Screening And Characterization Of DNA Aptamers For Human Bladder Cancer Cells

Bladder cancer is one of the most common malignant tumors in the urinary system,which holds the first incidence rate in the urological malignancies and bladder urothelial carcinoma accounts for more than 90% of bladder cancer.In general,the screening and early diagnosis of bladder cancer can help improve patient survival.At present,the early diagnosis of bladder cancer is based on cystoscopy and cytology as the gold standard.However,the cystoscopy is traumatic and expensive,and the sensitivity of urine cytology for early low-grade bladder cancer is extremely low.In addition,finding urinary tumor markers for bladder cancer diagnosis has also become a hot spots,but the application of urinary tumor markers is still lack of prospective in actual clinical practice.Therefore,the development of new and reliable methods for the early screening and treatment of bladder cancer and the prediction of the risk of disease is of great significance.Aptamers are short single-stranded oligonucleotide sequences obtained by screening in vitro,which bind to the target with a high specific affinity and high affinity through folding into a specific secondary structure.Since the aptamer has a series of characteristics superior to the antibodies including a small molecular weight,a wide range of targets and low immunogenicity,it has been rapidly developed in the fields of biomedicine and biochemistry.In particular,as a new and efficient tool for cancer research,aptamer has become the focus of modern molecular diagnostic medicine.To obtain a aptamer that specifically recognizes bladder urothelial carcinoma,human bladder urothelial carcinoma cell line 5637 was used as target cells for positive selection and the human immortalized urothelial cell line SV-HUC-1 was used as control cells for counter selection.Based on cell-SELEX technology,a series of studies were performed in this paper.(1)Screening of aptamer against bladder cancer cell line 5637We adopted the cell-SELEX strategy to obtain a DNA aptamer against 5637 cells.After 13 rounds of screening and enrichment,ssDNA pool was sequenced by a high-throughput sequencing device.Eight representative sequences were chosen and synthesized for further characterization.We found that aptamer spl3 can recognize 5637 cells but not SV-HUC-1 cells.In addition,the binding affinity of spl3 on target cells is less affected by temperature.(2)Identification and optimization of the properties of aptamer spl3To determine the binding specificity of aptamer spl3,we examined the bind ing of aptamer spl3 to different tissue-derived tumor cell lines and non-tumor cell lines by flow cytometry.We found that aptamer spl3 bound to bladder cancer cells with high specificity.The truncation analysis of the aptamer spl3 based on secondary structure simulation by the software showed that truncated sequence spl3 c had stronger binding ability to 5637 cells than full-length sequence spl3.The dissociation constants of the spl3 and spl3 c to 5637 cells were 23.08 nM and 18.99 nM,respectively.Target type analysis revealed that both aptamer spl3 and spl3 c might bind to cell membrane proteins,whereas competitive analysis showed that spl3 and spl3 c might share the same protein target.Meanwhile,we found the binding of aptamer spl3 c to target cells was not affected by temperature.Internalization experiments further showed that aptamer spl3 c can undergo a certain degree of internalization.The half-life of aptamer spl3 without any modification was 24 h in 10% FBS,and that of spl3 c was 10 h.Both of them showed stronger stability with potential for applications in vivo.In order to investigate the ability of aptamers to recognize bladder cancer under more complicated conditions,we characterized the ability of spl3 c to recognize the bladder cancer cell 5637 in tumor-bearing nude mice in vivo by tail vein injection and in vivo imaging system.We found that spl3 c was able to recognize bladder tumors in vivo with tissue recognition specificity.In summary,we successfully selected the aptamer spl3/spl3 c agains t bladder cancer cells 5637 with high specificity and high affinity.This aptamer provides new clues and evidence for non-invasive diagnosis and targeted therapy against bladder cancer.