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Biologia

体内Nanovector 心脏特异性 MicroRNA 海绵的交付

Published: June 15, 2018
doi:
10.3791/57845
, ,
1Princess Margaret Cancer Centre,University of Toronto, 2Department of Pathology, Department of Cardiology,Johns Hopkins University

Summary

组织特异性 microRNA 抑制是一种在 microRNA 领域发展不发达的技术。在这里, 我们描述了一个协议, 以成功地抑制 miR-181 microRNA 家族在骨骼肌细胞从心脏。Nanovector 技术是用来提供一个 microRNA 海绵, 表明重要的体内心脏特定 miR-181 家庭抑制。

Abstract

MicroRNA (miRNA) 是一种小的非编码 rna, 它抑制转录后信使 rna (mRNA) 的表达。人类疾病, 如癌症和心血管疾病, 已被证明激活组织和/或细胞特异的 miRNA 表达与疾病进展有关。miRNA 表达的抑制提供了治疗干预的潜力。然而, 传统的抑制 miRNAs 的方法, 使用 antagomir 寡核苷酸, 影响特定的 miRNA 功能, 在全球交付。本文提出了一种在大鼠模型中对 miR-181 家族进行心脏特异性抑制的协议。miRNA 海绵结构的设计包括10重复 anti-miR-181 绑定序列。心脏特异的α MHC 启动子被克隆成 pEGFP 骨干, 以驱动心脏特异的 miR-181 miRNA 海绵表达。为了创建一个稳定的细胞系表达 miR-181-sponge, 骨骼肌 H9c2 细胞被转染的α-MHC-EGFP 和平号-181 海绵结构, 并排序由荧光活化细胞分类 (资产管制署) 到 GFP 阳性 H9c2 细胞培养与新霉素(G418)。随着新霉素的稳定增长, 单克隆细胞的数量是由额外的和单细胞克隆建立的。由此产生的骨骼肌 H9c2-miR-181-sponge-GFP 细胞表现为 miR-181 家庭成员的功能丧失, 这是通过增加 miR-181 靶蛋白的表达来评估的, 而与表达非功能性海绵的 H9c2 细胞相比。此外, 我们还开发了一个 nanovector 的系统交付 miR-181-sponge 结构的络合正电荷脂质体纳米粒子和负电荷 miR-181-sponge 质粒。在活体成像的 GFP 显示, 多尾静脉注射的 nanovector 在三周的时间, 能够促进一个重要的表达 miR-181-sponge 在心脏特定的方式。重要的是, 在心脏组织, 而不是在肾脏或肝脏中观察到 miR-181 功能的丧失。miRNA 海绵是抑制组织特异 miRNA 表达的有力方法。驱动 miRNA 海绵表达从组织特定的启动器提供特异性的 miRNA 抑制, 这可以被限制在靶器官或组织。此外, 结合 nanovector 和 miRNA 海绵技术, 允许有效的分娩和组织特定的 miRNA 抑制在体内。

Introduction

在过去的两年里, 已经有许多研究指出 miRNAs 在人类疾病中的重要作用。大量文献表明, miRNAs 在疾病的病理生理学中具有不可否认的重要性, 如癌症1和心血管疾病2、3、4、5。例如, miR-21 是上调在许多癌症, 导致细胞周期增加和细胞增殖6。在丙型肝炎感染中, miR-122 在病毒7的复制中起着重要作用, 并表明 miR-122 抑制病毒负荷8。在心肌肥厚, miR-212/132 是上调在心脏和参与病理表型9。上调 miRNA 的下调或功能性抑制的明显重要性表明, 在几乎所有疾病中, 治疗利用 miRNA 生物学的机会。

在人类基因组的三个基因组中发现了四 miR-181 家庭成员, miR-181a/b/c/d。非编码 RNA 宿主基因 (MIR181A1-HG) 的内含子区域编码 miR-181-a/b-1 簇。NR6A1 基因的内含子区域对 miR-181-a/b-2 进行编码。miR-181-c/d 星团位于 uncharacterized 19 号染色体上。所有的 miR-181 家庭成员共有相同的 “种子” 序列, 所有四 miR-181 家庭成员可以有可能调节相同的 mRNA 目标。

我们3,4和其他10突出了 miR-181 家庭成员在末期心力衰竭的重要性。我们也认识到, miR-181c 上调发生在与心脏病风险增加有关的病理条件下, 如 II 型糖尿病、肥胖症和衰老3、4、5。据推测, miR-181c 的过度表达会导致氧化应激, 导致心脏功能障碍4。

有几个小组建议 miRNA 存在于线粒体11、12、13、14, 但我们是第一个证明 miR-181c 来源于核基因组、加工和随后移位到线粒体在 RISC3。此外, 我们检测到 miR-181a 和 miR-181b 的低表达在心脏线粒体室5。重要的是, 我们发现 miR-181c 压抑 mt-COX1 mRNA 表达, 从而证明 miRNAs 参与线粒体基因调控和改变线粒体功能3,4。

本文讨论了设计一个 miRNA 海绵, 以摧毁整个 miR-181 家庭在心肌细胞所需的方法。此外, 我们概述了在体内应用的 miR-181-sponge 的协议。

Protocol

所有实验程序均经约翰霍普金斯大学机构动物护理和使用委员会批准。 1. 海绵设计 MicroRNA 绑定 3 ‘ UTR注: miRNA 函数通过特定的基配对与部分互补点在其目标基因的 3 ‘ 未翻译区域 (UTR) 中的相互作用 (为全面审查, 见巴特尔)15。 设计 miRNA 表达抑制剂作为寡核苷酸, 对 miRNA 序列具有显著的互补性。通过广泛的基配对将 miRNA 在寡核苷?…

Representative Results

在稳定转染的 pEGFP-miR-181-sponge-expressing H9c2 细胞 (从步骤 4.2), 整个 miR-181 家族 (miR-181a, miR-181b, miR-181c 和 miR-181d) 的表达相对于 pEGFP–表达 H9c2 细胞有适度的减少。MiR-181-sponge 作为整个 miR-181 家族的一个竞争性抑制剂, 因此我们预计 miR-181c 线粒体靶基因的表达将会增加, mt-COX1。西方的印迹数据表明, pEGFP-miR-181-sponge-expressing H9c2 细胞中的 mt-COX1 表达比 pEGFP-爬行表达的 H9c2 细胞?…

Discussion

本文介绍了 miRNA 海绵的设计和合成, 并论证了海绵组织特异表达是抑制组织特异 miRNA 家族表达的有力工具。

我们已经证明, 一个 miR-181 的家庭靶向海绵可以克隆成表达质粒与心脏特定的启动子。该质粒可以有效地包装成 nanovector 颗粒, 用于在体外和体内使用电穿孔或尾静脉注射, 分别 (图 1)。miR-181 海绵可以抑制 miR-181 家族的心脏特异表达, ?…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

我们感谢彭博公共卫生学院生物化学与分子生物学系的安东尼 k.l. 梁, 他在设计 miR-181-sponge 结构方面的技术帮助。我们还感谢分子和比较病理部的宝琳娜 Sysa 和凯瑟琳 Gabrielson, 约翰霍普金斯医疗机构的技术援助, 通过体内成像的 miRNA 海绵分娩。

这项工作得到了 NIH、HL39752 (查尔斯 Steenbergen) 的赠款和美国心脏协会 14SDG18890049 (Samarjit Das) 的科学家发展补助金的支持。在辛辛那提儿童医院, 杰弗里 d. Molkentin 慷慨地提供了鼠心特异性启动子。

Materials

pEGFP-C1 vector Addgene 6084-1
In-fusion Clontech 121416
QIAprep Miniprep Qiagen 27104
QIAquick Gel Extraction Kit Qiagen 28704
miR-181-sponge synthesis Introgen GeneArt custome made
PCR primers Integrated DNA Technologies custome
EcoRI enzymes New Endland Biolabs R0101S
KpnI enzymes New Endland Biolabs R0142S
Rapid DNA Ligation Kit Sigma-Aldrich 11635379001
H9c2 cells ATCC CRL-1446
DMEM Media Thermo Fisher Scientific 11965092
Fetal Bovine Serum Thermo Fisher Scientific 10082139
Nucleofector 2b Device Lonza AAB-1001
Nucleofector Kits for H9c2 (2-1) Lonza VCA-1005
G418, Geneticin Thermo Fisher Scientific 11811023
FACSAria II Flow cytometer BD Bioscience 644832
Branson 450 sonifier Marshall Scientific EDP 100-214-239
The Xenogen IVIS Spectrum optical imaging device Caliper Life Sciences
Anti-MTCO1 antibody Abcam ab14705
α-tubulin antibody Abcam ab7291
Sequoia C256 ultrasound system Siemens
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Tags

In VivoNanovectorMicroRNA SpongeHeart-specificMicroRNA KnockdownEGFR PlasmidCMV PromoterAlpha-myosin Heavy Chain PromoterDNA PurificationBacterial TransformationLigation
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Citazione di questo articolo
Kent, O. A., Steenbergen, C., Das, S. In Vivo Nanovector Delivery of a Heart-specific MicroRNA-sponge. J. Vis. Exp. (136), e57845, doi:10.3791/57845 (2018).
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