| Background and objectives:Gastric cancer(GC)is the fifth most common cancer and the third common cause of cancer death worldwide.The incidence of GC is high in our country,which seriously threaten the health of Chinese people.The survival rate of GC patients is closely associated with early diagnosis and treatment,however,the diagnostic rate of early gastric cancer is less than 10%.Endoscopy followed by biopsy is the most effective method for GC diagnosis in clinical practice,but it is difficult to become a national screening method for GC due to its invasion and high cost.Serum biomarkers are simple and convenient tools for tumor screening,but no markers available were recommended for GC diagnosis.In recent years,with the development of serum proteomics,there are many potential serum markers of gastric cancer have been reported.Although rarely markers have been successfully applied to clinical practice,it is hopeful to discover novel serum markers for GC.Aptamers are artificial oligomeric nucleic acid ligands screened through an in vitro selective process known as Systematic Evolution of Ligands by Exponential enrichment(SELEX),with the function similar to antibodies but superior to antibodies in thermal stability and modifiability.Therefore,it has a favorable and prospective application in molecular diagnosis.The capture and analysis of target proteins of aptamers have become a new strategy to discover tumor biomarkers.In the previous work,we selected 86 aptamers against gastric cancer serum.In the present study,we aimed to evaluate their diagnostic values through the aptamer-based single-tube triple serum fluorescence assay.Then,we will capture the target proteins of aptamers with high diagnostic value,and followed by mass spectrometry analysis to detect novel serum biomarkers of GC.Methods:1.Collection of serum specimens: Serum specimens were collected from patients with GC,benign gastric disease(BGD)and normal controls(NC)for health check-up in the First Affiliated Hospital of Nanchang University from 2013 to 2016.Their clinical data available were also collected.2.Evaluation of diagnostic value of aptamers: The fluorescence intensity of serum autofluorescence,cell-free DNA-related fluorescence and aptamer-related fluorescence were successively measured at 8?,25?,37? and 42? in one tube using a real-time PCR system as a fluorometer in patients with GC or BGD and NC subjects(each 32 cases).The area under the receiver operating characteristic curve(AUROC)was used to evaluate the diagnostic value of single fluorescent indicators and a logistic regression model established by multiple fluorescent indicators for gastric cancer.The aptamers with good diagnostic value were selected to be validated in 64 cases of each group.The aptamers validated were further verified in large samples.Furthermore,we evaluated the value of combined analysis of aptamers in the diagnosis of GC by logistic regression,decision tree and neural network prediction method based on principal component analysis.3.The capture of target proteins with aptamer:(1)Aptamers with verified diagnostic value for GC were synthesized and labeled with biotin at 5' end.After the incubation of gradient concentrations of aptamers with fixed amount of streptavidin-coated magnetic beads,reaction liquid were analysized by 12% neutral polyacrylamide gel electrophoresis(PAGE)and silver staining to determine the optimal ratio of aptamers and magnetic beads;(2)Examining the reaction liquid of fixed amount of aptamers and gradient concentration of serum by PAGE and GelRed staining to determine the optimal ratio of aptamers and serum;(3)With the best ratios of aptamers,serum and magnetic beads,the target proteins of aptamers pooled sera of GC and NC were captured based on magnetic bead isolation and analyzed by SDS-PAGE and silver staining.4.Mass spectrometry analysis of target proteins of aptamers: Target proteins captured from pooled sera of GC and NC based on aptamer-magnetic bead isolation method were concentrated and lyophilized and sent to a biological company for LC-MS/MS analyses and Label free quantification.4.Analyze the differentially expressed proteins in serum by multiple reaction monitoring(MRM): Target proteins,which were specific or 3-flod differential expression in GC serum than NC group and associated with GC or other tumors,were selected as candidate proteins according to the reports published.The concentrations of these candidate proteins would be measured in serum samples of GC,NC,BGD and beging gastric tumor(BGT)by MRM analysis;moreover,their diagnostic values would be evaluated.Results:1.Subjects: The serum samples were collected from 236 patients with gastric cancer(male 167,female 69,average age of 59.7 year old),170 patients with benign gastric diseases(male 58,female 112,average age of 53.0 year old)and 184 healthy controls(male 74,female 110,average age of 48.7 year old).2.Diagnostic value of aptamers for GC: The diagnostic values of 86 aptamers for gastric cancer were evaluated in small serum specimens,in which 17 aptamers with the AUROC of single fluorescent indicators more than 0.8 and that of logistic regression models more than 0.9 were validated in 64 cases of each group.In the end,7 aptamers(AP-GCS-9-5,AP-GCS-9-15,AP-GCS-9-21,AP-GCS-15-16,AP-GCS-15-36,AP-GCS-17-25,AP-GCS-18-7)with potential value for GC detection were verified in a large sample of 590 cases.Results showed that most of the fluorescence variables have statistically significant difference between GC and control group,and the AUROCs of single fluorescence index were 0.59-0.74.Logistic regression,decision tree and neural network models based on principal component analysis probability can improve the diagnostic value of aptamers.In neural network models,AUROCs were 0.902-0.945,0.890-0.944,and 0.855-0.924,respectively,in discrimination GC from NC,BGD,and non-cancer cases(NC+BGD).Very High sensitivities and specificities in GC diagnosis were achieved when all seven aptamers were used.The sensitivity and specificity for detecting GC from NC,BGD and non-cancer cases was 100% and 99.5%,100% and 100%,97.5% and 97.7%,respectively.3.Capture and analysis of target proteins of aptamers against GC serum: Seven aptamers(AP-GCS-9-5,AP-GCS-9-15,AP-GCS-9-21,AP-GCS-15-16,AP-GCS-15-36,AP-GCS-17-25,AP-GCS-18-7)were used to capture target proteins in GC and normal serum specimens.The results showed that the total of captured target proteins from GC serum and normal serum in these seven aptamers were 44/46,80/75,61/64,114/101,52/59,79/89 and 79/68,respectively.And there were 165 proteins differentially expressed between GC and normal serum,in which 93 proteins were related to cancer according to literature reports.4.The results of MRM quantitative detection for target proteins: We select 40 proteins that may be closely related to GC to analyze by MRM from above 93 proteins,and 16 proteins could be detected by MRM method.Comparing the expression levels of them in serum samples from gastric cancer(n=120)and control(60 cases of normal,benign gastric disease and gastric tumor each group),11 proteins have significantly different expression among groups.AUROCs of these single proteins for diagnosing GC from non-GC were 0.56-0.66.The combination of 7 proteins(vWF,A1 AT,haptoglobin,ALS,ATIII,THBS-1 and C1 Inh)by a C5.0 decision tree model was valuable for GC diagnosis,with accuracies of 92.5%(111/120)for GC,85.0%(51/60)for BGT,83.3%(50/60)for BGD,96.7%(58/60)for NC and 90.0% for the total.Furthermore,81.8% of early GC cases(18/22)were correctly identified.Conclusions:1.Through the analyses of screening,validation and verification in clinical serum samples,we confirmed 7 of 86 aptamers against GC serum screened in previous study have good diagnostic value for GC,especially the combination analysis by neural network models.It shows that a group of serum aptamers with good diagnostic value for GC was successfully selected.2.The aptamer-based magnetic separation method for capturing serum target proteins of aptamers has successfully established,and 7 aptamers were used to capture target proteins in GC and normal serum.The MS analysis found 165 differentially expressed proteins,of which 93 proteins are associated with tumor,suggesting that some of them may be potential serum biomarkers of GC.3.The expression levels of 16 target proteins were analyzed in gastric cancer and control serum samples by the MRM quantitative method,and 11 proteins are differently expressed.With 7 of those proteins,a C5.0 decision tree model was constructed and shows a good diagnostic value for gastric cancer. |
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