| Background and aims:Primary liver cancer(Hereinafter referred to as liver cancer)has a high incidence and a poor prognosis.Early diagnosis and treatment is crucial to improve the prognosis of patients with liver cancer.Currently,the diagnosis of liver cancer mainly depends on imaging examination and alpha-fetoprotein(AFP)detection,but the diagnostic performance is unsatisfactory in clinical practice.It is necessary to establish simpler,more inexpensive and efficient novel methods for the diagnosis of liver cancer.Aptamers are single-stranded oligonucleotide biomolecular ligands selected from an artificially constructed random library by the Systematic Evolution of Ligands by Exponential Enrichment(SELEX)technology,which are similar to antibodies in function,but with many advantages that antibody does not possess and good application prospects in tumor diagnosis.Previously,we obtained a group of aptamers against liver cancer serum by SELEX technology.We found that some aptamers had a good diagnostic value for liver cancer by using polyacrylamide gel electrophoresis(PAGE)and gray value analysis,and the combination of aptamers could further improve the diagnostic efficiency.However,the operation of PAGE is cumbersome and the throughput is low,which limits its clinical application.The centrifugal ultrafiltration(CUF)method is a simple method that has been widely used in the laboratory purification,concentration and separation of a variety of biomolecules including DNA.Therefore,in the present study,we will establish a novel method for liver cancer diagnosis based on aptamer CUF separation and fluorescence quantitative analysis and evaluate the diagnostic value of single aptamer and mixed aptamers(array)for liver cancer,so as to lay the foundation for the development of a novel method for liver cancer diagnosis based on aptamer with clinical practical value.Methods:1.Collection of serum samples and clinical data: Serum samples of patients with primary liver cancer,cirrhosis and chronic hepatitis who were hospitalized in the First Affiliated Hospital of Nanchang University from September 2018 to March2021 were collected and stored in refrigerator at-80℃.Clinical data were collected by checking the corresponding medical records.2.Establishment of aptamer CUF separation and fluorescence quantitative analysis method: According to the previous study,mixed serum was used to optimize the factors that may affect the separation effect of aptamer CUF,including Ultrafiltration centrifuge tube cut-off value,centrifugation time,ultrafiltration system volume,aptamer and serum dosage.The optimal experimental conditions for aptamer CUF separation were based on the principles of good separation effect,time saving and economy.The fluorescence intensity of the ultrafiltrate was detected by a fluorescent quantitative PCR instrument using Eva Green dyes and conditions commonly used in previous studies.3.Selecting aptamers with higher diagnostic value for liver cancer by small sample size: Seven aptamers with higher diagnostic value for liver cancer confirmed by PAGE in the previous work were used in this study.Using above-mentioned aptamer CUF separation and fluorescence quantitative analysis method to detect 36 serum samples of liver cancer and cirrhosis,performing aptamer PAGE separation and gray value analysis as a control simultaneously.Receiver operating characteristic(ROC)curve analysis was used on the fluorescence analysis and gray value analysis indexes,to evaluate and compare the diagnostic value of each aptamer for liver cancer.Aptamers with a single fluorescent index for diagnosing liver cancer with an area under the ROC curve(AUROC)greater than 0.7 were selected for the subsequent research.4.Verifying the diagnostic value of the selected aptamers by enlarged sample size: The serum samples of liver cancer,cirrhosis and chronic hepatitis were enlarged to 108 cases each.Using CUF separation and fluorescence quantitative analysis method to detect serum samples with each selected aptamers.Perform aptamer PAGE separation and gray value analysis as a control simultaneously.ROC curve analysis was used on the fluorescence analysis and gray value analysis indexes to evaluate the diagnostic value of each selected aptamers for liver cancer.5.Evaluation of the diagnostic value of the selected aptamers combination:The binary logistic regression analysis models were established with the same fluorescence analysis indexes(SAE or SAE-SA)detected by the enlarged samples of the selected aptamers,and the diagnostic value of the combination of aptamers for liver cancer was evaluated by ROC curve analysis.6.Optimization of experimental conditions for aptamer array single tube CUF separation: Several aptamer arrays were constructed by permutation and combination of the selected aptamers,and the aptamers in each array were mixed in equal proportion for single tube detection.According to the above-mentioned system volume,serum dosage and total aptamer dosage of single aptamer CUF separation,the centrifugation speed and time of aptamer array single tube CUF separation were optimized by using mixed serum.7.Selecting aptamer array with the highest diagnostic value for liver cancer by small sample size: Under the optimized conditions of aptamer array single tube CUF separation and fluorescence quantitative analysis,each aptamer array was used to detect 36 serum samples of liver cancer and cirrhosis,performing ROC curve analysis on the fluorescence analysis indexes to evaluate the diagnostic value of them for liver cancer,and the most valuable aptamer array was selected.8.Verifying the diagnostic value of the optimal aptamer array by enlarged sample size: The serum samples of liver cancer,cirrhosis and chronic hepatitis were enlarged to 108 cases each,using aptamer array single tube CUF separation and fluorescence quantitative analysis method to detect serum samples with the optimal aptamer array,and the diagnostic value of the optimal aptamer array for liver cancer was evaluated by ROC curve analysis.9.Correlation analysis between the optimal aptamer array and commom blood test indexes: The commom blood test indexes(blood cell analysis,biochemistry,coagulation function and tumor markers)of 108 patients with liver cancer,108 patients with cirrhosis 108 patients with chronic hepatitis were collected.Pearson or Spearman correlation analysis was used to analyze the correlation between the best fluorescence analysis index of the optimal aptamer array and the blood test indexes.10.Evaluation of the diagnostic value of the optimal aptamer array and commom blood test indexes combination for liver cancer: On the basis of ROC curve analysis of the diagnostic value of each single blood index for liver cancer,the best fluorescence analysis index of the optimal aptamer array was combined with the commom blood test indexes to construct diagnostic models by multivariate logistic stepwise analysis(Forward: conditional).The diagnostic value of the models for liver cancer were evaluated by ROC curve analysis.Results:1.Optimization of experimental conditions of aptamer CUF separation and fluorescence analysis: Ultrafiltration centrifuge tube cut-off value was 100 KD,the system volume was 150μl(binding buffer 108.75μl,serum 11.25μl,aptamer 30μl,aptamer concentration was 0.1pmol/μl),centrifugation speed was 14000 ×g,and centrifugation time was 15 min.2.Selecting result of aptamers by small sample size: Through small sample size selecting,three aptamers(Ap-HCS-9-31,Ap-HCS-9-74 and Ap-HCS-9-90)were selected from the seven aptamers.3.Validation result of the selected aptamers by enlarged sample size:Enlarged sample size validation showed that Ap-HCS-9-31 had the best diagnostic value in distinguishing liver cancer from chronic hepatitis,the AUROCs of each fluorescence analysis index were 0.729 ～ 0.772;Ap-HCS-9-90 had the best diagnostic value in distinguishing liver cancer from cirrhosis,the AUROCs of each fluorescence analysis index were 0.621 ～ 0.837;while the AUROC of Ap-HCS-9-74 in distinguishing liver cancer from cirrhosis,chronic hepatitis or benign liver disease were all less than 0.7.4.Diagnostic value of the selected aptamers combination for liver cancer:The fluorescence analysis indexes of four combinations of the three selected optamers were analyzed respectively,the AUROCs of Ap-HCS-9-31 combined with Ap-HCS-9-90 or combination of Ap-HCS-9-31,Ap-HCS-9-74 and Ap-HCS-9-90 for distinguishing liver cancer from cirrhosis,chronic hepatitis or benign liver disease were all above 0.78,indicating that the combination of aptamers can improve the diagnostic efficiency of liver cancer.5.Optimization of experimental conditions for aptamer array single tube CUF separation: When the centrifugation speed was 16000 ×g and the centrifugation time was 20 min,the difference of the fluorescence analysis indexes between cirrhosis and liver cancer ultrafiltrate was the largest.6.Selecting result of aptamer array by small sample size: Four aptamer arrays of combination of the three selected aptamers were selected by small sample size,the aptamer array composed of Ap-HCS-9-31 and Ap-HCS-9-90 was the most valuable in the diagnosis of liver cancer.The AUROCs of each fluorescence analysis index to distinguish liver cancer from cirrhosis were 0.745 ～ 0.797.7.Validation result of the optimal aptamer array by enlarged sample size:Enlarged sample size validation showed that the optimal aptamer array composed of Ap-HCS-9-31 and Ap-HCS-9-90 had good effect in distinguishing liver cancer from cirrhosis,chronic hepatitis or benign liver disease,the AUROC was 0.890,0.796 and0.843,the sensitivity was 80.6%,69.4% and 58.3%,the specificity was 79.6%,72.2%and 87.5%,the accurary was 80.1%,70.8% and 77.8%,respectively.8.Correlation of the optimal aptamer array and commom blood test indexes: There was a moderate correlation between the best index of the optimal aptamer array single tube CUF separation and fluorescence analysis method diagnosing liver cancer and AFP(r =-0.409),but there was low correlation(| r | ≤0.258)or no correlation with other commom blood test indexes(P < 0.05).9.Diagnostic value of the optimal aptamer array and commom blood test indexes combination for liver cancer: Except for AFP,some commomly used blood test indexes had certain value in distinguishing liver cancer from cirrhosis,chronic hepatitis or benign liver disease(AUROC > 0.7).The AUROC of the optimal aptamer array combined with test indexes in the diagnosis of liver cancer was more than 0.95,and the sensitivity,specsificity and accuracy were more than 90%,which was significantly better than AFP alone in the diagnosis of liver cancer.Conclusions:1.A novel method for the diagnosis of liver cancer based on aptamer CUF separation and fluorescence quantitative analysis was successfully established.It is simple,fast,high throughput and of good practicability.2.Single aptamer detected by the novel method showed good diagnostic value for liver cancer.It was slightly inferior to the diagnostic value of aptamer PAGE analysis for liver cancer,but the method was simpler and more practical than PAGE.3.The optimal aptamer array,composed of Ap-HCS-9-31 and Ap-HCS-9-90,detected by the novel method showed good diagnostic value for liver cancer,which was better than aptamer alone and comparable to AFP.4.Diagnostic models constructed by combining the optimal aptamer array with AFP and other commom blood test indexes showed excellent diagnostic value for liver cancer,which complemented the deficiency of AFP. |
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