RNA荧光 原 位杂交在人骨肉瘤细胞中长链非编码RNA定位
Summary
本方案描述了一种RNA荧光 原位 杂交以在人骨肉瘤细胞中定位lncRNA的方法。
Abstract
已经研究了长链非编码RNA（lncRNA）在癌症中的重要作用，如调节癌细胞的增殖、上皮-间充质转化（EMT）、迁移、浸润和自噬。细胞中lncRNA的定位检测可以深入了解其功能。通过设计lncRNA特异性反义链序列，然后用荧光染料标记，RNA荧光 原位 杂交（FISH）可用于检测lncRNA的细胞定位。随着显微镜的发展，RNA FISH技术现在甚至可以对表达不良的lncRNA进行可视化。该方法不仅可以单独检测lncRNA的定位，还可以利用双色或多色免疫荧光检测其他RNA、DNA或蛋白质的共定位。在此，我们以人骨肉瘤细胞中的lncRNA小核仁RNA宿主基因6（SNHG6）为例，对RNA FISH的详细实验操作步骤和注意事项进行了介绍，以期为想要进行RNA FISH实验，尤其是lncRNA FISH的研究人员提供参考。
Introduction
近年来，全基因组技术的进步极大地扩展了我们对人类基因组的理解。大约93%的人类基因组可以转录成RNA，但只有2%的RNA可以翻译成蛋白质;其余 98% 没有蛋白质翻译功能的 RNA 称为非编码 RNA （ncRNA）1。长链 ncRNA （lncRNA） 是一类非编码 RNA （ncRNA） 中含有 200 多个核苷酸2，由于参与细胞的许多生理和病理过程，如分化、周期调控、凋亡、迁移和侵袭 3,4,5，因此越来越受到关注.LncRNA通过调节染色质结构和核基因表达、控制mRNA剪接过程和转录后修饰等多种机制发挥作用6。LncRNA在转录和转录后水平上调节恶性肿瘤的发生、发展和转移。转录调控是通过与染色体结构结合影响 RNA 转录在细胞核中实现的，而转录后调控是通过内源性竞争性 RNA （ceRNA） 机制控制靶基因在细胞质中实现的 5,7,8。CeRNA揭示了一种新的RNA相互作用机制，即lncRNA可以作为海绵吸附miRNA，抑制miRNA介导的相关靶基因降解9。因此，有关lncRNA的亚细胞定位的信息，无论特定的lncRNA位于细胞质还是细胞核中，对于帮助确定其生物学功能都很重要。
目前，lncRNA定位主要通过两种方法进行检测，一种是通过细胞核/细胞质组分分离试验，另一种是通过RNA FISH。在前者中，分别提取细胞质和细胞核组分中的RNA，然后用特异性lncRNA引物进行PCR扩增，以检测细胞质和细胞核中lncRNA的比例。这种方法的优点是时间效率高，而缺点是实际的lncRNA定位不能直接反映在细胞质和细胞核中lncRNA的相对比例上。RNA FISH可以通过设计lncRNA特异性反义链序列，然后用荧光染料标记来检测细胞中的lncRNA定位10。随着探针技术和检测方法的进步，RNA FISH方法得到了改进，包括荧光团标记的多个寡核苷酸探针组11、LNA探针12和支链DNA（bDNA）探针13。RNA FISH不仅可以检测lncRNA的定位，还可以使用双色或多色免疫荧光14检测其他RNA、DNA或蛋白质的共定位。
在这项工作中，我们以RNA FISH在骨肉瘤细胞（143B）中lncRNA小核仁RNA宿主基因6（SNHG6）的详细细胞内定位检测方案为例。SNHG6 是一种 600-730 核苷酸的成熟剪接形式的 lncRNA，被鉴定为多种人类癌症的新型致癌基因，包括结直肠癌、胃癌、卵巢透明细胞癌、骨肉瘤和肝细胞癌15、16、17、18。研究证实了 SNHG6 参与癌细胞的生物学行为，例如增殖、EMT 和自噬，并显示了 SNHG6 的细胞质定位，它可能通过结合（海绵化）miRNA15、16、17 来影响靶基因。本文介绍了通过RNA FISH进行SNHG6细胞内定位的详细检测方案。
Protocol
Representative Results
Discussion
该RNA FISH方案不仅可以检测lncRNA在细胞中的定位，还可以检测细胞中其他RNA、DNA或蛋白质的共定位，也可用于检测lncRNA在石蜡包埋组织中的位置。然而，在这种情况下的具体方案是不同的，因为石蜡包埋的组织需要脱蜡21。该实验程序可应用于 48 孔或 96 孔板，但 384 孔板太小，无法在此处使用。
需要注意该方法的几个关键步骤，包括无RNase环境、探针浓度、?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了（1）中国国家重点研发计划（2020YFE0201600）的资助;（2）国家自然科学基金（81973877 82174408）;（3）上海市医院中药制剂产业转化协同创新中心;（4）上海中医药大学预算内研究项目（2021LK047）。
Materials
| Automatic cell counter | Shanghai Simo Biological Technology Co., Ltd | IC1000 | Counting cells |
| Cell culture plate-12 | Shanghai YueNian Biotechnology Co., Ltd | 3513,corning | Place the coverslips in the plate |
| Cell line (143B) | Cell Bank of Chinese Academy of Sciences | CRL-8303 | osteosarcoma cancer cell line |
| Centrifuge tube (15 mL, 50 mL) | Shanghai YueNian Biotechnology Co., Ltd | 430790, Corning | Centrifuge the cells |
| Coverslips | Shanghai YueNian Biotechnology Co., Ltd | abs7026 | The cells are seeded on the coverslips |
| Cy3 label-SNHG6 DNA probe | Shanghai GenePharma Co.,Ltd | A10005 | Detect SNHG6 location |
| DMEM media | Shanghai YueNian Biotechnology Co., Ltd | LM-E1141 | Cell culture medium |
| Dry Bath Incubator | Haimen Kylin-Bell Lab Instruments Co.,Ltd. | DKT200-2 | Incubation at different high temperatures |
| Ethanol 100% | Sinopharm Chemical ReagentCo., Ltd | 10009218 | dehydration |
| Fluorescence microscope | Shanghai Waihai Biotechnology Co., LTD | Olympus BX43 equipped with a camera of Olympus U-TV0.5XC-3(SN:5J01719),olympus | Observation and positioning |
| Incubator | Shanghai Yiheng Scientific Instrument Co., LTD | DHP-9051 | The samples were incubated at 37 °C. |
| Mounting Medium | Sangon Biotech (Shanghai) Co., Ltd. | E675004 | Attach the coverslips to the slide |
| Shaker | Haimen Kylin-Bell Lab Instruments Co.,Ltd. | TS-8S | Washing sample |
| Slide | Shanghai YueNian Biotechnology Co., Ltd | 188105 | The coverslips is placed on the slide |
| Triton X-100 | Sangon Biotech (Shanghai) Co., Ltd. | A600198 | Permeable membrane and nuclear membrane |
| Trypsin (0.25%) | Shanghai YueNian Biotechnology Co., Ltd | 25200056, Gibco | trypsin treatment of cells |
| Tween-20 | Sangon Biotech (Shanghai) Co., Ltd. | A600560 | detergent |
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