基于多道DNA-Peptide探针/准定向蛋白质组学法的microRNA定量方法研究

Studies have demonstrated that determination of multiple miRNAs in tumor diagnosis is more efficient than a single miRNA, which is of great scientific value and research significance. However, one of the prerequisites for the use of multiple miRNAs in clinical diagnosis is the accurate quantification. On the basis of the previous research work and the pre-experimental results of high-responding peptides, we will convert the signal of multiple miRNAs into reporter peptides by synthesized and identified multiple DNA-Peptide Probe and the reporter peptides are ultimately quantified using LC-MS/MS-based targeted proteomics. Based on the method, we will further optimize the experimental parameters in each step and a number of breast cancer samples will be used for the validation of biological systems and the improvement of the methodology additionally. This method will redirect the advantages of proteomics such as high-throughput, high sensitivity, specificity and absolute quantification to the study of miRNAs, while possesses quick and simple operation, especially with no need of miRNA enrichment or amplification and so on. The research will help to provide an efficient, fast, and high throughput absolute quantification method for the detection of multiple miRNAs, also a new theoretical basis for the correlation between multiple miRNAs and clinical breast tumor accurate diagnosis.

研究表明，在肿瘤诊断中多组分miRNAs联合监测比单个miRNA效率更高，具有重要的科学价值和研究意义。然而，将多组分miRNAs用于临床诊断的先决条件之一就是精确定量其表达水平。本项目将以前期工作为研究基础，在预实验筛选多个高响应报告肽段的基础上，合成多道DNA-多肽探针，把多组分miRNAs的量化信号转化为对多肽的检测，利用定向蛋白质组学质谱多反应监测技术实现对它们的精确定量；并进一步探索优化各步骤实验参数，在一定数量的乳腺癌生物样本中进行系统验证和方法学的完善。该方法将定向蛋白质组学的高通量、高灵敏度、高特异性且能够绝对定量的优点成功地嫁接到 miRNAs 的分析研究，同时还兼具操作简单快速、无需对miRNA进行富集或扩增等优点。本项目有望为多组分miRNAs的检测提供一种高效、快捷、通量化的绝对定量新方法，并为研究多组分miRNAs与临床肿瘤精确诊断的相关性提供新的理论依据。

肿瘤诊断中，多组分miRNAs联合监测比单个miRNA效率更高，具有重要的科学价值和研究意义。然而，将多组分miRNAs用于临床诊断的先决条件之一就是精确定量其表达水平。针对目前多组分miRNAs精确定量的不足，我们开发了基于多道DNA-Peptide探针/准定向蛋白质组学法的microRNA定量方法研究。通过信息学预测和质谱实验筛选多个高响应报告肽段的基础上，合成多道DNA-多肽探针，利用DNA-多肽探针把多组分miRNAs的量化信号转化为对多肽的检测，利用靶向蛋白质组学质谱的多反应监测技术实现对它们的精确定量；在验证了探针的合成和稳定性的基础上，考察了杂交的特异性和敏感性。此外，我们利用了数学矩阵校正了DNA-多肽探针与非靶miRNA的非特异性杂交，并利用完善的方法学用来检测乳腺癌细胞系和一定数量的乳腺癌生物样本中。本项目为多组分miRNAs的检测提供一种高效、快捷、通量化的绝对定量新方法，并为揭示多组分miRNAs与乳腺癌发生发展的相关性提供数据支持。