Selection Of Aptamers For Clinical Kidney Cancer Tissue

Kidney cancer is the second most common malignant tumor in the urinary system,and its incidence is increasing in China.Due to the concealment of the kidney and the lack of specific tumor biomarkers,the early diagnosis of renal cancer is difficult.In addition,the advanced metastasis and the drug resistance makes poor prognosis and high recurrence rate of kidney.New molecular probes that can specifically recognize kidney cancer at early stage and the discovery of new tumor molecular biomarkers are important for the cancer diagnosis and treatment.Nucleic acid aptamers,obtained by an in vitro screening technique SELEX(systematic evolution of ligands by exponential enrichment)can form a specific spatial structure to bind to their target.Aptamers have a wide range of recognition targets,high specificity,low toxicity and low immunogenicity,and strong binding affinity,which play an important role in biomedical research,especially cancer-related research.In recent years the increasing number of aptamers are specifically recognized by Cell-SELEX technology and Protein-SELEX technology It has provided an potential tool for tumor research.However,since these two screening techniques use purified proteins or tumor cells cultured in vitro,the obtained aptamers are difficult to be applied for clinical tumor dianosis.Tissue-SELEX use clinical tumor tissues,and can obtain aptamers that specifically recognize clinical tumor tissues.This may be useful for kidney cancer early diagnosis,and new tumor biomarkers discovery.Based on the Tissue-SELEX technique,we used clinical kidney cancer tissue sections as experimental samples,and a new ssDNA library modified with affinity groups as a new screening scheme.There are 6 samples: Screening library and clinical kidney cancer tissue incubation samples,positive screening library and renal cancer adjacent tissue incubation samples,single negative screening library samples,negative screening library and clinical kidney cancer tissue incubation samples,negative screening library and renal cancer adjacent tissue incubation samples.Their sequencing analysis was performed to obtain aptamers that can specifically recognizes clinical kidney cancer tissues.In addition,we also investigated the properties of the aptamers obtained by screening.Our research contents are as follows:(1)Screening of aptamers in clinical Kidney camcerBased on Tissue-SELEX technology,the Kidney cancer tissues were used as targets,and the tissues adjacent to Kidney cancer were screened as control samples using X-Aptamer library with affinity group modification.After 2 rounds of screening,experimental groups and 5 control groups were subjected to second-generation sequencing and further comparative analysis,we picked eight sequences for further analysis.(2)Investigation of the characteristics of aptamer WCQ-5 in clinical Kidney cancerIn order to apply the selected aptamers to clinical diagnosis and treatment,we will study the characteristics of aptamers.Firstiy,we performed secondary structure prediction on candidate sequences,and also verified the binding ability of candidate sequences to kidney cancer cells by flow cytometry and confocal to determine the aptamer that specifically binds to kidney cancer cells.The affinity of the aptamer to 786-O cells was then detected by flow cytometry,wherein the dissociation constant of the aptamer WCQ-5 and 786-O cells was at the nanomolar level.And by detecting the electrophoresis bands of WCQ-5 at different time points,it was found that WCQ-5 without any modification had lower stability in serum.At the same time,we investigated the binding of kidney cancer cells and aptamers after different digestion treatments by flow,and determined that the binding target type of the aptamer and renal cancer cells was membrane protein.Finally,based on the experimental phenomenon that the aptamer WCQ-5 has internalization into the cell,we detected the localization of the aptamer in 786-O cells by fluorescence colocalization.According to the above research content,the aptamer WCQ-5 screened by Tissue-SELEX shows strong binding ability to 786-O and ACHN cells,which provides a powerful tool for the clinical diagnosis and treatment of renal cancer.