Construction And Application Of A New Type Of Tumor Marker MicroRNA Detection System

MicroRNA (miRNA) is a kind of noncoding small RNA, which can regulate gene expression at the post transcriptional level. MiRNA is ubiquitous in organisms and participates in regulation of many life processes, including cell proliferation, cell differentiation, cell apoptosis, individual development, disease occurrence, gene regulation and metabolism. The abnormality of miRNA is associated with human tumor disease. Therefore, quantitative detection of miRNA can provide a basis for the diagnosis, treatment and prognosis of tumor. Molecular beacon is one of the most ideal tools for quantitative detection of miRNA because of its high specificity, high sensitivity, etc. In this paper, we mainly focus on the detection of miRNA based on molecular beacon. The main contents are as follows:1. Establishment of a simple and rapid method for miRNA-204 detection in tumor samples based on molecular beaconIn this chapter, we designed a miRNA-204-targeted molecular beacon, through optimizing the condition of molecular beacon stability, hybrid performance and specific, constructing MB/miRNA-204 hybrid standard curve, we established a new method for quantitative detection of miRNA-204, which is fast, safe and specific, the detection limit reaches 1nM. We used this method to detect the expression level of miRNA-204 in tumor cells and found a decreased expression in MCF-7 and glioma tissues,which provide a reliable experimental basis for the further elucidation of the miRNA-204 in tumorigenesis.2. Ultrasensitive detection of miRNA-204 by combining molecular beacon with enzyme catalytic signal amplificationIn this chapter, we designed a molecular beacon fitted with RNA and DNA according to the characteristics of DSN enzyme. Through studying the influencing factors of enzyme promoting mediated signal amplification system and optimizing the experimental conditions, we established a sensitivity method for detecting miRNA-204 with a detection limit at PM level. Then we quantitatively detected the expression level of miR204 in tumor cells and glioma tissues using this system. The results is the same as that of direct detection, which indicated that the system was accurate and reliable. This method provides a new means for the detection of clinical samples, miRNA function, early diagnosis and prognosis of disease.3. Establishment of a simple and rapid method for Pre-miR-21 detection in tumor samples based on molecular beaconIn this chapter, through optimizing the hybridization conditions of MB/Pe-miR-21, studying the specificity of the MB and constructing standard curve for the detection of Pre-miR-21, we established a new method to detect hairpin precursor miRNA--Pre-miR-21 using molecular beacon. Then we detected the expression level of Pre-miR-21 in MCF-7 cells, glioma, gastric carcinoma, and verified the accuracy by qRT-PCR. The result showed that interfering Z38 gene expression in the MCF-7 cell can up regulate the expression of Pre-miR-21; the expression level of Pre-miR-21 in gastric cancer tissues is higher than adjacent tissues; the malignant degree of gliomas was positively correlated with the expression of Pre-miR-21. Molecular beacon has shown good application prospects in Pre-miR-21 detection.