Selection Of Highly Specific Aptamers Against KYSE-450 Cell Of Esophageal Squamous Cell Carcinoma By CELL-SELEX

ObjectiveEsophageal squamous cell carcinoma is the main type of esophageal cancer in China.Due to the clinical symptoms of early esophageal squamous cell carcinoma are not typical and there is no early tumor marker,most patients have been in advanced stage at the time of diagnosis.Until now,there is still no targeted therapeutic drugs for patients with esophageal squamous cell carcinoma,and the 5-year survival rate is still low.As a novel targeting molecule,nucleic acid aptamer have been proven to be useful as recognition tools for a variety of targets on the cell surface with high specificity as well as modifiability.Aptamers have been successfully used for targeted identification and intervention in a variety of tumors in vitro and in vivo.In this study,we intend to screen specific nucleic acid aptamer against esophageal squamous cell carcinoma KYSE-450 cell line by using CELL-SELEX protocol,which may provide a new strategy for targeting drug delivery to esophageal squamous cell carcinoma.Methods 1.Using HEEC cell line as control cells and KYSE-450 cell line as target cells,the secondary library with highly affinity and specificity to KYSE-450 cell line was screened.During the screening process,a Cell-Internalizing screening strategy was added,and the screening process was monitored in real time using fluorescence microscopy and flow cytometric analysis.After the optimal secondary library was determined,TA cloning,high-throughput sequencing,and homology analysis were performed to find candidate sequences,after which the candidate sequences were synthesized for further screening.2.The sequences with the highest binding and specificity were selected by fluorescence microscopy validation,flow cytometric analysis,as well as qPCR assays,and tested for affinity determination and internalization performance.3.The binding of the aptamers to other ESCC cell lines Eca-109 and TE-1 cell,as well as other related tumor cells,was verified using fluorescence microscopy assay and flow analysis.Results 1.After 18 rounds of CELL-SELEX screening,two nucleic acid aptamers specifically binding to esophageal squamous carcinoma cells KYSE-450 were obtained,named A14 and A24.2.Affinity measurements revealed that the dissociation constants of A14 and A24 for KYSE-450 cells were 108.4 nM and 51.32 nM,respectively;confocal microscopy revealed that A14 and A24 had internalization properties for KYSE-450 cells.3.The binding of aptamers A14 and A24 to other esophageal squamous cell carcinoma cell lines ECA-109 and TE-1 is different: A14 and A24 can bind to Eca-109 cells,but their binding to TE-1 cells is weak.Therefore,it is necessary to further determine the target of aptamer binding to cells.At the same time,A14 and A24 have weak binding to gastric cancer cells,gastric mucosal epithelial cells and other cancer cells.ConclusionsAfter 18 rounds of CELL-SELEX screening,two specific nucleic acid aptamers A14 and A24 against KYSE-450 of esophageal squamous carcinoma cells were obtained.Their dissociation constants for KYSE-450 cells were 108.4 nM and 51.32 nM,respectively,and the aptamers A14 and A24 were verified to have internalization properties.At the same time,the aptamers A14 and A24 could also bind to other esophageal squamous carcinoma cell lines such as Eca-109 cells.However,the specific binding target properties of A14 and A24 to KYSE-450 and Eca-109 cells have not been verified and still need further study.