合成mRNA转染后肿瘤细胞迁移和侵袭的实时定量测量
Summary
许多上调的基因刺激肿瘤细胞迁移和侵袭，导致预后不良。确定哪些基因调节肿瘤细胞迁移和侵袭至关重要。该协议提出了一种实时研究基因表达增加对肿瘤细胞迁移和侵袭的影响的方法。
Abstract
肿瘤细胞具有高度的运动性和侵袭性，并显示出基因表达模式的改变。了解基因表达的变化如何调节肿瘤细胞迁移和侵袭对于了解肿瘤细胞浸润到邻近健康组织和转移的机制至关重要。以前，已经证明基因敲低，然后基于阻抗的肿瘤细胞迁移和侵袭实时测量能够识别肿瘤细胞迁移和侵袭所需的基因。最近，针对 SARS-CoV-2 的 mRNA 疫苗增加了人们对使用合成 mRNA 进行治疗的兴趣。本文对合成mRNA方法进行了修正，研究了基因过表达对肿瘤细胞迁移和侵袭的影响。这项研究表明，通过合成 mRNA 转染和基于阻抗的实时测量提高基因表达可能有助于识别刺激肿瘤细胞迁移和侵袭的基因。本方法论文提供了有关检查基因表达改变对肿瘤细胞迁移和侵袭影响的程序的重要细节。
Introduction
肿瘤细胞运动在转移中起着至关重要的作用 1,2。肿瘤细胞扩散到邻近和遥远的健康组织使癌症治疗变得困难，并导致复发3,4。因此，了解肿瘤细胞运动的机制并制定相关的治疗策略至关重要。由于许多肿瘤细胞改变了基因表达谱，因此了解基因表达谱的哪些变化导致肿瘤细胞运动性改变至关重要 5,6。
已经开发了几种测定法来测量 体外细胞迁移。由于只允许在特定时间点进行测量，一些检测仅提供有限的信息，而另一些则实时提供有关肿瘤细胞运动的全面信息7。尽管这些细胞运动测定中的许多可以在给定时间或终点提供定量结果，但它们无法提供有关实验期间细胞迁移速率动态变化的足够详细的信息。此外，根据实验设计、细胞类型和细胞数量，可能很难检查细胞迁移速率的潜在变化。此外，可以通过传统运动测定的简单定量来研究简单处理的效果，但可能需要更复杂的定量来研究各种联合处理的复杂效果8。
已经开发出一种用于监测覆盖有微电极的微量滴定板孔底电流的仪器9。细胞与孔表面的粘附阻碍了电子流动，阻抗与细胞的定量和定性结合相关。孔底存在微电极，可以测量细胞粘附、扩散和增殖。微电极存在于上腔室的微孔膜下方，允许测量细胞迁移和侵袭到下腔室，上腔室涂有细胞外基质 （ECM） 蛋白以允许侵袭10。
此前，已经证明，基于阻抗的肿瘤细胞迁移和侵袭的实时测量在整个实验过程中提供了实时数据，以及在各种实验条件下的即时比较和量化11。在该方法论文中，诱导基因敲低以测试目标蛋白在肿瘤细胞迁移和侵袭中的作用。由于在测试的实验条件下，使用小干扰 RNA （siRNA） 8 电穿孔后需要 3-4 天才能完全实现基因敲低效应，因此在电穿孔后重新接种细胞，并在 3 天后重新收获，以进行基于阻抗的肿瘤细胞迁移和侵袭实时测量。
激酶 （Crk） 和 Crk 样 （CrkL） 的 CT10 调节因子是介导各种生长因子受体激酶通路和非受体酪氨酸激酶通路下游的蛋白质-蛋白质相互作用的接头蛋白12。Crk 和 CrkL 蛋白水平升高会导致几种人类癌症（包括胶质母细胞瘤13）的预后不良。然而，目前尚不清楚 Crk 和 CrkL 蛋白升高如何导致预后不良。因此，确定 Crk 和 CrkL 过表达对肿瘤细胞功能的影响非常重要。此前，进行了一项基因敲低研究，以证明胶质母细胞瘤细胞迁移和侵袭需要内源水平的 Crk 和 CrkL 蛋白8。在这里，已经开发了一种改进的测定系统来解决 Crk 和 CrkL 过表达对肿瘤细胞迁移和侵袭的影响。
最近，由于针对 SARS-CoV-2 的 mRNA 疫苗的开发，mRNA 的体外合成及其治疗应用再次受到关注（由 Verbeke 等人审查 14）。此外，在癌症和其他疾病中使用合成mRNA方面也取得了显着进展15,16。细胞电穿孔是递送合成 mRNA 和诱导瞬时基因修饰的有效方法（Campillo-Davo 等人 17 评论），使用合成 mRNA 可在永生化成纤维细胞中快速有效地表达基因18。本方法论文将使用合成mRNA的基因过表达与实时细胞分析相结合，以研究肿瘤细胞迁移和侵袭。然而，用于 siRNA 的实验方案不适用于合成 mRNA 转染，因为外源蛋白的水平在合成 mRNA 转染时迅速增加并逐渐降低18。因此，该方法已被修改为在转染后立即进行细胞迁移和侵袭的实时分析，而无需额外培养细胞。
本方法论文表明，将基于阻抗的实时测量与用合成mRNA转染肿瘤细胞相结合，可以快速、全面地分析基因上调对肿瘤细胞迁移和侵袭的影响。本方法论文描述了测量胶质母细胞瘤细胞迁移和侵袭如何受 Crk 和 CrkL 过表达影响的详细程序。通过研究合成mRNA对肿瘤细胞迁移的浓度依赖性影响，该论文清楚地描述了蛋白质水平的增加如何刺激肿瘤细胞迁移。此外，还提出了一种改变ECM凝胶浓度的方法，以评估基因表达变化对肿瘤细胞侵袭的影响。
Protocol
1. mRNA的合成 注意：对于mRNA合成，所有试剂和设备必须在使用前经过特殊处理以灭活RNase。有关本协议中使用的所有材料、仪器和试剂的详细信息，请参阅 材料表 。 DNA的线性化注：将 CrkI 和 CrkL 的小鼠 cDNA 克隆到 pFLAG-CMV-5a 表达载体中，以在 C 末端添加 FLAG 表位标签，并将亚克隆到 pcDNA3.1/myc-His 载体中以掺入 T7 启动子?…
Representative Results
Crk 和 CrkL 蛋白在许多细胞类型的运动中起着重要作用，包括神经元22、T 细胞23、成纤维细胞 18,19 和多种肿瘤细胞13。由于已报道 Crk 和 CrkL 蛋白在胶质母细胞瘤24,25,26 中升高，因此本研究研究了 CrkI（Crk 的剪接变体）过表达对胶质母细胞…
Discussion
迁移和侵袭是肿瘤细胞的重要特征。测量肿瘤细胞的运动性并了解控制肿瘤细胞运动的潜在机制为治疗干预提供了重要的见解2,27。已经开发了几种方法来研究细胞迁移7.使用划痕或培养插入物的伤口愈合测定是一种简单且常用的方法，可提供间隙闭合的对比图像。单个细胞追踪测定需要通过延时成像监测单个细胞，为此可以用荧光染?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
作者感谢堪萨斯城儿童慈善中心的医学写作中心编辑了这份手稿。这项工作得到了娜塔莉的 A.R.T. 基金会（对 TP）的支持，并得到了儿童慈善医院 （CMH） 和堪萨斯大学癌症中心 （KUCC） 的 MCA 合作伙伴咨询委员会资助（对 TP）。
Materials
| AlphaImager HP | ProteinSimple | 92-13823-00 | Agarose gel imaging system |
| α-Tubulin antibody | Sigma | T9026 | Used to detect α-tubulin protein (dilution 1:3,000) |
| CIM-plate 16 | Agilent Technologies, Inc | 5665825001 | Cell invasion and migration plates |
| Crk antibody | BD Biosciences | 610035 | Used to detect CrkI and CrkII proteins (dilution 1:1,500) |
| CrkL antibody | Santa Cruz | sc-319 | Used to detect CrkL protein (dilution 1:1,500) |
| Dulbecco’s Modified Eagle’s Medium (DMEM) | ATCC | 302002 | Cell culture medium |
| Dulbecco's phosphate-buffered saline (DPBS) | Corning | 21-031-CV | Buffer used to wash cells |
| Fetal bovine serum (FBS) | Hyclone | SH30910.03 | Culture medium supplement |
| Heracell VIOS 160i CO2 incubator | Thermo Scientific | 51030285 | CO2 incubator |
| IRDye 800CW goat anti-mouse IgG secondary antibody | Li-Cor | 926-32210 | Secondary antibody for Western blot analysis (dilution 1:10,000) |
| IRDye 800CW goat anti-rabbit IgG secondary antibody | Li-Cor | 926-32211 | Secondary antibody for Western blot analysis (dilution 1:10,000) |
| Lithium chloride | Invitrogen | AM9480 | Used for RNA precipitation |
| Matrigel matrix | Corning | 354234 | Extracellular matrix (ECM) gel |
| MEGAscript T7 transcription kit | Invitrogen | AM1334 | Used for RNA synthesis |
| Millennium RNA markers | Invitrogen | AM7150 | Used for formaldehyde agarose gel electrophoresis |
| Mini centrifuge | ISC BioExpress | C1301P-ISC | Used to spin down cells |
| Mouse brain QUICK-Clone cDNA | TaKaRa | 637301 | Source of genes (inserts) for cloning |
| NanoQuant | Tecan | M200PRO | Nucleic acid quantification system |
| Neon electroporation system | ThermoFisher Scientific | MPK5000 | Electroporation system1 |
| Neon transfection system 10 µL kit | ThermoFisher Scientific | MPK1025 | Electroporation kit |
| Neon transfection system 100 µL kit | ThermoFisher Scientific | MPK10096 | Electroporation kit |
| NorthernMax denaturing gel buffer | Invitrogen | AM8676 | Used for formaldehyde agarose gel electrophoresis |
| NorthernMax formaldehyde load dye | Invitrogen | AM8552 | Used for formaldehyde agarose gel electrophoresis |
| NorthernMax running buffer | Invitrogen | AM8671 | Used for formaldehyde agarose gel electrophoresis |
| Nuclease-free water | Teknova | W3331 | Used for various reactions during mRNA synthesis |
| Odyssey CLx Imager | Li-Cor | Imager for Western blot analysis | |
| pcDNA3.1/myc-His | Invitrogen | V80020 | The vector into which inserts (mouse CrkI and CrkL cDNAs) were cloned |
| pFLAG-CMV-5a | Millipore Sigma | E7523 | Source of the FLAG epitope tag |
| Phenol:chloroform:isoamyl alcohol | Sigma | P2069 | Used for DNA extraction |
| PmeI | New England BioLabs | R0560L | Used to linearize the plasmids for mRNA synthesis |
| Poly(A) tailing kit | Invitrogen | AM1350 | Used for poly(A) tail reaction |
| Polystyrene tissue culture dish (100 x 20 mm style) | Corning | 353003 | Used for culturing cells before transfection |
| Polystyrene tissue culture dish (35 x 10 mm style) | Corning | 353001 | Used for culturing transfected cells |
| Proteinase K | Invitrogen | 25530049 | Used to remove protein in the reaction mixture |
| Purifier Axiom Class II, Type C1 | Labconco Corporation | 304410001 | Biosafety cabinet for sterile handling of cells |
| Resuspension Buffer R | ThermoFisher Scientific | A buffer included in the electroporation kits, MPK1025 and MPK10096. The buffer is used to resupend cells before electroporation, and its composition is proprietary information. | |
| RNaseZap | Invitrogen | AM9780 | RNA decontamination solution |
| Scepter | Millipore | C85360 | Handheld automated cell counter |
| ScriptCap 2'-O-methyltransferase kit | Cellscript | C-SCMT0625 | Used for capping reaction |
| ScriptCap m7G capping system | Cellscript | C-SCCE0625 | Used for capping reaction |
| Sodium dodecyl sulfate solution | Invitrogen | 15553-035 | Detergent used for the proteinase K reaction |
| Sorvall Legend XT centrifuge | Thermo Scientific | 75004532 | Benchtop centrifuge to spin down cells |
| Trypsin-EDTA | Gibco | 25300-054 | Used for dissociation of cells |
| U-118MG | ATCC | HTB15 | An adherent cell line derived from a human glioblastoma patient |
| Vinculin antibody | Sigma | V9131 | Used to detect vinculin protein (dilution 1:100,000) |
| xCELLigence RTCA DP | Agilent Technologies, Inc | 380601050 | Instrument used for real-time cell analysis |
| 1Electroporation parameters and other related information for various cell lines are available on the manufacturer's homepage (https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/neon-transfection-system/neon-transfection-system-cell-line-data.html?). |
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Citer Cet Article
Park, T., Large, N. Real-Time Quantitative Measurement of Tumor Cell Migration and Invasion Following Synthetic mRNA Transfection. J. Vis. Exp. (196), e64274, doi:10.3791/64274 (2023).