Study On Quantitation Of Potential Biomarker Circulating Micrornas In Liver Cancer

microRNAs (miRNAs) are a highly conserved group of non-coding RNAs containing approximately 22 nucleotides. They regulate gene expression at the post-transcriptional level by binding to the 3’-untranslated region (3’-UTR) of messenger RNAs (mRNAs) and perform key functions in diverse cellular processes. Emerging evidence has demonstrated that the dysregulation of some specific miRNAs is associated with various clinicopathological conditions in liver diseases, and miRNAs have attracted tremendous attention as promising noninvasive diagnostic biomarkers. Recently, a number of miRNAs are observed circulating outside cells in biological fluids with dramatic stability in the form of encapsulated in lipid vesicles and/or in complexes of miRNA-protein. The lipid vesicles and miRNA-binding-proteins protect miRNAs from RNase digestion. Circulating miRNAs have close correlation with disease prognosis, indicating that the cell-free miRNAs serve as informative biomarkers for diverse hepatic pathological status.Precise quantitative analysis of miRNAs in specific cells, tissues and biological fluids is extremely important in the clinical application of miRNA-biomarkers. This thesis establishes a novel miRNAs quantitative strategy with the advantages of high specificity, low detection limit and wide linear range. This method is feasible in miRNAs detection from clinical serum samples with good accuracy, and provides reliable and valuable information for the early detection of miRNA-related diseases. The main contents are as follows:1. Establishment and application of miRNAs detection method based on magnetic nanoparticles and chemiluminescenceWe report a new approach for miRNAs analysis based on the capture probe conjugated with magnetic nanoparticls (MNPs) and the reporter probe labeled with biotin. Through the biotin-streptavidin interaction, the alkaline phosphatase (ALP) is conjugated to MNPs, and then interacts with its substrate 3-(2’-spiroadamantane)-4-methoxy-4-(3’’-phosphoryloxy) phenyl-1,2-dioxetane (AMPPD) to generate chemiluminescence (CL) signals. The proposed assay displays a good specificity with low detection limit of 60 fM and a wide linear relationship in the range from 10-2 to 104 pM. Moreover, the proposed approach shows great feasibility in analyzing miRNAs from serum samples and has potential in practical application.2. Application of miRNAs detection method based on quantitative real-time polymerase chain reactionWe use the quantitative real-time polymerase chain reaction (qRT-PCR) to detect miR-21 in serum samples. The obtained quantitative results are compared with what from the proposed approach. The copy numbers from these two methods are excellently consistent, indicating that the developed assay offers unambiguous accuracy and repeatability in miRNAs quantitation.3. Chemiluminescent detection of relative expression levels of miRNAs in liver cancerWe use the proposed method to detect relative expression levels of miR-21, miR-122 and miR-221 in serum samples (normalized to miR-16) and evaluate their diagnostic values. These three miRNAs have great diagnostic power for liver cancer against healthy group and show potentials as biomarkers in clinical application.