Screening Of Serum Nucleic Acid Aptamers In Liver Cancer And Preliminary Study On Their Specific Binding Proteins

Liver cancer is one of the most common malignant tumors with a high incidence,and it is often found in the middle and late stages.The 5-year survival rate of patients with liver cancer is less than 20%.Early diagnosis is the most effective way to reduce liver cancer mortality.Traditional alpha-fetoprotein(AFP)detection and ultrasound imaging analysis are insufficient in sensitivity and specificity,which is difficult to achieve early diagnosis.A nucleic acid aptamer is a single-stranded DNA or RNA capable of forming a specific three-dimensional structure by intramolecular folding to bind to a target molecule with high specificity and high affinity.The nucleic acid aptamer has high sensitivity and specificity in binding to the target molecule,and has great potential and superiority in biomedicine,new drug development,targeted therapy and disease detection.In this paper,two-way thermal cycle subtraction SELEX technology was used to screen liver cancer serum and healthy human serum-specific nucleic acid aptamers,which laid a foundation for the establishment of new technology for liver cancer tumor detection.At the same time,the serum-specific protein of liver cancer patients was obtained by using the selected nucleic acid aptamers,and a preliminary analysis was carried out on it.The research content and results are as follows.1.This experiment used liver cancer serum and healthy human serum as the target,using two-way thermal cycle reduction SELEX technology.After 19 rounds of screening,more than 1000 sequences of liver cancer patient serum-specific nucleic acid aptamers and healthy human serum-specific nucleic acid aptamers were obtained.Four high-abundance nucleic acid aptamer sequences were picked and synthesized,and their specific detection was performed.50 cases of liver cancer patients and healthy people were tested for specificity.The results showed that the positive rate of liver cancer aptamer was 92%,and the positive rate of serum nucleic acid aptamers in healthy people was 94%.2.Secondary analysis of the selected nucleic acid aptamers was performed using the online analysis software NUPACK.The results showed that the eight nucleic acid aptamer sequences were rich in G and C bases,and the secondary structure showed astem-loop structure.3.A magnetic bead-aptamer complex was prepared by combining the obtained nucleic acid aptamers with streptavidin magnetic beads.Using the prepared magnetic bead-aptamer complex,a nucleic acid aptamer-specific binding protein was obtained from the liver cancer serum,and high-throughput sequencing was performed to obtain a protein group that can specifically bind to the nucleic acid aptamer.4.Bioinformatics analysis was performed on the obtained proteome by software such as KEGG.Among them,growth differentiation factor 11(GDF11)is involved in the interaction between cytokine-cytokine receptors.Serum angiopoietin-2(Ang-2)is involved in signaling pathways such as MAPK signaling pathway,Ras signaling pathway and Rap1 signaling pathway.Alpha-fetoprotein AFP is involved in TGF-?signaling pathway,Wnt signaling pathway,and HIPPO signaling pathway.Fucosidase AFU is involved in the degradation of other polysaccharides and some pathways in lysosomes.