| Background and objectives:Gastroscopy and biopsy are currently principal method for the diagnosis of gastric cancer, but it is impossible for them to be used in screening of gastric cancer due to patient suffering and high cost. Serum tumor marker detection is a simple and practical method for cancer screening and diagnosis, but lack of markers of gastric cancer for clinical application. SELEX (systematic evolution of ligands by exponential enrichment) is a new combinatorial chemistry technology, by which nuclear acid ligands (aptamers) binding to targets with high specificity and affinity could be screened out from a synthetic single-stranded random oligonucleotide library. Aptamers is similar to or better than antibodies in function and more stable than antibodies in chemical properties, so there is a good prospect for disease diagnosis. In the present study, aptamers against gastric cancer serum were selected by SELEX, and their specificity and affinity in binding to gastric cancer serum were analyzed, for the purpose of providing the foundation of a new gastric cancer diagnostic technology based on aptamers.Methods:1. Serum specimen collection:Serum specimens were collected from patients with gastric cancer and benign gastric diseases and healthy people for medical checkup in the XX Hospital of Nanchang University.2. The diagnostic value evaluation of conventional serum tumor markers in gastric cancer:the diagnostic value of serum tumor markers AFP, CEA, CA125 and CA19-9, single or combined, in the patients with gastric cancer was analyzed and evaluated by conventional statistical analysis, receiver operating characteristic (ROC) curve and the area under curve (AUC), univariate/multivariate Logistic regression analysis.3. Selection of aptamers against gastric cancer sera:(1) Target serum preparation: 15 serum samples from preoperative patients with gastric cancer confirmed by pathology and 15 serum samples from healthy subjects were selected to prepared the pooled sera of gastric cancer and normal subjects by mixture with equal proportion, respectively (to eliminate individual differences), and used as target sera of SELEX. (2) Library and primers synthesis:single-stranded random DNA library and primers designed and applied in our previous studies were synthesized by a biotechnology company. (3) Subtractive SELEX with normal sera:the library was incubated with pooled normal serum, unbound ssDNA was recovered and purified by gel excision after 12% neutral polyacrylamide gel electrophoresis (PAGE) and GelRed staining, then the sub-libraries were prepared by asymmetric PCR amplification, and used as next round of selection. Three rounds of subtractive SELEX were carried out. (4) The development of single-primer-limited amplification method to prepare sub-library:we established the method for severe non-specific amplification appeared in the preparation of sub-library by asymmetric PCR. The ssDNA sub-library prepared in the third round of subtractive selection was used as template to amplify the minus-stranded DNA with single downstream primer which was 5 times of templates quantity. And then the minus-stranded DNA was used as template to amplify the plus-stranded DNA with single upstream primer which was 10 times of template quantity. The products were compared with asymmetric PCR amplification by PAGE and silver staining and real-time PCR quantification. (5) SELEX with gastric cancer sera as target:the pooled gastric cancer serum was incubated with the sub-library generated after 3 rounds of subtractive selection, nucleic acid binding to serum were separated and recovered by 12% PAGE, then the sub-library were prepared by single-primer-limited amplification and used as next round of selection. nine rounds of SELEX were carried out.4. Aptamer isolation and secondary structure analysis:the bond nucleic acids recovered at the ninth round of selection were used as a template for symmetric PCR amplification, and the products were sent to a biological company to clone and isolate nucleic acid fragment and sequence. The sequence of the separated nucleic acid fragments was identical with libraries as aptamers. The secondary structures of aptamers were imitated and analyzed by RNA Structure 4.6 software.5. Specificity analysis of the aptamers:an aptamers in gradient concentration was incubated with pooled gastric cancer sera and pooled normal sera, respectively, and the binding bands were observed after PAGE and GelRed staining, to analyze the specificity of aptamers in binding to pooled gastric cancer sera.6. Affinity analysis of the aptamers:an aptamer in gradient concentration was incubated with pooled gastric cancer sera, and then the gray values of specific band was measured by Quantity One image analysis software after 12%PAGE and GelRed staining. The Kds were calculated by Graphpad prism 5.01 software.Results:1. Subjects:the serum samples were collected from 149 patients with gastric cancer (male 115, female 34, average age of 59.1 year old),111 patients with benign gastric diseases (male 76, female 35, average age of 52.9 year old) and 124 healthy controls (male 72, female 52, average age of 53.0 year old).2. Diagnostic value of conventional serum tumor markers for gastric cancer: Serum levels of CEA, CA125, and CA19-9 in gastric cancer group were higher than that in the benign gastric disease group and the healthy control group. By using normal cut-off value, the sensitivity of AFP, CEA, CA125 and CA19-9 in the diagnosis of gastric cancer was 4.7-20.8%, and increased to 40.3% when combined. By using optimal cut-off value, the sensitivity of CEA, CA125 and CA19-9 in the diagnosis of gastric cancer was improved, such as CEA from 17.4% up to 58.4% but the specificity from 99.1% down to 83.4%, and the combined use of four markers up to 69.1% but specificity down to 80.4%, and the positive and negative likelihood ratios increased slightly. These results demonstrate that value of single and combined conventional serum tumor markers is poor for the diagnosis of gastric cancer.3. Selection and separation of aptamers to gastric cancer sera:After 3 rounds of subtractive SELEX with pooled normal sera and 9 rounds of SELEX with pooled gastric cancer sera,22 nucleic acid fragments were isolated by cloning. The sequences were identical to the library at two sites and different each other in the middle. It is indicated that a group of aptamers against gastric cancer sera were selected successfully.4. Secondary structure characteristics of the aptamers:Imitating analysis by computer software showed that the secondary structures were various in the aptamers, including pocket, hairpin, bar, bat. It is suggested that these aptamers are against different targets or binding sites in the gastric cancer sera.5. Specificity of the aptamers:Most of aptamers showed stronger bound bands in pooled gastric cancer sera than in pooled normal sera. Four aptamers had a specific bound band in gastric cancer sera, worthy of further studying.6. Affinity of the aptamers:Two aptamers with obvious bound bands were analyzed for their affinity of binding to pooled gastric cancer sera. Their Kds were 3.798nM and 0.871nM, respectively, showing that they bind to the gastric cancer sera with high affinity.7. Single-primer-limited amplification (SPLA) to amplify the random ssDNA sub-library:non-specific products in the SPLA appeared late and therefore the target product was pure, while non-specific products in the asymmetric PCR appeared early and the dominant non-specific products were much stronger than the target products. The yield of ssDNA by SPLA was 16.1 times that by the asymmetric PCR.Conclusions:(1) The diagnostic value of classic serum tumor markers AFP, CEA, CA125 and CA19-9, single or combined, is poor in gastric cancer, indicating that it is required to develop new simple and practical methods in diagnosis of gastric cancer.(2) 22 aptamers against gastric cancer sera were generated by use of complex-subtractive SELEX. The secondary structures of the aptamers were various in imitating analyses by software. Four of the aptamers were capable of binding to gastric cancer sera with high specificity and high affinity. These results suggest that aptamers against gastric cancer sera were successfully selected out and has potential diagnostic value.(3) A novel method termed single-primer-limited amplification was developed to generate random ssDNA sub-library. This method is superior to asymmetric PCR in preparing sub-library for SELEX due to its efficient inhibition of non-specific amplification during the sub-library amplification. It is valuable in the generation of random ssDNA sub-library for SELEX. |
