CELL-SELEX筛选膀胱癌核酸适配体的细胞学研究

Cytological study on aptamer selection for bladder cancer through CELL-SELEX

目的基于膀胱癌标志物的早期诊断和术后复发监测方法是目前的研究热点。文中利用CELL-SELEX技术筛选出可特异性识别膀胱癌细胞系(EJ、T24、BIU87)的核酸适配体。方法以人膀胱癌细胞系EJ、T24和BIU87为阳性质控细胞,以HCV29(人正常尿路上皮细胞系)、293T(人胚肾细胞系)、huh7(人肝癌细胞系)为阴性质控细胞,进行CELL-SELEX筛选。PCR上游引物5’端标记FITC,下游引物5’端标记Biotin,将每一轮次筛选得到的ssDNA作为模板进行PCR扩增、纯化。利用生物素-链亲和素磁珠分离法将PCR纯化产物进行分离后获得次级FITC-ssDNA用于下一轮次的筛选,流式细胞仪监测CELL-SELEX筛选进程。选取与膀胱癌细胞系EJ、T24和BIU87结合率最高轮次的ssDNA文库进行PCR扩增、产物纯化、分子克隆和测序;根据测序结果,利用Dnaman软件模拟核酸适配体的二级结构。体外合成FITC标记的适配体,流式细胞仪检测适配体与EJ、T24和BIU87的结合率。结果随着CELL-SELEX流程的推进,FITC-ssDNA与EJ、T24和BIU87细胞的结合率逐渐增加;第15轮FITC-ssDNA与EJ结合率最高,文库中apt1富集度最高;第18轮FITC-ssDNA与T24、BIU87结合率最高,文库中apt2和apt3富集度最高。DNA结构预测分析显示apt1、apt2和apt3的二级结构主要为茎环结构。流式细胞仪检测:FITC-apt1可特异性识别EJ细胞,阳性率达71.5%。FITC-apt2与T24细胞的结率明显高于BIU87细胞。FITC-apt3与BIU87细胞的结合率明显高于T24细胞。FITC-apt2可特异性识别T24细胞,阳性率达63.9%。FITC-apt3可特异性识别BIU87细胞,阳性率达74.7%。结论利用CELL-SELEX技术筛选出可识别膀胱癌细胞系的适配体。其中适配体apt1可特异性识别EJ细胞,适配体apt2可特异性识别T24细胞,适配体apt3可特异性识别BIU87细胞,为膀胱癌早期诊断和靶向治疗技术的研发提供了实验依据。

Objective The methods based on bladder cancer markers which could be applied to early diagnosis and postoperative recurrence monitoring of bladder cancer were current research hotspots.This study aims to screen aptamers that specifically recognize human bladder cancer cell lines(EJ,T24,BIU87)through cell-based systematic evolution of ligand by exponential enrichment(CELL-SELEX).Methods For CELL-SELEX screening,bladder cancer cell lines EJ,T24,and BIU87 were used as positive control cells.HCV 29(human normal urothelial cell line),293 T(human embryonic kidney cell line),huh7(human hepatocellular carcinoma cell line)were used as negative control cells.PCR upstream primers were labeled with FITC,downstream primer was labeled with Biotin.ssDNA fragments collected from each round were amplified by PCR,and the amplified product was then purified using a DNA purification Kit.The biotin-streptavidin magnetic separation methods were used to isolate the PCR product to obtain secondary FITC-ssDNA for the next CELL-SELEX round.The screening process was monitored by flow cytometry.ssDNA pool with the highest binding rates to bladder cancer cell lines(EJ,T24,and BIU87)was selected to PCR amplification,product purification,molecular cloning,and sequencing.According to the sequencing results,the secondary structure of the aptamer was pre-simulated by Dnaman software.Aptamer labeled with FITC was synthesized in vitro,flow cytometry was used to detect the binding rate of the aptamer to bladder cancer cell lins(EJ,T24 and BIU87).Results With the advance of the CELL-SELEX process,the binding rate of FITC-ssDNA to bladder cancer cell lins(EJ,T24,and BIU87)increased gradually.By the 15 th round,the binding rate of FITC-ssDNA to EJ cells reached the highest level.The apt1 had the highest enrichment among the 15 th round ssDNA pool.By the 18 th round,the binding rate of FITC-ssDNA to T24 or BIU87 cells reached the highest level.The apt2 and apt3 had the highest enrichment among the 18 th round ssDNA pool.DNA structure prediction showed that the secondary structure of apt1,apt2,and apt3 was mainly stem-loop structure.Flow cytometry showed that the highest binding rate was FITC-apt1 to EJ cells,FITC-apt2 to T24 cells,and FITC-apt3 to BIU87 cells,respectively.There is no significant combination between these aptamers with the negative cells.Conclusion In this study,three kinds of aptamers with high specificity for bladder cancer cell lines were successfully screened by CELL-SELEX.The apt1 can specifically recognize EJ cells,apt2 can specifically recognize T24 cells and apt3 can specifically recognize BIU87 cells,all of which provide experimental evidence for early diagnosis and targeted therapy technology research of bladder cancer.