Development of diagnostic platforms for extracellular protease activity and intracellular lncRNA in cancer cells

Abstract

With the recent advances in cancer imaging and diagnosis, a number of biomarkers identified from the genome and proteome have been targeted to assess cancer presence, tumor stage, and aggressiveness. To date, emerging evidence has been accumulating that new reliable biomarkers-extracellular enzymes or long non-coding RNAs (lncRNAs)-are strongly involved in cancerous microenvironment or tumorigenesis. However, despite the recent efforts to detect these biomarkers, reliable and sensitive detection methods still remain challenging due to the lack of technological development. In this thesis, I proposed two newly developed methods for efficiently detecting potential biomarkers: an imaging method for detection of metastasis-related enzymes in the extracellular matrix (ECM) and a diagnostic method for detection of lncRNA in the urine of prostate cancer patients. In the first part, a novel extracellular fluorescence resonance energy transfer (FRET) reporter was demonstrated, which can visualize matrix metalloproteinase-2 (MMP2) activity as an indicator of tumor metastasis characteristics of HeLa cells in two dimensional and three dimensional culture conditions. The extracellular reporter comprises a collagen-binding protein and a FRET coupler of enhanced green fluorescent protein and small dye molecule. The stable, matrix-sticky, and protease-sensitive extracellular reporter via split intein-mediated protein trans-splicing is able to adhere to collagen matrices, leading to fluorescence changes by MMP2 activity during living cell culture without impeding cell viability. In the second part, a sensitive and convenient method was demonstrated to assay prostate cancer antigen 3 (PCA3) and prostate-specific antigen (PSA) genes using single-stranded (ss) DNAzyme-facilitated chemiluminescence for prostate cancer (PC) diagnosis. By tethering peroxidase-mimicking ssDNAzyme sequence to PSA or PCA3-targeting primers, target genes were amplified through only PCR process, which subsequently enabled the chemiluminescence (CL) detection in the presence of hemin and luminol substrate. A ratiometric CL analysis of PCA3/PSA expression effectively discriminated the difference between patient and normal groups in human urine. The proposed methods are immediately amenable for monitoring cancer biomarkers and will open the new route to image or diagnose many diseases in the biomedical field.