一种报告基因检测法，用于分析哺乳动物细胞中内旋微RNA成熟
Summary
我们开发了一种内旋microRNA生物发生报告基因检测方法，用于具有四种质粒 的体外 细胞：一种具有内旋性miRNA，一种具有靶标，一种用于过表达调节蛋白，另一种用于Renilla荧光素酶。miRNA经过处理，可以通过与靶序列结合来控制荧光素酶的表达。
Abstract
微RNA（miRNA）是短RNA分子，在真核生物中广泛存在。大多数miRNA是从内含子转录而来的，它们的成熟涉及细胞核中不同的RNA结合蛋白。成熟的miRNA经常介导基因沉默，这已成为理解转录后事件的重要工具。除此之外，它还可以被探索为一种有前途的基因疗法方法。然而，目前缺乏评估哺乳动物细胞培养物中miRNA表达的直接方法。在这里，我们描述了一种高效而简单的方法，该方法通过确认其与靶序列的相互作用来帮助确定miRNA生物发生和成熟。此外，该系统允许使用多西环素诱导的启动子将外源性miRNA成熟与其内源性活性分离，该启动子能够以高效率和低成本控制初级miRNA（pri-miRNA）转录。该工具还允许在单独的质粒中用RNA结合蛋白进行调节。除了与各种不同的miRNA及其各自的靶标一起使用外，只要这些细胞系适合转染，它还可以适应不同的细胞系。
Introduction
前体mRNA剪接是真核生物1中基因表达调控的重要过程。成熟RNA中内含子的去除和外显子的结合是由剪接体催化的，剪接体是一种2兆达尔顿的核糖核蛋白复合物，由5个snRNA（U1，U2，U4，U5和U6）以及100多个蛋白质2，3组成。剪接反应以共转录方式发生，并且剪接体在每个新内含子处组装，由外显子 – 内含子边界和内含子4内的保守剪接位点的识别引导。不同的内含子可能具有不同的剪接速率，尽管剪接体复合物及其组分具有显着的守恒。除了剪接位点保存的差异外，分布在内含子和外显子上的调节序列可以引导RNA结合蛋白（RBP）并刺激或抑制剪接5，6。HuR是一种普遍表达的RBP，是控制mRNA稳定性7的重要因素。我们小组先前的结果表明，HuR可以与含有miRNA的内含子结合，表明该蛋白可能是促进miRNA加工和成熟的重要因素，也导致产生替代剪接同种型6，8，9。
许多微纳（miRNA）是从内旋序列编码的。虽然有些是内含子的一部分，但其他的被称为“mirtrons”，由整个内含子10，11形成。miRNA是短的非编码RNA，长度为12的18至24个核苷酸。它们的成熟序列与mRNA中的靶序列具有部分或全部互补性，因此影响翻译和/或mRNA衰变率。miRNA和靶标的组合驱动细胞获得不同的结果。几种miRNA可以驱使细胞产生促肿瘤或抗肿瘤表型13。致癌性miRNA通常靶向触发抑制特征的mRNA，导致细胞增殖，迁移和侵袭增加14。另一方面，肿瘤抑制性miRNA可能靶向致癌性mRNA或与细胞增殖增加相关的mRNA。
miRNA的加工和成熟也取决于它们的来源。大多数内源性miRNA是在微处理器的参与下处理的，微处理器由核糖核酸酶Drosha和蛋白质辅助因子12形成。米氏物以剪接体的活性进行加工，独立于Drosha15。考虑到在内含子中发现的miRNA的高频，我们假设参与剪接的RNA结合蛋白也可以促进这些miRNA的加工和成熟。值得注意的是，RBP hnRNP A2 / B1已经与微处理器和miRNA生物发生16相关联。
我们之前已经报道过，几种RNA结合蛋白，如hnRNP和HuR，通过质谱17与内源性miRNA相关。使用免疫沉淀和计算机分析9证实了HuR（ELAVL1）与miR-17-92内旋簇的miRNA的关联。miR-17-92是一种内源性miRNA簇，由6个miRNA组成，在不同癌症中表达增加18，19。该簇也称为“oncomiR-1”，由 miR-17、miR-18a、miR-19a、miR-20、miR-19b 和 miR-92a组成。HuR表达的增加刺激了miR-19a和miR-19b合成9。由于该簇两侧的内源性区域与HuR相关，我们开发了一种方法来研究该蛋白是否可以调节miR-19a和miR-19b的表达和成熟。对我们的假设的一个重要预测是，作为一种调节蛋白，HuR可以促进miRNA生物发生，导致表型改变。一种可能性是miRNA是通过HuR的刺激处理的，但不会成熟和功能化，因此，蛋白质的作用不会直接影响表型。因此，我们开发了一种剪接报告基因检测法，以研究像HuR这样的RBP是否会影响内源性miRNA的生物发生和成熟。通过确认miRNA处理和成熟，我们的测定显示了与靶序列的相互作用以及成熟和功能性miRNA的产生。在我们的测定中，我们将内旋性miRNA簇的表达与荧光素酶质粒偶联，以检查培养细胞中的miRNA靶标结合。
Protocol
Representative Results
Discussion
前mRNA剪接是基因表达调控的重要过程，其控制可以触发对细胞表型修饰的强烈影响22，23。超过70%的miRNA是从人类内含子转录而来的，我们假设它们的加工和成熟可以通过剪接调节蛋白24，25来促进。我们开发了一种分析内源性miRNA处理和功能的方法。我们的测定使用四种不同的质粒，并允许我们测试内源性和?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
作者感谢E.马凯耶夫（新加坡南洋理工大学）的海拉克雷细胞和pRD-RIPE和pCAGGS-Cre质粒。我们感谢埃德娜·木村、卡罗琳娜·珀塞尔·戈伊斯、吉塞拉·拉莫斯、露西亚·罗塞蒂·洛佩斯和安塞尔莫·莫里斯科特的支持。
Materials
| Recombinant DNA | |||
| pCAGGS-Cre (Cre- encoding plasmid) | A kind gift from E. Makeyev from Khandelia et al., 2011 | ||
| pFLAG-HuR | Generated during this work | ||
| pmiRGLO-RAP-IB | Generated during this work | ||
| pmiRGLO-scrambled | Generated during this work | ||
| pRD-miR-17-92 | Generated during this work | ||
| pRD-RIPE-donor | A kind gift from E. Makeyev from Khandelia et al., 2011 | ||
| pTK-Renilla | Promega | E2241 | |
| Antibodies | |||
| anti-B-actin | Sigma Aldrich | A5316 | |
| anti-HuR | Cell Signaling | mAb 12582 | |
| IRDye 680CW Goat anti-mouse IgG | Li-Cor Biosciences | 926-68070 | |
| IRDye 800CW Goat anti-rabbit IgG | Li-Cor Biosciences | 929-70020 | |
| Experimental Models: Cell Lines | |||
| HeLa-Cre | A kind gift from E. Makeyev from Khandelia et al., 2011 | ||
| HeLa-Cre miR17-92 | Generated during this work | ||
| HeLa-Cre miR17-92-HuR | Generated during this work | ||
| HeLa-Cre miR17-92-HuR-luc | Generated during this work | ||
| HeLa-Cre miR17-92-luc | Generated during this work | ||
| HeLa-Cre miR17-92-scrambled | Generated during this work | ||
| Chemicals and Peptides | |||
| DMEM/high-glucose | Thermo Fisher Scientific | 12800-017 | |
| Doxycycline | BioBasic | MB719150 | |
| Dual-Glo Luciferase Assay System | Promega | E2940 | |
| EcoRI | Thermo Fisher Scientific | ER0271 | |
| EcoRV | Thermo Fisher Scientific | ER0301 | |
| Geneticin | Thermo Fisher Scientific | E859-EG | |
| L-glutamine | Life Technologies | ||
| Opti-MEM I | Life Technologies | 31985-070 | |
| pFLAG-CMV™-3 Expression Vector | Sigma Aldrich | E6783 | |
| pGEM-T | Promega | A3600 | |
| Platinum Taq DNA polymerase | Thermo Fisher Scientific | 10966-030 | |
| pmiR-GLO | Promega | E1330 | |
| Puromycin | Sigma Aldrich | P8833 | |
| RNAse OUT | Thermo Fisher Scientific | 752899 | |
| SuperScript IV kit | Thermo Fisher Scientific | 18091050 | |
| Trizol-LS reagent | Thermo Fisher | 10296-028 | |
| trypsin/EDTA 10X | Life Technologies | 15400-054 | |
| XbaI | Thermo Fisher Scientific | 10131035 | |
| XhoI | Promega | R616A | |
| Oligonucleotides | |||
| forward RAP-1B pmiRGLO | Exxtend | TCGAGTAGCGGCCGCTAGTAAG CTACTATATCAGTTTGCACAT |
|
| reverse RAP-1B pmiRGLO | Exxtend | CTAGATGTGCAAACTGATATAGT AGCTTACTAGCGGCCGCTAC |
|
| forward scrambled pmiRGLO | Exxtend | TCGAGTAGCGGCCGCTAGTAA GCTACTATATCAGGGGTAAAAT |
|
| reverse scrambled pmiRGLO | Exxtend | CTAGATTTTACCCCTGATATAGT AGCTTACTAGCGGCCGCTAC |
|
| forward HuR pFLAG | Exxtend | GCCGCGAATTCAATGTCTAAT GGTTATGAAGAC |
|
| reverse HuR pFLAG | Exxtend | GCGCTGATATCGTTATTTGTG GGACTTGTTGG |
|
| forward pre-miR-1792 pRD-RIPE | Exxtend | ATCCTCGAGAATTCCCATTAG GGATTATGCTGAG |
|
| reverse pre-miR-1792 pRD-RIPE | Exxtend | ACTAAGCTTGATATCATCTTG TACATTTAACAGTG |
|
| forward snRNA U6 (RNU6B) | Exxtend | CTCGCTTCGGCAGCACATATAC | |
| reverse snRNA U6 (RNU6B) | Exxtend | GGAACGCTTCACGAATTTGCGTG | |
| forward B-Actin qPCR | Exxtend | ACCTTCTACAATGAGCTGCG | |
| reverse B-Actin qPCR | Exxtend | CCTGGATAGCAACGTACATGG | |
| forward HuR qPCR | Exxtend | ATCCTCTGGCAGATGTTTGG | |
| reverse HuR qPCR | Exxtend | CATCGCGGCTTCTTCATAGT | |
| forward pre-miR-1792 qPCR | Exxtend | GTGCTCGAGACGAATTCGTCA GAATAATGTCAAAGTG |
|
| reverse pre-miR-1792 qPCR | Exxtend | TCCAAGCTTAAGATATCCCAAAC TCAACAGGCCG |
|
| Software and Algorithms | |||
| Prism 8 for Mac OS X | Graphpad | https://www.graphpad.com | |
| ImageJ | National Institutes of Health | http://imagej.nih.gov/ij |
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