Selection and appraisement of nucleic acid aptamers for castration resistant prostate cancer cells based on Cell-SELEX / 西安交通大学学报(医学版)

ABSTRACT

【Objective】 To screen nucleic acid aptamers that can specifically recognize castration-resistant prostate cancer （CRPC） cells by Cell-SELEX. 【Methods】 For Cell-SELEX selection， CRPC cell lines C4-2 were used as positive control cells， while androgen-dependent prostate cancer cell lines LNCap were used as negative control cells. The 5’end of the upstream primers was labeled with FITC， and the 5’end of the downstream primers was labeled with biotin. Single-stranded DNA （ssDNA） collected from each round of screening was used as template for PCR amplification and purification. The biotin-streptavidin magnetic bead separation was used to isolate PCR product for the next round of screening. The process of Cell-SELEX was analyzed by flow cytometry. ssDNA products of the last round were collected for PCR amplification， purification， cloning and DNA sequencing. The secondary structure of selected DNA aptamers was predicted. Dissociation constants of the two aptamers were calculated. Flow cytometry and confocal microscopy were used to evaluate selective binding of aptamers to CRPC cells and tissues. 【Results】 The binding rate of DNA products to CRPC cells gradually increased with the increase of selection cycles， reaching the highest in the last round. DNA structure prediction analysis showed that the secondary structure of aptamers CRPC-1 and CRPC-2 was mainly stem-loop structure. Flow cytometry analysis and confocal images showed that both CRPC-1 and CRPC-2 could specifically target C4-2 cells. In addition， immunohistofluorescence assay showed that CRPC-1 could specifically target CRPC tissues. 【Conclusion】 Cell-SELEX can be used to screen aptamers that specifically target CRPC cells and tissues， which provides experimental basis for early screening and targeted diagnosis of CRPC. It is of significance for treatment plan adjustment and prognosis improvement of prostate cancer.