| Gastric cancer is one of the most common malignant tumors,its high incidence and mortality seriously threaten human life,causing widespread concern.Gastric adenocarcinoma accounts for 95% of the pathological types of gastric cancer,and its pathogenesis has not been clarified yet.The patients with gastric adenocarcinoma show no obvious symptoms in the early stage,usually develope into the middle and late stages once diagnosed.Therefore,early diagnosis and early treatment of gastric adenocarcinoma are of great significance for reducing the incidence and mortality rates.Aptamers are short DNA or RNA oligonucleotides that evolved from the Systematic Evolution of Ligands by Exponential Enrichment(SELEX)that can form a specific structure to bind to the target.Compared with antibodies,aptamers have many unique advantages,including stable properties,easy to modification,easy in vitro synthesis,small particle size,low immunogenicity and low cost.Aptamers can be used as a molecular probe in the early diagnosis and treatment of gastric adenocarcinoma,which make them exhibit a great application prospect in the early diagnosis of gastric adenocarcinoma.In this study,the Serum-SELEX technique was used to select aptamers targeting gastric adenocarcinoma using carboxyl agarose magnetic beads as carrier.Using ss DNA as aptamers library,serum from patients with gastric adenocarcinoma as positive screening targets,and serum from healthy people and patients with cervical cancer as anti-screening targets,aptamers for gastric adenocarcinoma were selected out after 7 rounds of positive selections and 2 rounds of subtractive screening.The screening was completed when the mass ratio between ss DNA library input to the gastric adenocarcinoma serum tended to be stable.After high-throughput sequencing,the homology of the aptamers was analyzed by homology of the aptamers was analyzed by DNAMAN software further,combined with manual selection,seven candidate aptamers(Apt-3,Apt-56,Apt-67,Apt-69,Apt-77,Apt-88,Apt-101)were screened out finally.During the process of aptamer sequences alignment,we found that the middle random region of the aptamer sequences were rich in GC bases,which leading to form stem-loop structure in secondary structure easily.Circular Dichroism analysis result showed that these candidate aptamers were all right-handed helix.After simulating their spatial structures by RNAstrucure software,the affinities of the candidate aptamers to target gastric adenocarcinoma serum was measured by q-PCR technique.The linear relationship between concentration gradient and ct value of candidate aptamers was simulated by Graph Pad Prism 8 software.The results showed that the concentration gradient of candidate aptamers was positively correlated with ct value,and the affinity was at the level of nanomoles.Using the serum samples from healthy people,cervical cancer patients,colon cancer patients and gastric cancer patients as negative control groups,the specificity of aptamer to the serum samples from gastric adenocarcinoma was further verified by q-PCR,Quantum Dots Labeling and ELASA methods.These results showed that Apt-101 exhibited higher affinity and stronger specificity for the gastric adenocarcinoma serum target than Apt-77,and the equilibrium dissociation constant of Apt-101 was 6.444±1.128 nm,and the positive rate of specificity was more than 70%.In summary,in the results of screening gastric adenocarcinoma aptamers using Ser-SELEX technology,Apt-101 has high affinity and specificity for gastric adenocarcinoma targets,achieving the screening purpose and has great application prospects in the early diagnosis of gastric adenocarcinoma. |
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