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

描述(申请人提供)：非编码RNA，如microRNAs(MiRNAs)，已被确定为潜在的癌症检测生物标记物，因为它们的特征反映了许多肿瘤类型的发育谱系和分化状态。由于miRNAs比血液或其他体液中的mRNA等其他核酸更稳定，因为它们可以通过结合内源性RNase蛋白或包含在细胞分泌的纳米粒子(即外体)中而受到保护，因此使用稳定的miRNAs作为体液中有效的生物标记物为实现早期癌症的非侵入性检测提供了一种很好的手段。虽然已经使用定量逆转录聚合酶链式反应(qRT-PCR)结合超速离心或ExoQuickTM外切体沉淀试剂盒(SBI Inc.)等预浓缩技术对人血清或血浆中的miRNAs进行了定量检测，但这种方法繁琐、昂贵且耗时。在临床上，如果能开发一种简单、快速和低成本的方法作为常规的预先筛查工具，以便在癌症早期进行更深入但更具侵入性和更昂贵的乳房X光或磁共振成像(MRI)检测方法和疾病治疗，将是非常有价值的。我们最近开发了一种简单的链接型阳离子脂质体纳米颗粒(TCLN)生物芯片，该芯片以脂质体纳米颗粒中预加载的分子信标(MBS)为探针，无需任何样品前或样品后处理即可捕获和检测人血浆和细胞培养液中的靶向miRNAs。带负电的miRNAs，无论是与内源性RNase蛋白结合还是包含在外切体内，都可以很容易地被带正电的脂质体捕获并与其融合，从而可以通过MBS进行检测。由于靶向miRNAs和MB探针都被限制在纳米级的脂质体颗粒中(检测体积比聚合酶链式反应小1012倍)，因此该方法具有很高的检测灵敏度，而不需要像聚合酶链式反应那样进行扩增。类似的MB探针也能够捕获和鉴定同一样本中的mRNA。该tCLN生物芯片以miR-21锁定核酸(LNA)MB和TTF-1mRNA MB为生物标记物，成功地比较了肺癌细胞株A549和正常上皮细胞系细胞培养液中的外切体。使用肺癌患者血液样本的初步结果也证明了有希望的结果。肺癌是全球癌症死亡的主要原因，总的5年生存率为15%，令人失望。对患者友好的早期发现和监测 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。我们已经组建了一个强大的跨学科团队来进一步开发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个靶向miRNAs和mRNAs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本进行中试规模测试。

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.