Duplex-Specific Nuclease-Mediated Amplification Strategy for Mass Spectrometry Quantification of MiRNA-200c in Breast Cancer Stem Cells
Duplex-Specific Nuclease-Mediated Amplification Strategy for Mass Spectrometry Quantification of MiRNA-200c in Breast Cancer Stem Cells
复制标题
用于乳腺癌干细胞中 miRNA-200c 质谱定量的双链体特异性核酸酶介导的扩增策略
DOI：
10.1021/acs.analchem.8b04468
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发表时间：
2019-07-16
影响因子：
7.4
通讯作者：
Chen, Yun
中科院分区：
文献类型：
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作者：
Kuang, Yuqiong;Cao, Jianxiang;Chen, Yun
microRNA（miRNAs）在许多生物学过程中发挥重要作用，并与包括乳腺癌在内的一系列癌症有关。MiRNA具有成为临床实践中生物标志物的潜力，因为它们的扭曲和独特表达，特别是它们存在于癌症干细胞（CSC）中，可应用于癌症诊断和治疗。因此，miRNA表达水平的绝对确定是探索其应用的先决条件。然而，目前可用的方法可能不足以检测CSC中的miRNA，这是由于这些细胞的固有低群体。因此，我们结合了一种特异性核酸酶（DSN）介导的扩增策略与液相色谱-串联质谱（LC-MS/MS）用于本研究。我们设计了底物肽GDKAVLGVDPFR，其含有报告肽AVLGVDPFR和胰蛋白酶切割位点（在位置3的赖氨酸）以及与靶miRNA互补的生物素化DNA序列（即，miR-200c）。然后，将这种新合成的DNA-肽探针与靶miRNA杂交。在引入DSN后，酶将探针降解成两部分，其中靶miRNA被整合，从而触发进一步切割。最终用胰蛋白酶消化积累的肽片段以释放报告肽用于LC-MS/MS定量。在这些情况下，miRNA信号被转换并放大为报告肽的质量响应。在优化参数（包括温度、杂交/DSN时间、DSN和链霉亲和素琼脂糖珠的量）后，我们证明了miR-200 c的线性检测范围在1 fM和200 fM之间。得到的检测限比以前报道的低3个数量级。最后，对乳腺癌干细胞（BCSC）和从乳腺肿瘤分离的干细胞中的miR-200 c进行定量。我们还将这些数据与定量逆转录PCR（qRT-PCR）结果进行了比较。
MicroRNAs (miRNAs) play a significant role in numerous biological processes and are implicated in a range of cancers, including breast cancer. MiRNAs have the potential to be biomarkers in clinical practice because of their distorted and unique expression, especially with regard to their presence in cancer stem cells (CSCs) that have applications in cancer diagnosis and treatment. Thus, the absolute determination of miRNA expression levels is a prerequisite for exploring their applications. Nevertheless, currently available methods may not be adequate for the detection of miRNAs in CSCs due to the inherently low population of these cells. Therefore, we combined a duplex-specific nuclease (DSN)-mediated amplification strategy with liquid chromatography-tandem mass spectrometry (LC-MS/MS) for use in this study. We designed the substrate peptide GDKAVLGVDPFR, which contains the reporter peptide AVLGVDPFR and a tryptic cleavage site (lysine at position 3) in combination with a biotinylated DNA sequence that was complementary to a target miRNA (i.e., miR-200c). Then, this newly synthesized DNA-peptide probe was hybridized with the target miRNA. Upon the introduction of the DSN, the enzyme degraded the probe into two parts where the target miRNA was integrated and thereby triggered further cleavage. The accumulated peptide fragments were ultimately digested with trypsin to release the reporter peptide for LC-MS/MS quantification. Under these circumstances, the miRNA signal was converted and amplified into a mass response of the reporter peptide. After optimization of the parameters, including temperature, hybridization/DSN time, and the amounts of DSN and streptavidin agarose beads, we demonstrated a linear detection range between 1 fM and 200 fM for miR-200c. The detection limit obtained was 3 orders of magnitude lower than those previously reported. Finally, quantification of miR-200c in breast cancer stem cells (BCSCs) and in stem cells isolated from breast tumors was performed. We also compared these data with quantitative reverse transcription PCR (qRT-PCR) results.