应用单链特异性核酸酶检测血清miRNA标志物进行肺癌早期诊断

Early diagnosis is important for the prevention and treatment of lung cancer. Studies have shown that specific changes in the serum miRNA of lung cancer patients can be used as a novel biomarker, which has important value in the early diagnosis, staging, and prognosis evaluation of lung cancer. However, there are many defects in current miRNA detection techniques, such as high cost and complexity of operation, which hinder their clinical application. This project intends to develop a novel high-throughput miRNA detection technique with low-cost and easy to operate by applying a single-strand-specific nuclease (SSSN). Depending on the ability of the nuclease to specifically recognize and cleave unpaired single-stranded nucleotides and mismatched double-stranded nucleotides, we can check partially complementary and mismatched nucleotides in the hybridization between specific nucleic acid probes (fixed onto a 96-well enzyme-linked immunosorbent assay (ELISA) plate via the amide reaction) and target miRNA (extracted from the serum samples). Afterwards, the enzymatic reaction between horseradish peroxidase conjugated to the nucleic acid probes and a chemiluminescent substrate allows quick amplification of detectable fluorescence signals. The concentration of target miRNA can subsequently be determined from the fluorescence intensity. In this project, we also aim to utilize this novel technique to detect the serum miRNA biomarkers in the samples of nonsmall cell lung cancer patients and define its optimal application conditions, which assesses the sensitivity, the specificity, the practicability and the value of the technique in the early diagnosis of lung cancer. This project would provide a new method of clinical cancer diagnosis (e.g. lung cancer) for the future.

肺癌的早期诊断是肺癌防治的关键。研究显示肺癌患者血清中存在特异性变化的miRNA可作为一种新生物标志物，对肺癌的早期诊断、分期及预后评估具有重要价值。但是，现阶段miRNA检测技术存在成本高、操作复杂等缺陷，阻碍了其临床应用。本项目拟应用单链特异性核酸酶，开发一种低成本、操作简便、可高通量检测miRNA的新技术。通过核酸酶能够特异性识别并切割未配对单链核酸和碱基错配双链核酸的能力，检查核酸探针(酰胺反应连接至96孔酶标板)与目标miRNA(提取自血清)杂交中发生的错配和部分互补；然后利用结合到核酸探针上的辣根过氧化物酶与化学发光底物发生酶促反应产生荧光，快速放大检测信号；根据荧光强度确定目标miRNA含量。本项目拟用这一技术检测非小细胞肺癌患者血清样本中miRNA标志物，确定技术的最佳应用条件，并评估其灵敏度、特异性、实用性以及在肺癌早期诊断中的价值；为临床癌症(肺癌)的诊断提供新方法。

对于肺癌的防治来说，肺癌的早期诊断与检测是关键。目前研究发现血清中循环miRNA可作为一种新生物标志物，用于肿瘤的早期诊断和预后监测。但是临床上尚缺乏准确可靠、操作简便的血清循环miRNA检测技术。本项目首先应用了一种单链特异性核酸酶(SSSN)并结合核酸探针以及多孔板检测手段，开发了一套可直接检测血清中miRNA的化学发光检测新技术(DCDS)。根据已有文献报道并结合本项目的预定设计方案，我们先用配置的处理液处理血清样品从而使血清中的循环miRNA释放出来，从而无需进行miRNA提取。同时，我们将核酸探针通过酰胺反应和核酸杂交连接到96孔酶标板表面，再将处理好的样本加入，进行核酸杂交；然后应用一种SSSN核酸酶(RNase ONE 核糖核酸酶)来检查核酸探针与目标miRNA杂交后发生的错配或部分互补。最后，我们利用核酸探针上修饰的生物素连接链霉亲和素标记的辣根过氧化物酶，加入底物产生荧光，利用化学发光手段确定血清目标miRNA浓度。我们使用此DCDS技术检测了16例非小细胞肺癌(NSCLC)患者与10例健康人血清样本中的三种目标miRNA (miR-16、miR-25、miR-223)，发现NSCLC患者血清中的miR-25与miR-223表达水平比健康人血清高，与已有文献报道相符。与PCR技术相比，该检测技术的特异性高，得到的结果准确可靠，操作简便易行；其检测线性范围在10 fM至50 pM之间，最低能检测3.020 fM浓度的miRNA。在此基础上，我们又根据一种核酸部分互补的策略，重新设计了核酸探针，又开发出一套血清循环miRNA检测技术(iMEC)。此技术通过核酸探针与目标miRNA互补的严格性来保证结果的准确，而无需利用SSSN酶。此技术最低能检测1.248 fM浓度的miRNA，其线性检测范围在10 fM至50 pM之间。我们同样使用了iMEC技术检测了18例NSCLC患者与9例健康人血清样本中的两种目标miRNA (miR-16与miR-21)，得到的结果与PCR技术结果相比有很好的一致性(P > 0.05)。综上所述，我们开发了两套能够检测血清中循环miRNA标志物的新技术。这两套技术的检测结果准确可靠，操作简便易行；具有成本低、高通量、无需进行核酸提取与扩增等特点，将为未来基于循环miRNA标志物的肿瘤检测与诊断奠定了技术基础。