Selection And Characterization Of DNA Aptamer For Prostate Cancer

Prostate cancer is the most common cancer except lung cancer in men. The main method of early screening and diagnosis is to detect the level of prostate specific antigen (PSA), however, it lacks specificity due to the PSA also can change when the prostate cells are pathological. Furthermore, the metastasis of cancer is the main cause of death, which is a huge challenge to cure prostate cancer. Therefore, it is important to find some specific molecules for early screening and diagnosis of prostate cancer. Aptamers are some 20-80nt ssRNA or ssDNA, which bind target molecules by three-dimensionally. Aptamers own the advantage of high affinity and specificity and it is easy to synthesis and modify, it develop rapid in biochemistry and biomedicine recently and become a novel and efficient method to study malignant tumor. It is promising to apply aptamers in biomedical and clinical area. We use cell-SELEX to selected an aptamer which target prostate cancer cells, it provided a new clue and direction for prostate cancer study.In this paper, we selected an aptamer against AR-negative human prostate cancer cells DU145 by cell-SELEX and analysis the characteristic of it. The aptamer selected in this paper is expected to be a new biomarker for prostate cancer diagnosis and treatment. The research content mainly conclude:(1) We adopted the cell-SELEX strategy to obtain a DNA aptamer, termed DML-7. DML-7 binds to the classical DU145 metastatic prostate cancer cell line with high affinity. The human prostatic stromal myofibroblast cell line, WPMY-1 is used as the negative cell, which aptamer DML-7 couldn’t bind specifically. In selection process, the selection conditions were optimized, and the stringency was gradually increased according change cell numbers, the incubation time with target cells and negative cells. We conducted 18 round screening and the ssDNA is used for sequencing.(2) To make aptamer DML-7 better used in prostate cancer diagnosis and treatment, we investigate different property of aptamer DML-7. The sequence DML-7 was truncated by gradually removing the nucleotides at the 5’and 3’termini, the results showed that all sequences in DML-7 is indispensable. The dissociation constant is 49.4 ±5.1 nM which is in nanomolar scale, showing high binding affinity towards DU145 cells. It also demonsrates good temperature stability. The aptamer can internalize into cells in physiological state which indicate that it is a good candidate for molecular probe. When deal with different proteinase the binding ability of DML-7 and is not affected. We suggested that maybe the target of DML-7 are some protein which are resisted to proteinase or it has high glycosylation degree. We also characteize DML-7 binding to other cell lines and the results showed that DML-7 could bind to AR-negative PCa cells and some adherent cancer cells but not AR-positive cell lines and suspension cancer cells, so we concluded that the target of DML-7 may related with cell adhesion and metastasis. What’s more, we test the ability of DML-7 to recognize targets under complex situations such as in clinical tissues, the results showed that DML-7 displayed strong binding and recognition ability for clinical metastatic Pca tissues.