固定化DNA-多肽探针-准定向蛋白质组学定量研究乳腺癌相关的microRNAs

microRNAs（miRNAs）的异常表达与乳腺癌的发生密切相关。寻找和发现这些miRNAs作为一类新型的生物标志物，对建立乳腺癌诊断新模式具有重要的意义。要实现这一目标，基本前提就是准确地定量测定miRNAs的表达水平。然而由于miRNAs自身的一些特点，目前采用的分析方法均存在一定缺陷。因此，我们提出了DNA-多肽探针-准定向蛋白质组学的方法理念。该方法通过DNA-多肽探针，首次将miRNAs的量化信息转变成报告多肽的信号，再用定向蛋白质组学测定。但是，我们之前采用的体系是先固定miRNAs，再与DNA-多肽探针反应，稳定性差、副反应多和干扰大。本项目中，我们将先共价固定DNA-多肽探针，然后与目标miRNAs杂交，并通过调整多肽序列、优化探针的固定方法以及杂交条件，提高检测准确度。最后，采用人乳腺癌和癌旁组织为模型，定量和比较目标miRNAs的水平，揭示与乳腺癌相关的miRNAs。

The distorted and unique expression of microRNAs (miRNAs) in breast cancer makes them an attractive source of biomarkers for early diagnosis. One of prerequisites for the application of miRNAs in clinical practice is to accurately profile their expression. However, most of the currently available assays for the quantification of miRNAs are ill-suited in complex biological mixtures. Thus，we are the first to develop a LC-MS/MS-based quasi-targeted proteomics assay. This approach actually converted the signal of target miRNAs into reporter peptide by a DNA-peptide probe and the reporter peptide was ultimately quantified using targeted proteomics. In our previous work, the DNA-peptide probe was hybridized with target miRNA, which was immobilized in advance. But this process was susceptible to several potential factors. Thus, we will covalently immobilize the DNA-peptide probe first in this study, followed by peptide sequence adjustment and optimization of immobilization and hybridization conditions. Finally, the developed assay will be applied to determine the amounts of target miRNAs in matched pairs of breast tissue samples. The results could facilitate the discovery of new biomarkers for breast cancer early detection.