Screening And Identifiction Of Lung Adenocarcinoma Cell H-1975 Aptamers

Lung cancer is a complex and diverse disease,which is the most common cause of cancer-related deaths worldwide.Lung cancer patients have low five-year survival rate and poor prognosis.The current diagnosis and treatment methods do not meet the treatment needs,and the overall efficacy is relatively low.Therefore,early diagnosis of lung cancer is the key to achieving better treatment effect.Nucleic acid aptamers are single-stranded DNA or RNA molecules that specifically binding to its targeting molecule.Previous studies have shown that nucleic acid aptamers have a good application prospect in the diagnosis and treatment of cancer.Hence,we used the Cell-SELEX technology,screened the nucleic acid aptamers of lung adenocarcinoma cell line H-1975,and identified the specificity and affinity,which would lay the foundation for the discovery of tumor markers and the establishment early diagnosis and treatment techniques for lung cancer based on nucleic acid aptamers.The research content and result mainly conclude:(1)Selection of DNA aptamers of lung adenocarcinoma cell line H-1975We adopted artificial random oligonucleotide library as the DNA initial library,comprised a randomized region of 52(N52)nucleotides flanked by 24 nt primer,and lung adenocarcinoma cell line H-1975 was used as target selection,human alveolar epithelial cell HPAEpi C was used as counter selection.After 18 rounds of screening,four nucleic acid aptamers that bind to lung adenocarcinoma cell line H-1975 were obtained and named T1,T2,T3,T4 by optimizing the screening conditions.(2)Prediction of the secondary structure of nucleic acid aptamersThe secondary structure of T1,T2,T3 and T4 was predicted by NUPACK online software.The results showed that T1,T2,T3 and T4 have abundant secondary structure,mainly composed of stem-loop structures and stem-convex loop structures.(3)Identification on the specificity and affinity of aptamers binding lung adenocarcinoma cell line H-1975.Flow cytometry was used to identify the affinity of four high-abundance aptamers(T1,T2,T3,T4)and lung adenocarcinoma cell line H-1975.The results showed that the dissociation constant of the four nucleic acid aptamers were 92.3±7.6n M,108.6±16.2 n M,66.25±12.6 n M,and 36.3±5.8 n M,all within the nanomolarlevel,indicating that the four aptamers have higher affinity for lung adenocarcinoma cell line H-1975.Flow cytometry was used to identify the specificity of the nucleic acid aptamer T4 and lung adenocarcinoma cell line H-1975.The results showed that after the combination with the nucleic acid aptamer T4,the fluorescence signal was obviously increased of the lung adenocarcinoma cell line H-1975,while the fluorescence signal with other cells showed little change compared with the initial.This indicates that the nucleic acid the aptamer T4 is capable of specifically binding to the lung adenocarcinoma cell line H-1975 without binding to control cells and other human tumor cells or normal cells,and is expected to be a novel molecular probe for lung adenocarcinoma cell line H-1975.(4)Effect of trypsin treatment on the binding ability of nucleic acid aptamers and lung adenocarcinoma cell line H-1975Flow cytometry was used to analyze the binding ability of the nucleic acid aptamer T4 and the lung adenocarcinoma cell line H-1975 with trypsin treatment.The result showed that after combination with the nucleic acid aptamer T4,the fluorescent signal was gradually decreased of the lung adenocarcinoma cell line H-1975 with the increase of trypsin treatment time.After 5 minutes of trypsin treatment,the fluorescence curve of the lung adenocarcinoma cell line H-1975 almost coincided with the initial library.This indicates that the nucleic acid aptamer T4 can bind to a certain protein on the surface of lung adenocarcinoma cell H-1975,which would provide a new idea for the discovery of lung cancer unknown tumor markers.