外来体miRNA的分析非小细胞肺癌（NSCLC）患者的血浆通过定量PCR：一种可行的液体活检工具
Summary
This protocol describes the feasibility to perform miRNA profiling in exosomes, released in plasma of NSCLC patients, through a commercial exosome isolation kit with Proteinase K and RNAse treatments, in order to avoid circulating miRNAs contamination and evaluate their biomarker features in NSCLC.
Abstract
The discovery of alterations in the EGFR and ALK genes, amongst others, in NSCLC has driven the development of targeted-drug therapy using selective tyrosine kinase inhibitors (TKIs). To optimize the use of these TKIs, the discovery of new biomarkers for early detection and disease progression is mandatory. These plasma-isolated exosomes can be used as a non-invasive and repeatable way for the detection and follow-up of these biomarkers. One ml of plasma from 12 NSCLC patients, with different mutations and treatments (and 6 healthy donors as controls), were used as exosome sources. After RNAse treatment, in order to degrade circulating miRNAs, the exosomes were isolated with a commercial kit and resuspended in specific buffers for further analysis. The exosomes were characterized by western blotting for ALIX and TSG101 and by transmission electron microscopy (TEM) analysis, the standard techniques to obtain biochemical and dimensional data of these nanovesicles. Total RNA extraction was performed with a high yield commercial kit. Due to the limited miRNA-content in exosomes, we decided to perform retro-transcription PCR using an individual assay for each selected miRNA. A panel of miRNAs (30b, 30c, 103, 122, 195, 203, 221, 222), all correlated with NSCLC disease, were analyzed taking advantage of the remarkable sensitivity and specificity of Real-Time PCR analysis; mir-1228-3p was used as endogenous control and data were processed according to the formula 2–ΔΔct 13. Control values were used as baseline and results are shown in logarithmic scale.
Introduction
非小细胞肺癌（非小细胞肺癌）患者生存率低的主要原因是在先进的疾病和不良的早期检测1治疗的疗效有限。在EGFR基因活化突变，已在非小细胞肺癌的患者的亚群被发现是已知赋予灵敏度酪氨酸激酶抑制剂（TKI中）。不幸的是，大多数EGFR突变的非小细胞肺癌患者的发展TKI阻力位2。特别是在这种情况下，正确的诊断是必需的。在一般情况下，由于肿瘤的定位，在非小细胞肺癌获得活检组织并不总是可行的。因此，一些研究是使用非侵入性的方法来获得这些生物数据正在进行。外来体被描述为可能，以获得与非侵入性技术3的诊断和预后值进行调查液体活检组件。
外来体是纳米囊泡 – 电子商务（40 100纳米直径）ndocytic原点由既在生理和病理条件4种不同类型的细胞释放。它们可以携带蛋白质，脂质，mRNA和微小RNA。此外，一些研究表明，肿瘤衍生的外来体，其可影响肿瘤细胞，血管生成，基质细胞和胞外基质重构和耐药5的生长和存活的多效性作用。
微RNA（miRNA）是所涉及的转录后调控，结合靶mRNA的3'-UTR以及导致mRNA的降解或到非翻译6短的非编码RNA。 miRNA的选择性排序成外来体已经描述。在这种情况下，最近证实， 在体外和体内中，miR-21（公知的miRNA用致癌效应）的姜黄素治疗7后由慢性髓性白血病细胞系释放外来的选择性包装。
该外来体miRNA谱是类似的原发肿瘤的miRNA谱和这个特征可在早期诊断和预后3被利用。具体地说，是表征，通过实时PCR一种可能性，与疾病的分子轮廓，相关选择的miRNA 例如 ，除其他8 EGFR突变。
这里，描述了非小细胞肺癌相关的miRNA 8-9的选定面板的分析，认为是从非小细胞肺癌患者的血液中释放的外来体分离。获得来自患者的知情同意报告后，12个NSCLC患者和6名健康对照的血浆，进行了分析。外来体分离按照生产商的方案用市售的试剂盒进行。外来体分离之前，血浆样品用RNA酶处理，以降低污染物循环的miRNA。我们决定来执行该分析用市售试剂盒，由于其他TECHNI有限标准化疑问句（ 例如 ，超离心）的临床样品时这是特别重要的。这种试剂盒包含一个蛋白酶K处理，这将降低RNA酶，并因此降低外来体裂解后降解外来体的miRNA的风险。被敏感，特异的实时PCR分析进行分析的microRNA;的mir-1228-3p用作内源的控制和数据进行标准化根据公式2 – ΔΔCT。控制值被用作基准和结果在对数标度示出。
Protocol
Representative Results
Discussion
用该组合协议是可能的从非小细胞肺癌患者血浆执行外来体miRNA的分析。这些数据可反映疾病的状态，但是这需要通过进一步研究加以证实。
在这篇文章中所使用的协议是基于切体隔离商业套件。其原因是，其它已知的分离方法（ 例如 ，密度梯度超速离心）需要更多的人工程序，从而导致有限的标准化。然而，该协议的局限性之一是，与用于外来体隔离密度梯度?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This study was also financed by MOCA (Multidisciplinair Oncologisch Centrum Antwerpen) grant 2014.
Materials
| Total exosome isolation kit (from plasma) | Invitrogen | 4484450 | Use Proteinase K treatment |
| Ribonuclease A | Sigma-Aldrich | R4875-100MG | |
| ALIX Antibody (3A9) | Cell Signaling | 2171S | |
| TSG-101 Antibody (C-2) | SantaCruz Biotecnology | SC-7964 | |
| Anti-Mouse HRP 1ml | Cell Signaling | 7076P2 | |
| RNAspin Illustra mini kit 50 | GE HealthCare | 25-0500-71 | |
| Taqman MicroRNA Reverse Transcription Kit | Life Technologies | 4366596 | |
| hsa-miR-1228-3p TaqMan MicroRNA assay | Life Technologies | 002919 | |
| hsa-miR-30b TaqMan MicroRNA assay | Life Technologies | 000602 | |
| hsa-miR-30c TaqMan MicroRNA assay | Life Technologies | 000419 | |
| hsa-miR-122 TaqMan MicroRNA assay | Life Technologies | 002245 | |
| hsa-miR-195 TaqMan MicroRNA assay | Life Technologies | 000494 | |
| hsa-miR-203 TaqMan MicroRNA assay | Life Technologies | 000507 | |
| hsa-miR-103 TaqMan MicroRNA assay | Life Technologies | 000439 | |
| hsa-miR-221 TaqMan MicroRNA assay | Life Technologies | 000524 | |
| hsa-miR-222 TaqMan MicroRNA assay | Life Technologies | 002276 | |
| TaqMan Universal Master Mix II, no UNG | Life Technologies | 4440043 |
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