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

• 批准号: 8735903
• 负责人: Ly James Lee
• 金额: $ 19.49万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-17 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8735903
• 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

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，例如 microRNA (miRNA) 已被确定为癌症检测的潜在生物标志物，因为它们的概况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 mRNA 等其他核酸更稳定，因为它们可以通过与内源性 RNase 蛋白结合或包含在细胞分泌的纳米颗粒（即外泌体）中而受到保护，因此使用稳定的 miRNA 作为体液中可行的生物标志物提供了实现早期癌症检测的非侵入性检测的绝佳方法。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（例如超速离心或 ExoQuickTM 外泌体沉淀试剂盒（SBI Inc.））对人血清或血浆中的 miRNA 进行了定量测量，但这种方法繁琐且昂贵并且耗时。对于临床使用来说，如果能够开发出一种简单、快速且低成本的方法作为常规预筛查工具，以便进行更深入但更具侵入性和更昂贵的乳房X光检查或磁共振成像（MRI）检测，那将非常有价值癌症早期的后续治疗方法和疾病治疗都可以进行。我们最近开发了一种简单的系留阳离子 Lipoplex 纳米颗粒 (tCLN) 生物芯片，在 lipoplex 纳米颗粒中预装了分子信标 (MB) 作为探针，可捕获和检测人血浆和细胞培养基中的目标 miRNA，而无需任何预加载或后加载。样品处理。带负电荷的 miRNA，无论是与内源性 RNase 蛋白结合还是包含在外泌体中，都可以轻松地被带正电荷的脂质体捕获并与其融合，从而允许 MB 进行检测。由于目标miRNA和MB探针都被限制在纳米大小的脂质体颗粒内（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，而不需要像PCR那样进行扩增。类似的 MB 探针也能够捕获和识别同一样本中的 mRNA。使用 miR-21 锁核酸 (LNA) MB 和 TTF-1 mRNA MB 作为生物标志物，该 tCLN 生物芯片已成功比较从肺癌细胞系 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 年生存率仅为 15%，令人失望。对患者友好的早期检测和监测 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) 使用明确的人类血浆样本进行中试规模测试来自早期肺癌患者和吸烟者。