene expression (DGE) across experimental/biological conditions, RNA-seq data can also be utilized to explore other complex regulatory mechanisms at the exon level. Alternative splicing and polyadenylation play a crucial role in the functional diversity of a gene by generating different isoforms to regulate gene expression at the post-transcriptional level, and limiting analyses to the whole gene level can miss this important regulatory layer. Here, we demonstrate detailed step by step analyses for identification and visualization of differential exon and polyadenylation site usage across conditions, using Bioconductor and other packages and functions, including DEXSeq, diffSplice from the Limma package, and rMATS.

Alternative splicing (AS) and alternative polyadenylation (APA) expand the diversity of transcript isoforms and their products. Here, we describe bioinformatic protocols to analyze bulk RNA-seq and 3' end sequencing assays to detect and visualize AS and APA varying across experimental conditions.

RNA-seq数据中替代剪接和聚腺苷酸化的鉴定

As well as the typical analysis of RNA-Seq to measure differential gene expression (DGE) across experimental/biological conditions, RNA-seq data can also ...

RNA Splicing

Splicing is the process by which eukaryotic RNA is edited before its translation into protein. The RNA strand transcribed from eukaryotic DNA is called the primary transcript. The primary transcripts that become mRNAs are called precursor messenger RNAs (pre-mRNAs). Eukaryotic pre-mRNA contains alternating sequences of exons and introns. Exons are nucleotide sequences that code for proteins, whereas introns are the non-coding regions. In RNA splicing, introns are removed and exons are bonded together.
Splicing is Mediated by the Spliceosome
Splicing occurs in the nucleosome and is mediated by a complex of proteins and small RNAs called small nuclear ribonucleoproteins (snRNPs). snRNPs, together with other proteins, form the spliceosome, which recognizes specific nucleotide sequences at the ends of the exon and intron. First, it binds to a GU-containing sequence at the 5&#39; end of the intron and to a branch point sequence containing an A towards the 3&#39; end of the intron. In a number of carefully-orchestrated steps, other snRNPs then bring the branch point close to the 5&#39; splice site. Subsequently, a chemical reaction cleaves the 5&#39; end of the intron from its upstream exon and attaches it to the branch point, forming a loop called a lariat. To release the lariat, the AG-containing sequence of the intron near the 5&#39; end of the downstream exon reacts with the 3&#39; end of the upstream exon. This reaction patches the two exons together, concluding the splicing process.
Splicing Allows the Expression of Several Proteins from a Single Gene
Typically, exons are joined together in the order in which they appear in a gene. However, during alternative splicing, different combinations of exons in pre-mRNA are combined to form mature mRNA. This produces several different proteins from a single pre-mRNA transcript.
Different patterns of alternative splicing include exon skipping, alternative 5&#39; or 3&#39; splice sites, and intron retention. These patterns are guided by the length of exons or introns and the strength of the splicing signal at the splice sites. Because of this, exons that are shorter than other exons may be overlooked by the spliceosome and omitted from the mature mRNA. In contrast, introns that are significantly shorter than other introns may evade removal by the spliceosome and are retained in the mature mRNA. As a result, alternative splicing generates variants of mature mRNA that were copied from the same stretch of DNA. The RNA sequence variants produce different proteins with additional or fewer amino acids, shifts in the reading frame, or a premature stop codon. These protein isoforms have different biological properties, including function, cellular localization, and interaction with other proteins, thereby playing a vital role in tissue- and environment-specific gene expression.
Abnormal Splicing Can Cause Diseases
Errors in splicing can produce aberrant protein isoforms, which may contribute to diseases, including cancer. For instance, alternative splicing of the BCL2L1 gene generates a long and short protein isoform&#8212;BCL-XL and BCL-XS, respectively&#8212; through the use of alternative 5&#39; splice sites. The longer BCL-XL isoform promotes cell survival and is highly expressed in several types of cancers (e.g., blood, breast, and liver cancers). Expression of the short BCL-XS isoform that promotes cell death is suppressed in cancer.

RNA Splicing, Lariat and the Spliceososme

RNA 剪接

Splicing is the process by which eukaryotic RNA is edited before its translation into protein. The RNA strand transcribed from eukaryotic DNA is called ...