School of Pharmacy , Nanjing Medical University , Nanjing 211166 , China.
State Key Laboratory of Reproductive Medicine , Nanjing Medical University , Nanjing 210029 , China.
Anal Chem. 2019 Jul 16;91(14):8820-8826. doi: 10.1021/acs.analchem.8b04468. Epub 2019 Jun 27.
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.
微小 RNA（miRNA）在许多生物过程中发挥着重要作用，并与包括乳腺癌在内的多种癌症有关。由于 miRNA 表达的扭曲和独特性，特别是它们在具有癌症诊断和治疗应用的癌症干细胞（CSC）中的存在，miRNA 有可能成为临床实践中的生物标志物。因此，绝对确定 miRNA 表达水平是探索其应用的前提。然而，由于这些细胞的固有低群体，目前可用的方法可能不足以检测 CSCs 中的 miRNA。因此，我们结合了双特异性核酸酶（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，它包含报告肽 AVLGVDPFR 和一个胰蛋白酶切割位点（位置 3 的赖氨酸），并与互补于靶 miRNA（即 miR-200c）的生物素化 DNA 序列结合。然后，这种新合成的 DNA-肽探针与靶 miRNA 杂交。引入 DSN 后，酶将探针降解成两部分，其中靶 miRNA 被整合并由此触发进一步切割。积累的肽片段最终用胰蛋白酶消化以释放用于 LC-MS/MS 定量的报告肽。在这种情况下，miRNA 信号被转化并放大为报告肽的质量响应。在优化包括温度、杂交/DSN 时间以及 DSN 和链霉亲和素琼脂糖珠的量等参数后，我们证明了 miR-200c 的线性检测范围在 1 fM 至 200 fM 之间。获得的检测限比以前报道的低 3 个数量级。最后，在乳腺癌干细胞（BCSCs）和从乳腺癌肿瘤中分离的干细胞中进行了 miR-200c 的定量。我们还将这些数据与定量逆转录 PCR（qRT-PCR）结果进行了比较。
