在肉瘤细胞间质上皮转变的诱导
Summary
我们在这里提出用于诱导基于微小RNA-200家族成员及grainyhead样2（GRHL2）的合并的异位表达肉瘤细胞间质上皮转换（MET）的细胞培养方法。这种方法适用于更好地理解可塑性的癌症侵袭性和治疗方法的生物学影响。
Abstract
表型可塑性是指一种现象，其中细胞中瞬时获得另一谱系的性状。在癌的进展，表型可塑性驱动入侵，传播和转移。事实上，虽然大多数表型可塑性的研究一直在上皮来源的癌的情况下，原来的肉瘤，这是起源于间质，也表现出表型可塑性，有肉瘤的一个子集进行一个类似于间充质现象上皮转化（MET）。在这里，我们分别开发了一种方法，其包括与miR-200家族和grainyhead样2（GRHL2）以模仿肉瘤患者samples.We观察GRHL2和所述miR-200家族使用细胞转导和转染顺序表达这种MET-状现象， ，以更好地理解肉瘤细胞表型，这些转变的分子基础。表达的miR-200和GRHL2肉瘤细胞显示增强的上皮characterist集成电路细胞形态和上皮和间充质标志物的改变。使用这些方法今后的研究可以被用来更好地了解肉瘤细胞MET状突起，如迁移，侵入，转移倾向，和治疗的抗性的表型的影响。
Introduction
可塑性是指细胞表型之间的可逆转变，并且通常分为两种类型，上皮 – 间质（EMT）的转变和间充质到上皮转变（MET）。此可塑性起着多细胞生物体，如发育和伤口愈合1的正常过程中起重要作用;然而，这些相同的途径和基因表达的方案也可导致疾病，如纤维化（评价的2，3，4）和癌转移（参考文献5，6，7审查，8）。期间转移，例如，EMT破坏细胞极性，细胞-细胞相互作用，并且促进侵袭9,10。总之，EMT贡献s至促进癌细胞的传播表型状态。此外，EMT也导致了驱动侵袭性表型的表型的其它改变，包括癌细胞代谢6的失调，药物抗性11,12的发展，增加的肿瘤引发能力13,14和宿主免疫逃避15的主机。
可塑性已经很好地研究了癌进展;然而，肉瘤还表现出表型可塑性。有趣的是，它看起来好像有些可塑性的癌相同的驱动程序也有助于肉瘤可塑性和侵略性。例如，从肉瘤患者的循环肿瘤细胞（CTC）已经显示出表达的EpCAM，是通常在上皮细胞上发现的16的细胞表面蛋白。阿迪倚重，250个软组织肉瘤样品被归类为上皮样或间充质样基于基因表达。在上皮样生物标记物标志患者比患者的间充质样生物标记物标志17预后较好。这与许多癌，在与患者多间充质样肿瘤18例相比，具有更多上皮样癌有更好的结果是一致的。
虽然一些肉瘤显示生物标记物和基因表达的途径与MET一致，此表型可塑性的分子基础仍然知之甚少。为了研究MET的机制和司机肉瘤我们开发使用两种特定的上皮细胞因子诱导MET的模型中，微小RNA（MIR）-200家庭和grainyhead样2（GRHL2）。与miR-200S是通过结合至MESSEN的3' 非编码区调节基因表达的小的非编码RNA家族蒙古包RNA并防止翻译成蛋白质。与miR-200家族包括两个子组的 – 一种含的miR-141和miR-200a中，和另一种包括的miR-200b中，的miR-200c中，和miR-429。所述miR-200家族的成员富含上皮组织，和miR-200S的损失与转移癌19相关联。与miR-200家族也下调在软组织肉瘤与正常组织相比20。类似的miR-200S，GRHL2是上皮发展21重要的关键调节。所述GRHL2转录因子作用于两种方式上调上皮基因，如E-钙粘蛋白：1）在上皮细胞，GRHL2直接阻遏EMT主调节器，ZEB1 22;和2）GRHL2直接激活上皮基因23的转录。我们以前的研究已显示，在肉瘤细胞的miR-200和GRHL2的组合表达诱导MET样表型24。在这里，我们提出了一个详细的协议来创建肉瘤细胞使用的miR-200和GRHL2的异位表达MET诱导的体外模型。
Protocol
1.试剂的配制通过添加胎牛血清（FBS）和5毫升青霉素 – 链霉素（5000 U / mL）添加到500mL的DMEM中的50毫升制备DMEM用于细胞培养。此介质可以被储存在4℃下进行长达六个月。 重悬无核酸酶水冻干引物以10μM的终浓度。在-20℃储存再悬浮的引物。 制备放射免疫沉淀测定（RIPA）缓冲液（150mM NaCl的，0.5％脱氧胆酸钠，0.1％SDS，50毫摩尔Tris，pH8.0）中。有蛋白酶抑制剂混合物补…
Representative Results
架构MET诱导细胞肉瘤对于在肉瘤细胞MET样改变感应的一般时间线示于图1。该协议开始于转导GRHL2（ 图1A），随后所述miR-200家族（ 图1B）的转染。 GRHL2或miR-200家族成员未能单独表达时冲击RD细胞的出现，但GRHL2和miR-200S的异位表达一起导致在RD细胞上皮样形态学变化。从一个纺锤状的形式，以?…
Discussion
肉瘤是间充质细胞系的很少见，但高发性癌。尽管他们的间充质细胞系，肉瘤的一个子集似乎经历一个更上皮样状态的表型转化。这MET-像开关具有预后相关性，因为患者多上皮样的肿瘤较少侵略性24。尽管他们的临床相关性，很少有研究解决的分子机制肉瘤推动这些表型的转变。
为了检查肉瘤细胞MET样的转变，我们通过结合上皮因素GRHL2，且所述miR-200家族?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
JAS确认来自杜克大学癌症研究所，杜克大学的泌尿生殖系肿瘤学实验室，骨科的杜克大学部的支持。 HL是由美国国家科学基金会（NSF）中心理论生物物理的支持（NSF PHY-1427654）和NSF DMS-1361411，并作为CPRIT（癌症预防和得克萨斯州的研究所）学者在得克萨斯州癌症研究在莱斯大学。 KEW由NIH F32 CA192630 MKJ和HL从与玛丽·C·法拉克-卡森，JN Onuchic，萨米尔·M·汉什，肯内特·J·皮塔，和唐纳德·S·科费有益的讨论中受益的支持。
Materials
| Countess automated counter | Life technologies | AMQAX1000 | |
| Countess cell counting chamber slides | Invitrogen | C10283 | |
| SimpliAmp Thermal Cycler | Thermo Fisher | A24811 | |
| Odyssey Fc | LI-COR Inc | ||
| ViiA7 Real Time PCR System | Thermo Fisher | 4453536 | |
| PCR microplate | Corning | 321-29-051 | |
| KAPA SYBR Fast Universal qPCR Kit | KAPA Biosystems | KK4602 | |
| Starting Block (PBS) Blocking Buffer | Thermo Fisher | 37538 | BSA-based blocking buffer |
| Agarose General Purpose LE | Genesee Scientific | 20-102 | |
| 10X Tris/Glycine/SDS Buffer | Bio-Rad Laboratories Inc | 161-0732 | Running buffer |
| 10X Tris/Glycine Buffer | Bio-Rad Laboratories Inc | 161-0734 | Transfer buffer |
| RIPA Buffer | Sigma Life Sciences | SLBG8489 | |
| Amersham Protran 0.45 μm nitrocellulose | GE Healthcare Lifesciences | 10600012 | |
| Quick-RNA MiniPrep Kit | Genesee Scientific | 11-358 | |
| Laemmli Sample Buffer (4X) | Bio-Rad Laboratories Inc | 1610747 | |
| Mini Trans-Blot Cell | Bio-Rad Laboratories Inc | 1703930 | |
| Mini-Protean Tetra Cell | Bio-Rad Laboratories Inc | 1658005EDU | |
| DPBS | Life technologies | 14190-144 | |
| 0.05% Trypsin-EDTA | Life technologies | 11995-065 | |
| DMEM | Life technologies | 11995-065 | |
| Lipofectamine RNAi Max | Thermo Fisher | 13778150 | |
| Lipofectamine 2000 Ragents | Thermo Fisher | 11668019 | |
| Penicillin Streptomycin | Life technologies | 15140-122 | |
| miRVana miRNA mimic negative control #1 | Thermo Fisher | 4464058 | neg miRNA |
| hsa-miR-200 mirVana miRNA mimic | Thermo Fisher | 4464066 | miR200A |
| has-miR-200 mirVana miRNA mimic | Thermo Fisher | 4404066 | miR200B |
| has-miR-200 mirVana miRNA mimic | Thermo Fisher | 4404066 | miR200C |
| Opti-MEM | Life technologies | 11088-021 | serum-free media |
| anti-Ecadherin antibody | BD Bioscience | 610182 | |
| anti-beta actin | Santa Cruz Biotechnology | sc-69879 | |
| anti-EpCam | Ab Serotec | MCA18706 | |
| anti-ZO1 | Invitrogen | 402200 | |
| IRDye 800W | LI-COR Inc | 925-32210 | |
| IRDye 680 | LI-COR Inc | 926-32223 | |
| anti-mouse AlexaFluor 647 | Thermo Fisher | A211241 | |
| anti-rabbit AlexaFluor 647 | Thermo Fisher | ab150075 | |
| Halt Protease and Phosphatesse Inhibitor | Thermo Fisher | 1861281 | |
| Precision Plus Protein Dual Color | Bio-Rad Laboratories Inc | 161-0374 | |
| Partec CellTrics | Sysmex | 04-004-2326 | 30 μm filter for flow |
| GAPDH-F | IDT | AGCCACATCGCTCAGACAC | |
| GAPDH-R | IDT | GCCCAATACGACCAAATCC | |
| Ecadherin-F | IDT | TGGAGGAATTCTTGCTTTGC | |
| Ecadherin-R | IDT | CGCTCTCCTCCGAAGAAAC | |
| ZEB1-F | IDT | GCATACAGAACCCAACTTGAACGTC | |
| ZEB1-R | IDT | CGATTACACCCAGACTGC | |
| NOTCH-F | IDT | GGCAATCCGAGGACTATGAG | |
| NOTCH-R | IDT | CTCAGAACGCACTCGTTGAT | |
| nitro blue tetrazolium | Sigma | N5514 | |
| hexadimethrine bromide | Sigma | H9268 | polybrene |
| 3 mL syringe | BD Bioscience | 309657 | |
| Sterile syringe filter | VWR | 28145-505 | |
| 5mL polypropylene round-bottom tube | 352063 | flow cytometry tubes | |
| High-Capacity cDNA Reverse Transcription Kit | Thermo Fisher | 4368814 | reverse transcription kit |
| 4% paraformaldyhyde | Santa Cruz Biotechnology | sc-281612 | |
| Triton-X100 | Sigma | 93443 | |
| bovine serum albumin | Sigma | A7906 |
References
- Weber, C. E., Li, N. Y., Wai, P. Y., Kuo, P. C. Epithelial-mesenchymal transition, TGF-beta, and osteopontin in wound healing and tissue remodeling after injury. J Burn Care Res. 33 (3), 311-318 (2012).
- Galichon, P., Finianos, S., Hertig, A. EMT-MET in renal disease: should we curb our enthusiasm. Cancer Lett. 341 (1), 24-29 (2013).
- Carew, R. M., Wang, B., Kantharidis, P. The role of EMT in renal fibrosis. Cell Tissue Res. 347 (1), 103-116 (2012).
- Willis, B. C., Borok, Z. TGF-beta-induced EMT: mechanisms and implications for fibrotic lung disease. Am J Physiol Lung Cell Mol Physiol. 293 (3), L525-L534 (2007).
- Ye, X., Weinberg, R. A. Epithelial-Mesenchymal Plasticity: A Central Regulator of Cancer Progression. Trends Cell Biol. 25 (11), 675-686 (2015).
- Li, L., Li, W. Epithelial-mesenchymal transition in human cancer: comprehensive reprogramming of metabolism, epigenetics, and differentiation. Pharmacol Ther. 150, 33-46 (2015).
- Tsai, J. H., Yang, J. Epithelial-mesenchymal plasticity in carcinoma metastasis. Genes Dev. 27 (20), 2192-2206 (2013).
- Bitting, R. L., Schaeffer, D., Somarelli, J. A., Garcia-Blanco, M. A., Armstrong, A. J. The role of epithelial plasticity in prostate cancer dissemination and treatment resistance. Cancer Metastasis Rev. 33 (2-3), 441-468 (2014).
- Schaeffer, D., Somarelli, J. A., Hanna, G., Palmer, G. M., Garcia-Blanco, M. A. Cellular Migration and Invasion Uncoupled: Increased Migration Is Not an Inexorable Consequence of Epithelial-to-Mesenchymal Transition. Mol Cell Biol. 34 (18), 3486-3499 (2014).
- Mathow, D., et al. Zeb1 affects epithelial cell adhesion by diverting glycosphingolipid metabolism. EMBO Rep. 16 (3), 321-331 (2015).
- Ware, K. E., et al. A mechanism of resistance to gefitinib mediated by cellular reprogramming and the acquisition of an FGF2-FGFR1 autocrine growth loop. Oncogenesis. 2, e39 (2013).
- Yauch, R. L., et al. Epithelial versus mesenchymal phenotype determines in vitro sensitivity and predicts clinical activity of erlotinib in lung cancer patients. Clin Cancer Res. 11 (24 Pt 1), 8686-8698 (2005).
- Jolly, M. K., et al. Towards elucidating the connection between epithelial-mesenchymal transitions and stemness. J R Soc Interface. 11 (101), 20140962 (2014).
- Mani, S. A., et al. The epithelial-mesenchymal transition generates cells with properties of stem cells. Cell. 133 (4), 704-715 (2008).
- Chen, L., et al. Metastasis is regulated via microRNA-200/ZEB1 axis control of tumour cell PD-L1 expression and intratumoral immunosuppression. Nat Commun. 5, 5241 (2014).
- Nicolazzo, C., Gradilone, A. Significance of circulating tumor cells in soft tissue sarcoma. Anal Cell Pathol (Amst). , 697395 (2015).
- Somarelli, J. A., et al. Mesenchymal-epithelial transition in sarcomas is controlled by the combinatorial expression of miR-200s and GRHL2. Mol Cell Biol. , (2016).
- Bae, Y. K., Choi, J. E., Kang, S. H., Lee, S. J. Epithelial-Mesenchymal Transition Phenotype Is Associated with Clinicopathological Factors That Indicate Aggressive Biological Behavior and Poor Clinical Outcomes in Invasive Breast Cancer. J Breast Cancer. 18 (3), 256-263 (2015).
- Humphries, B., Yang, C. The microRNA-200 family: small molecules with novel roles in cancer development, progression and therapy. Oncotarget. 6 (9), 6472-6498 (2015).
- Renner, M., et al. MicroRNA profiling of primary high-grade soft tissue sarcomas. Genes Chromosomes Cancer. 51 (11), 982-996 (2012).
- Petrof, G., et al. Mutations in GRHL2 result in an autosomal-recessive ectodermal Dysplasia syndrome. Am J Hum Genet. 95 (3), 308-314 (2014).
- Werner, S., et al. Dual roles of the transcription factor grainyhead-like 2 (GRHL2) in breast cancer. J Biol Chem. 288 (32), 22993-23008 (2013).
- Werth, M., et al. The transcription factor grainyhead-like 2 regulates the molecular composition of the epithelial apical junctional complex. Development. 137 (22), 3835-3845 (2010).
- Somarelli, J. A., et al. Mesenchymal-Epithelial Transition in Sarcomas Is Controlled by the Combinatorial Expression of MicroRNA 200s and GRHL2. Mol Cell Biol. 36 (19), 2503-2513 (2016).
- Varma, S., et al. The transcription factors Grainyhead-like 2 and NK2-homeobox 1 form a regulatory loop that coordinates lung epithelial cell morphogenesis and differentiation. J Biol Chem. 287 (44), 37282-37295 (2012).
- Pruitt, S. C., Mielnicki, L. M., Stewart, C. C. Analysis of fluorescent protein expressing cells by flow cytometry. Methods Mol Biol. 263, 239-258 (2004).
- Zhao, Z., et al. A high-content morphological screen identifies novel microRNAs that regulate neuroblastoma cell differentiation. Oncotarget. 5 (9), 2499-2512 (2014).
- Borowicz, S., et al. The soft agar colony formation assay. J Vis Exp. (92), e51998 (2014).
- Yang, J., et al. Integrated proteomics and genomics analysis reveals a novel mesenchymal to epithelial reverting transition in leiomyosarcoma through regulation of slug. Mol Cell Proteomics. 9 (11), 2405-2413 (2010).
- Alba-Castellon, L., et al. Snail1 expression is required for sarcomagenesis. Neoplasia. 16 (5), 413-421 (2014).
- Takaishi, M., Tarutani, M., Takeda, J., Sano, S. Mesenchymal to Epithelial Transition Induced by Reprogramming Factors Attenuates the Malignancy of Cancer Cells. PLoS One. 11 (6), e0156904 (2016).
Citer Cet Article
Ware, K. E., Gilja, S., Xu, S., Shetler, S., Jolly, M. K., Wang, X., Bartholf Dewitt, S., Hish, A. J., Jordan, S., Eward, W., Levine, H., Armstrong, A. J., Somarelli, J. A. Induction of Mesenchymal-Epithelial Transitions in Sarcoma Cells. J. Vis. Exp. (122), e55520, doi:10.3791/55520 (2017).