Study On Detection Methods For Epigenetic Nucleic Acid In Breast Cancer

Breast cancer is the most common malignant disease in women. Its prevalence rate has exhibited a clear increase in China. Furthermore, the mortality rate of breast cancer has risen by 38.9% in the last 20 years. Current prognostic criteria only poorly predict the metastasis risk for an individual breast cancer patient due to the lack of specific biomarkers. Thus, new prognostic biomarkers are urgently needed to identify breast cancer patients and those at the highest risk for developing metastasis. With the development of molecular biology, cell biology, analytical chemistry, new biomarker and detecting technology are used in breast cancer research.1. Diagnostic and prognostic value of plasma and tissue UHRF1 in breast cancer patientsUHRF1 (ubiquitin-like, containing PHD and RING finger domains 1) has been reported to play an important role in breast carcinogenesis. This work investigated the correlation of UHRF1 DNA level in plasma with clinical characteristics of breast cancer and its clinical significance in breast cancer diagnosis. The expression of UHRF1 in primary breast cancer tissue was examined by Western blot. The UHRF1 DNA levels in plasma and UHRF1 mRNA expression in tissues were determined by accurate real-time quantitative PCR. The associations of UHRF1 levels with clinical variables were evaluated using standard statistical methods. The UHRF1 DNA in plasma of 229 breast cancer patients showed higher expression than healthy controls, which showed high specificity up to 76.2% at a sensitivity of 79.2%, and was significantly associated with c-erbB2 positive status, cancer stage and lymph node metastasis. High UHRF1 DNA level in plasma was significantly associated with short progression-free survival.The UHRF1 DNA level in plasma is highly correlative with breast cancer and its status and stage, and may be a potential independent diagnostic and prognostic factor for both breast cancer and the survival of breast cancer patients.2. Cellular delivery of quantum dot-bound hybridization probe for detection of intracellular pre-microRNA using chitosan/poly(y-glutamic acid) complex as a carrierA quantum dot (QD)-bound hybridization probe was designed for detection of intracellular pre-miRNA using chitosan (CS)/poly(y-glutamic acid) (y-PGA) complex as a gene vector. The probe was prepared by assembling thiolated RNA to gold nanoparticle (Au NP) via Au-S bond and then binding 3’-end amine of the RNA to the carboxy group capped on quantum dot surface. The QD-RNA-Au NP probe was assembled on the vector by mixing with aqueous y-PGA solution and then CS solution to construct a gene delivery system for highly effective cellular uptake and delivery. After the probe was released from CS/y-PGA complex to the cytoplasm by electrostatic repulsion at intracellular pH, it hybridized with pre-miRNA precursor as target. The formed product was then cleaved by RNase III Dicer, leading to the separation of QDs from Au NPs and fluorescence emission of QDs, which could be detected by confocal microscopic imaging to monitor the amount of the intracellular pre-miRNA precursor. The in vitro assays revealed that the QD-RNA-Au NP was a robust, sensitive and selective probe for quantitative detection of target pre-miRNA. Using MDA-MB231 and MCF-7 breast cancer cells as models, the relative amount of pre-miRNA let-7a could be successfully compared. Since the amount of miRNA is related to the progress and prognosis of cancer, this strategy could be expected to hold promising application potential in medical research and clinical diagnostics.3. Colorimetric methylation analysis of p16/CDKN2 promoter with hyperbranched rolling circle amplificationDNA methylation is a hallmark of the epigenetic regulation of gene expression. High efficient DNA methylation analysis is emergently demanded for early cancer detection. We developed a simple, fast, sensitive and economical assay for DNA methylation analysis with hyperbranched rolling circle amplification (HRCA) and enzyme-based colorimetric detection. The assay was carried out on an aptamer modified 96 cell microplate with 4 steps, including target recognition, methylation-sensitive endonuclease digestion, isothermal HRCA process, and colorimetric readout. We examined the p16 methylation in cell-free DNA of 72 breast cancer patients and 43 healthy individuals. The proposed DNA methylation assay could be accomplished within 2.5 h. The strategy exhibited excellent detection specificity and showed a log-linear response in methylated DNA concentration from 100 fM to 10 nM. The p16 promoter was methylated in 57 (79.2%) of 72 patients with breast cancer, and in 4 (9.3%) of 43 healthy individuals. The median concentrations of the methylated p16 for the cancer and healthy groups were 1.2×1012 and 5.5×1011 copies/L, respectively(P<0.0001, Wilcoxon test). There was no significant association between the methylated p16 concentration and the clinical parameters, such as age, menopausal, grade, histology grade, stage and lymph node metastasis, of the patients. The colorimetric DNA methylation analysis combining an isothermal HRCA-based signal enhancement allowed efficient methylation detection with simplicity, rapidness, low cost and high sensitivity, showing great promise for further applications in early diagnosis of methylation-related diseases as well as high-throughput analyses.