Aptamers Against The Serum Of AFP-negative Hepatoma:Selection,Diagnostic Evaluation And Target Protein Capture And Proteomic Analysis

Background and objectives:The diagnosis of alpha-fetoprotein(AFP)negative primary hepatic carcinoma(ANHC)is a big challenge.The development of new diagnostic methods with high sensitivity and specificity is crucial for solving this problem.Many new ANHC biomarkers have been discovered with the development and application of proteomics technology,however,few has been used in clinic.Aptamers,selected by systematic evolution of ligands by exponential enrichment(SELEX),are artificial nucleic acid ligands of biological molecules,which are like antibodies in function but with advantages of thermal stability and easy preparation and therefore valuable in molecular detection and biomarker discovery.In previous work,we selected a group of aptamers against primary hepatic carcinoma(PHC),and identified potential PHC biomarkers by capturing and analyzing serum target proteins of these aptamers.In this study,we will take the methodology advantages of the previous work to select specific aptamers against ANHC serum,evaluate their diagnostic values,capture their protein targets in serum by magnetic beads followed by proteomic analysis,by which we attempt to facilitate the development of new approach for ANHC diagnosis.Methods:1.Collection of serum specimens:Serum specimens prior to therapy were collected from patients with ANHC,AFP positive PHC,liver cirrhosis(LC),chronic hepatitis(CH)hospitalized in the First Affiliated Hospital of Nanchang University and normal subjects(NS)for health check-up in this hospital from 2011 to 2015.Their clinical data available were collected.2.Preparation of initial library for the selection of aptamers against ANHC serum:The sub-library of random single-stranded DNA(ssDNA)previously generated from the last round selection of aptamers against PHC serum and cryopreserved for 18 mouths was used as the initial library for selecting aptamers against ANHC serum.We first verified the integrity of the sub-library using the PCR amplification and then prepared the initial library for aptamer selection of ANHC serum by asymmetric PCR amplification and recovered by gel cutting.3.Selection of aptamers against ANHC serum:(1)Preparation of target serum:pooled sera of ANHC and LC were prepared by mixing 10 serum specimens with same volume(elimination of heterogeneity among individuals)as target serum for SELEX.(2)Positive SELEX:The initial library was incubated with pooled ANHC serum,and then the bound ssDNA was separated by 12%neutral polyacrylamide gel electrophoresis(PAGE)with GelRed staining and recovered by gel cutting.The recovered ssDNAs were amplified by single-primer-limited amplification(SPLA)and used as the sub-library for the next round of selection.The positive selection was repeated until the binding band with ANHC serum was no longer densified in two successive rounds.(3)Negative SELEX:The sub-library prepared after last positive selection was incubated with pooled LC sera,and then the unbound ssDNAs were separated by 12%PAGE and stained with GelRed and recovered by gel cutting,followed by the amplification by SPLA for the next round of selection.The negative selection was repeated until the binding band with LC serum was no longer decreased.4.Isolation and structure analysis of individual aptamers:The recovered ssDNAs after SELEX were amplified by symmetric PCR.The PCR products were sent to a biological company for cloning and sequencing.The sequences in accordance with the length and fixed sequences at both ends of the library were aptamers.The secondary structures of the isolated sequences were imitated by RNA Structure 4.6 software.5.Evaluation of specificity of aptamers:Aptamers were artificially synthesized,incubated with pooled ANHC and normal sera and separated by 12%PAGE with staining by GelRed to observe specificity of aptamers to pooled ANHC sera first,and then incubated with single ANHC and control serum(LC,CH and NS)to further validate specificity of aptamers for ANHC serum.6.Evaluation of diagnostic value of aptamers:Some aptamers with good specificity were incubated with ANHC and LC serum samples(32 cases for each),and the fluorescence intensity was measured using the aptamer-serum triple fluorescence detection method established previously.The areas under the receiver operating characteristic curve(AUC)were utilized to evaluate the diagnostic value of aptamers for ANHC,the optimum detection condition was the one that with maximum AUC for each aptamer.Aptamers with good diagnostic value were incubated with a large sample size of ANHC,AFP positive PHC,LC,CH and NS serum specimens at their corresponding optimum detection conditions and the fluorescence intensities were detected and diagnostic values were evaluated for ANHC.7.Capture and proteomic analysis of aptamer targets in serum:Aptamers with perfect diagnostic value for ANHC in a large sample size were used to capture target proteins in ANHC pooled sera as well as the pooled sera of control groups(LC,CH and NS)based on magnetic bead isolation method established previously.Captured proteins were freeze-dried and sent to a biological company for proteomic analyses.Results1.Integrity of previous sub-library and preparation of initial library for aptamer selection:The previous sub-library cryopreserved for 18 months worked well,and we successfully prepared the initial library for the selection of aptamers against ANHC serum.2.Selection and isolation of aptamers against ANHC serum:After 6 rounds of positive SELEX with pooled ANHC serum,the sub-library was bound to the target pooled ANHC serum stronger than control serum.After 3 rounds of negative SELEX with pooled LC serum,the sub-library was bound to pooled LC serum weaker than ANHC pooled serum.A total of 120 sequences were isolated from the sub-libraries generated for the 6th round of positive selection and the second round of negative selection,which indicating that we successfully selected 120 aptamers against ANHC serum.Secondary structure imitating analyses of these aptamers showed lots of structure types,including pocket,hairpin,bar,bat,etc,indicating that they are specific to different targets in ANHC serum.3.Specificity of aptamer for ANHC:Some aptamers showed stronger bound bands to pooled and single ANHC serum than control serum in PAGE gel,indicating that some aptamers were specific to ANHC serum.4.Diagnostic value of aptamers for ANHC:Sixteen aptamers had their own optimum detection conditions;and they were valuable for diagnosing ANHC,under their optimum detection conditions,with AUROCs 0.724-0.897 for single fluorescence indicators and 0.838-0.935 for the diagnostic models combining 3 fluorescence indicators in small sample size.Three aptamers,AP-ANHC-6-2-33,AP-ANHC-6-1-26 and AP-ANHC-6-2-20,remaining with perfect diagnostic value in a large sample size,with AUROCs 0.713-0.902 for single fluorescence indicators and 0.863-0.933 for the diagnostic model established with multiple fluorescence indicators.Moreover,when combining the three aptamers,the AUROCs were higher than 0.95,and the sensitivity,specificity and accuracy for differentiating ANHC from all non-hepatoma were all more than 90%,indicating that those aptamers can complement with each other and specific to different targets or different parts of a target.5.Capture and proteomic analysis of aptamer targets in sera:Three aptamers with perfect diagnostic value for ANHC were used to capture target proteins in sera.The results of proteomic analysis showed that 21,21 and 3 target proteins were only expressed in ANHC serum captured by aptamer AP-ANHC-6-2-33,AP-ANHC-6-1-26,and AP-ANHC-6-2-20,respectively,and captured proteins were mainly cancer-related extracellular binding proteins involved in posttranslational modification,protein turnover and chaperones,indicating that target proteins of ANHC serum aptamers are mainly extracellular proteins for molecule binding.Conclusions:1.A total of 120 ANHC serum aptamers were isolated after positive and negative SELEX with ANHC serum and LC serum.Imitated secondary structures of these aptamers were various and some aptamers were highly specific to ANHC serum,indicating that we successfully selected a group of aptamers against ANHC serum.2.Three aptamers were excellent for diagnosing ANHC in a large sample size,especially in combination analyses,indicating that we successfully selected a group of ANHC serum aptamers with great diagnostic value.3.We successfully captured serum target proteins of three aptamers and proteomic analysis showed that 45 target proteins expressed only in ANHC serum and they are mainly extracellular molecules with binding functions and most of them are cancer-related proteins,indicating that some of them may be potential serum biomarkers of ANHC.4.The sub-library prepared previously during SELEX worked well after cryopreserved 18 months,which can be used to prepare new library by PCR amplification for SELEX,indicating that ssDNA library is stable and can be cryopreserved for a long time.