JoVE Journal > Genetics
Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
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유전학

用于评估肿瘤细胞中的微RNA水平、功能和相关靶基因的体外协议

Published: May 21, 2019
doi:
10.3791/59628
, , ,
1Department of Biomedical Science and Research Institute for Bioscience & Biotechnology,Hallym University

Summary

该协议使用基于探针的实时聚合酶链反应(PCR)、磺胺胺B(SRB)测定、3’未翻译区域(3’UTR)克隆和荧光素酶测定来验证感兴趣的miRNA的目标基因,并了解miRNA的功能。

Abstract

微RNA (miRNA) 是小型调节RNA,可识别调节包括癌症在内的多种疾病中的许多细胞内信号通路。这些小型调节RNA主要与其目标信使RNA(mRNA)的3’未翻译区域(3’UTR)相互作用,最终导致mRNA解码过程的抑制和目标mRNA降解的增强。基于表达水平和细胞内功能,miRNA能够作为致癌和肿瘤抑制性mRNA的调节因子。在数百甚至数千个计算预测目标中识别 miRNA 的真正目标基因是识别感兴趣的 miRNA 的作用和基本分子机制的关键步骤。各种miRNA目标预测程序可用于搜索可能的miRNA-mRNA相互作用。然而,最具挑战性的问题是如何验证感兴趣的miRNA的直接靶基因。该协议描述了如何识别与miRNA功能相关的miRNA靶点的关键方法的可重复策略。该协议提供了一个实用的指南,用于使用基于探针的实时聚合酶链反应(PCR)、磺胺B(SRB)测定,在miRNA模拟转染后发现miRNA水平、功能和相关目标mRNA。,剂量反应曲线生成,和荧光素酶测定以及基因的3’UTR克隆,这是正确理解单个miRNA的作用所必需的。

Introduction

微RNA(miRNA)是一种小型调节RNA,主要通过对真实靶基因1中的3’未翻译区域(3’UTR)做出反应来调节信使RNA(mRNA)的转换和降解过程。miRNA的表达可以通过转录和转录后机制调节。这种调节机制的不平衡导致包括癌症在内的众多疾病中不受控制和独特的miRNA表达水平。单个 miRNA 可以与不同的 mRNA 进行多种交互。相应地,单个 mRNA 可以由各种 miRNA 控制。因此,细胞内信号网络受到明显表达的miRNA的复杂影响,通过这种影响,生理疾病和疾病可以启动和恶化2,3,45,6.虽然在各种癌症中观察到了miRNA的改变表达,但调节癌细胞与miRNA一起进行的分子机制在很大程度上仍不为人所知。

累积的证据表明,miRNA的致癌或肿瘤抑制作用取决于癌症的类型。例如,通过瞄准叉头盒o3(FOXO3),miR-155促进结肠直肠癌7、8的细胞增殖、转移和化学抗药性。相比之下,胶质瘤细胞入侵的限制性是由miR-107通过神经原位位同源蛋白2(NOTCH2)表达9的调节引起的。评估miRNA-目标相互作用与miRNA功能是一个不可或缺的部分,更好地了解miRNA如何调节各种生物过程在健康和疾病状态10。此外,miRNA真正靶点的发现,可以进一步为基于miRNA的抗癌药物治疗提供微调的策略。然而,miRNA领域的主要挑战是确定miRNA的直接目标。在这里,详细的方法作为可重复的实验方法,用于miRNA靶基因的测定。miRNA目标鉴定的成功实验设计涉及各种步骤和注意事项(图1)。比较肿瘤细胞和正常细胞中的成熟miRNA水平是选择感兴趣的miRNA的常见程序之一(图1A)。对选定的miRNA进行功能研究,以检测miRNA对细胞增殖的影响,对于缩小感兴趣的miRNA最佳潜在候选靶点列表非常重要(图1B)。基于miRNA的实验验证功能,需要用miRNA靶向预测程序对公司文献和数据库进行系统审查,以搜索与基因功能最相关的信息(图1C)。通过实施荧光素酶测定以及基因3’UTR的克隆、实时PCR和西方印迹(图1D),可以识别感兴趣的miRNA的真实目标基因。当前协议的目标是提供关键实验、基于探针的实时聚合酶链反应 (PCR)、磺胺 B (SRB) 分析后 miRNA 模拟转染、剂量-反应曲线生成以及荧光酶测定以及基因3’UTR的克隆。目前的协议对于更好地了解单个 miRNA 的功能以及 miRNA 在癌症治疗中的含义非常有用。

Protocol

1. 成熟微RNA (miRNA) 表达分析 成熟 miRNA 补充 DNA (cDNA) 合成 加入总RNA的254纳克和4.5μL的脱氧核糖核酸I(DNase I)混合物,然后将超纯水加入PCR带管,以补制成高达18μL(图2A)。根据反应总数,使用足够量的DNase I混合物,制备从多个细胞系纯化的每个总RNA样品的反应。注:DNase I混合物由DNase I(1.8 μL)、核糖酸酶抑制剂(0.3 μL)和25 mM MgCl 2(2.4 ?…

Representative Results

成功和准确地确认miRNA水平对于解释基于miRNA在疾病发展和进展中预期作用的miRNA分类的数据非常重要。使用基于探针的定量PCR在三个胰腺细胞系中测量了miRNA-107和miRNA-301的水平。在相同反应中合成特定miRNA和参考基因的cDNA可以提高数据的可重复性。PANC-1和CAPAN-1是人类胰腺导管腺癌细胞系,而HPNE是一种不朽的胰腺导管细胞系,通过携带人类端粒酶逆转录酶(hTERT)基因的逆转录病毒表达载体进行转导。?…

Discussion

确定具有目标miRNA功能的真正miRNA靶点的策略对于理解miRNA的多重作用是必不可少的。miRNA靶基因的识别可以作为解释由miRNA在细胞中调节的细胞信号事件的指南。揭示miRNA的功能重要的靶基因可以为开发基于miRNA的癌症治疗提供基本知识。

微阵列、小RNA库测序、深度测序、原位PCR逆转录酶和北方印迹等多种方法,利用从细胞系和组织中分离出的总RNA,可用于探索m…

Disclosures

The authors have nothing to disclose.

Acknowledgements

这项研究得到了韩国国家科学研究基金会(NRF)基础科学研究计划的支持,该基金会由韩国教育部资助(2017R1D1A3B03035662);和Hallym大学研究基金,2017年(HRF-201703-003)。

Materials

15 mL conical tube SPL Life Sciences 50015
24-well plate Thermo Scientific 142475
50 mL conical tube SPL Life Sciences 50050
6-well plate Falcon 353046
6X DNA loading dye Real Biotech Corporation RD006 1 mL
8-cap strip Applied Biosystems N8010535 For cDNA synthesis
8-tube strip Applied Biosystems N8010580 For cDNA synthesis
96-well plate Falcon 353072
Acetic acid Sigma A6283-1L 1 L
Agarose A Bio Basic D0012 500 g
Alkaline phosphatase New England Biolabs M0290S 10,000 U/mL
Ampicillin Bio basic Canada Inc AB0028 25 g
AriaMx 96 tube strips Agilent Technologies 401493 For real time PCR
AriaMx real-time PCR system Agilent Technologies G8830A qPCR amplification, detection, and data analysis
AsiSI New England Biolabs R0630 10,000 units/mL
CAPAN-1 cells ATCC HTB-79
Cell culture hood Labtech Model: LCB-1203B-A2
Counting chambers with V-slash Paul Marienfeld 650010 Cells counter
CutSmart buffer New England Biolabs B7204S 10X concentration
DMEM Gibco 11965-092 500 mL
DNA gel extraction kit Bionics DN30200 200 prep
DNA ladder NIPPON Genetics EUROPE MWD1 1 Kb ladder
DNase I Invitrogen 18068015 100 units
Dual-luciferase reporter assay system Promega E1910 100 assays
Fetal bovine serum Gibco 26140-079 500 mL
HIT competent cells Real Biotech Corporation(RBC) RH617 Competent cells
HPNE cells ATCC CRL-4023
LB agar broth Bio Basic SD7003 250 g
Lipofectamine 2000 Invitrogen 11668-027 0.75 mL
Lipofectamine RNAiMax Invitrogen 13778-075 0.75 mL
Luminometer Promega Model: E5311
Microcentrifuge tube Eppendorf 22431021
Microplate reader TECAN Infinite F50
miRNA control mimic Ambion 4464058 5 nmole
miRNA-107 mimic Ambion 4464066 5 nmole
miRNeasy Mini Kit Qiagen 217004 50 prep
Mupid-2plus (electrophoresis system) TaKaRa Model: AD110
NotI New England Biolabs R3189 20,000 units/mL
Oligo explorer program GeneLink For primer design
Optical tube strip caps (8X Strip) Agilent Technologies 401425 For real time PCR
Opti-MEM Gibco 31985-070 500 Ml
PANC-1 cells ATCC CRL-1469
Penicillin/streptomycin Gibco 15140-122 100 mL
Phosphate buffer saline Gibco 14040117 1000 mL
Plasmid DNA miniprep S& V kit Bionics DN10200 200 prep
PrimeSTAR GXL DNA polymerase TaKaRa R050A 250 units
Shaker TECAN Shaking platform
Shaking incubator Labtech Model: LSI-3016A
Sigmaplot 14 software Systat Software Inc For dose-response curve generation
Sulforhodamine B powder Sigma S1402-5G 5 g
SYBR green master mix Smobio TQ12001805401-3 Binding fluorescent dye for dsDNA
T4 DNA ligase TaKaRa 2011A 25,000 U
TaqMan master mix Applied Biosystems 4324018 200 reactions, no AmpErase UNG
TaqMan microRNA assay (hsa-miR-107) Applied Biosystems 4427975 Assay ID: 000443 (50RT, 150 PCR rxns)
TaqMan microRNA assay (hsa-miR-301) Applied Biosystems 4427975 Assay ID: 000528 (50RT, 150 PCR rxns)
TaqMan miR RT kit Applied Biosystems 4366597 1000 reactions
Thermo CO2 incubator (BB15) ThermoFisher Scientific 37 °C and 5% CO2 incubation
Trichloroacetic acid Sigma 91228-100G 100 g
Trizma base Sigma T4661-100G 100 g
Ultrapure water Invitrogen 10977-015 500 mL
Veriti 96 well thermal cycler Applied Biosystems For amplification of DNA (or cDNA)
XhoI New England Biolabs R0146 20,000 units/mL
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Tags

Keywords: MicroRNAMicroRNA TargetIn Vitro ProtocolTumor CellsCell Viability AssayTransfectionMicroRNA MimicMicroRNA-107
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Seo, H. A., Hwang, C. Y., Moeng, S., Park, J. K. An In Vitro Protocol for Evaluating MicroRNA Levels, Functions, and Associated Target Genes in Tumor Cells. J. Vis. Exp. (147), e59628, doi:10.3791/59628 (2019).
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