Screening Of Specific DNA Aptamers For Breast Cancer Cell ZR-75-1

Background:Breast cancer is the most common malignant tumor in women.Luminal B(HER2+)type breast cancer often shows early local recurrence,frequent lymph node metastasis,endocrine therapy tolerance and poor prognosis,which has a great impact on patient survival rate.At present,the diagnosis of breast cancer is mainly based on imaging methods,but for some micro-lesions,it is difficult to determine the nature of the lesion by imaging diagnosis alone,and there are false positives and false negatives.Therefore,it is urgent to develop high specific tumor marker molecular probes for early diagnosis and targeted drug delivery for treatment of breast cancer.Due to aptamers are a class of molecular probes with high affinity,specificity and stability,they have been widely applicated in early diagnosis and treatment of tumors.Therefore,in this study,Cell-SELEX technology was used to screen nucleic acid aptamers that specifically bind to target cells,that is,Luminal B(HER2~+)type breast cancer cell line ZR-75-1 was used as the target cell,and human normal mammary epithelial cell line Hs578Bst was used as control cell.Methods:In order to amplify specific ssDNA,symmetric PCR and asymmetric PCR reaction conditions were first optimized.ZR-75-1 and Hs578Bst cells were cultured in logarithmic phase.Cell-SELEX was used to select aptamers which binding to ZR-75-1 cells with high specificity and affinity.To monitor the enrichment of ssDNA by Flow cytometry,5'-FAM-primer was used to amplify ssDNA in asymmetric PCR reaction.The ssDNA library selected in the last round was cloned and sequenced.Ten highly enriched sequences were selected from the sequencing results and the secondary structure prediction and analysis were performed using NUPACK online analysis software.Three of them with lower free energy was selected to chemical synthesis.Then,the affinity was determined by flow cytometry,and the binding of the aptamer to the target cells was observed by fluorescence microscopy.Results:1.Optimizated conditions of PCR:the ratio of downstream primers was 4/1000of the asymmetric PCR reaction,and annealing temperature was 60°C.2.A number of ssDNA that specifically bind to target cells were successfully selected through 20 rounds of Cell-SELEX cyclic selection.3.Among the three aptamers with lower free energy,the aptamer ZR5 had the strongest binding ability to ZR-75-1 cells,when compared to the aptamer ZR6 and ZR7,and the equilibrium dissociation constant was 59.17±4.37 nM.4.Fluorescence microscopy imaging confirmed that ZR5 has strong binding ability to ZR-75-1 cells,whereas,without binding ability to Hs578Bst cells.Conclusions:The aptamer ZR5 was selected through Cell-SELEX technology that binded to ZR-75-1 cells with high specificity and affinity,which provide a possible for finding of biomarker,the early diagnosis,and targeted treatment of Luminal B(HER2~+)type breast cancer.