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Plasma RNA based Early Lung Cancer Detection by Tethered Cationic Lipoplex Assay

通过系留阳离子脂质复合物检测进行基于血浆 RNA 的早期肺癌检测

基本信息

批准号：
8570641
负责人：
Ly James Lee
金额：
$ 16.7万
依托单位：
OHIO STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-09-17 至 2015-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8570641
关键词：
A549 Address Area Binding Biological Assay Biological Markers Biology Blood Blood Circulation Blood specimen Body Fluids Cancer Detection Cancer Etiology Cancer Patient Cancer cell line Cell Culture Techniques Cell Line Cells Centrifugation Cessation of life Charge Clinic Clinical Comprehensive Cancer Center Country Culture Media Detection Development Devices Diagnostic Diagnostic Neoplasm Staging Disease Disease model Doctor of Medicine Doctor of Philosophy Early Diagnosis Enrollment Epithelial Cells Evaluation Fluorescence Functional RNA Heterogeneity Human Knowledge Liposomes Magnetic Resonance Imaging Malignant Neoplasms Malignant neoplasm of lung Mammography Measures Messenger RNA Methods MicroRNAs Molecular Nanotechnology Nucleic Acids Ohio Patients Performance Phenotype Plasma Plasma Cells Polymerase Chain Reaction Precipitation Process Proteins RNA Research Personnel Reverse Transcription Ribonucleases Role Sampling Screening for cancer Serum Signal Transduction Smoker Staging Surveillance Methods Survival Rate Techniques Technology Testing Time Tumor Biology Universities Untranslated RNA Woman base biochip clinical decision-making cohort cost design experience follow-up high risk imaging modality locked nucleic acid lung cancer screening men mortality nanoparticle nanoscale novel particle prognostic programs public health relevance research clinical testing screening socioeconomics statistics tool treatment planning tumor

项目摘要

DESCRIPTION (provided by applicant): Non-coding RNAs such as microRNAs (miRNAs) have been identified as potential biomarkers for cancer detection because their profiles reflect developmental lineage and differentiation state of many tumor types. Since miRNAs are more stable than other nucleic acids such as mRNA in blood or other body fluids because they could be protected by bounding to endogeneous RNase proteins or contained within cell secreted nanoparticles, i.e. exosomes, using stable miRNAs as viable biomarkers in body fluids provide an excellent means to achieve noninvasive assays for early cancer detection. Although miRNAs have been quantitatively measured in human sera or plasma using quantitative reverse transcription polymerase chain reaction (qRT-PCR) with pre-concentration techniques such as ultra-centrifugation or ExoQuickTM exosome precipitation kit (SBI Inc.), this approach is tedious, expensive and time consuming. For clinic use, it would be highly valuable if a simple, fast and low-cost method can be developed as a routine pre-screening tool such that more in-depth but also more invasive and expensive mammography or magnetic resonance imaging (MRI) detection methods as well as disease treatment can be conducted as follow-up in early stage of cancer. We recently developed a simple tethered Cationic Lipoplex Nanoparticle (tCLN) biochip with pre-loaded molecular beacons (MBs) in the lipoplex nanoparticles as probes to capture and detect targeted miRNAs in human plasma and cell culture medium without any need of pre- or post-sample treatment. The negatively charged miRNAs, whether being bounded to endogeneous RNase proteins or contained within exosomes, can be easily captured by and fused with positively charged liposomes, allowing detection by MBs. Since both targeted miRNAs and MB probes are all confined within nanometer sized liposome particles (detection volume 1012 times smaller than that in PCR), this method provides very high detection sensitivity without the need of amplification as in PCR. Similar MB probes are also able to capture and identify mRNA in the same sample. This tCLN biochip has been successfully demonstrated in comparing exosomes isolated from cell culture media of a lung cancer cell line, A549 and a normal epithelial cell line using miR-21 locked nucleic acid (LNA) MB and TTF-1 mRNA MB as biomarkers. Preliminary results using lung cancer patient blood samples also demonstrate promising results. Lung cancer is the leading cause of cancer deaths worldwide with a disappointing 15% overall 5-year survival rate. A patient-friendly early detection and surveillance method would substantially reduce the mortality in this serious disease. The same technique may also be applied to many other cancers. We have assembled a strong interdisciplinary team to further develop the tCLN biochip method and to evaluate its feasibility in lung cancer patients. Our primary objectives are (1) to optimize the tCLN biochip and to evaluate its performance for capture and detection of 5-6 targeted miRNAs and mRNAs in lung cancer patient plasma, and (2) to conduct pilot scale test using well defined human plasma samples from early lung cancer patients and smokers.

描述（由申请人提供）：非编码RNA如microRNA（miRNA）已被鉴定为癌症检测的潜在生物标志物，因为它们的谱反映了许多肿瘤类型的发育谱系和分化状态。由于miRNA比血液或其他体液中的其他核酸如mRNA更稳定，因为它们可以通过结合到内源性RNA酶蛋白质或包含在细胞分泌的纳米颗粒（即外来体）内而受到保护，因此使用稳定的miRNA作为体液中的可行生物标志物提供了实现用于早期癌症检测的非侵入性测定的极好手段。尽管已经使用定量逆转录聚合酶链反应（qRT-PCR）和预浓缩技术如超离心或ExoQuickTM外泌体沉淀试剂盒（SBI Inc.）定量测量了人血清或血浆中的miRNA，这种方法是乏味、昂贵和耗时的。对于临床应用，如果能够开发一种简单、快速和低成本的方法作为常规的预筛选工具，使得能够在癌症的早期阶段进行更深入但也更侵入性和昂贵的乳腺X射线摄影或磁共振成像（MRI）检测方法以及疾病治疗作为随访，则将是非常有价值的。我们最近开发了一种简单的拴系阳离子Lipoplex纳米颗粒（tCLN）生物芯片，在Lipoplex纳米颗粒中预加载分子信标（MB）作为探针，以捕获和检测人血浆和细胞培养基中的靶向miRNA，而无需任何样品前或后处理。带负电荷的miRNA，无论是与内源性RNA酶蛋白结合还是包含在外来体内，都可以容易地被带正电荷的脂质体捕获并与之融合，从而允许通过MB进行检测。由于靶向的miRNA和MB探针都被限制在纳米尺寸的脂质体颗粒内（检测体积比PCR小1012倍），因此该方法提供了非常高的检测灵敏度，而不需要PCR中的扩增。类似的MB探针也能够捕获和鉴定相同样品中的mRNA。该tCLN生物芯片已成功地证明了使用miR-21锁核酸（LNA）MB和TTF-1 mRNA MB作为生物标志物比较从肺癌细胞系A549和正常上皮细胞系的细胞培养基中分离的外泌体。使用肺癌患者血液样本的初步结果也显示出有希望的结果。肺癌是全球癌症死亡的主要原因，总体5年生存率为15%，令人失望。对患者友好的早期检测和监测这种方法将大大降低这种严重疾病的死亡率。同样的技术也可以应用于许多其他癌症。我们组建了一支强大的跨学科团队，进一步开发tCLN生物芯片方法，并评估其在肺癌患者中的可行性。我们的主要目标是（1）优化tCLN生物芯片并评估其捕获和检测肺癌患者血浆中5-6种靶向miRNA和mRNA的性能，以及（2）使用来自早期肺癌患者和吸烟者的明确定义的人血浆样品进行中试规模测试。