Screening,Identification,Validation And Biological Function Of Serum Non-inflammatory Biomarker Of Gastric Adenocarcinoma

BackgroundGastric adenocarcinoma,a type of gastric cancer,is derived from the deterioration of gastric glandular cell and accounts for 95%of the overall incidence of gastric cancer.Although early diagnosis can significantly improve the efficacy of cancer treatment,advanced or distant metastasis has already occurred when they visit hospital due to the lack of early diagnosis,which delay the disease and miss the best timing of treatment.Currently,the common clinical tumor markers used for the diagnosis of gastric cancer are carcinoembryonic antigen(CEA),carbohydrate antigen 125(CA125)and carbohydrate antigen 19-9(CA19-9),as serum tumor markers have become ideal indicators of gastric cancer patients because of its advantages such as simplicity,quickness,and painlessness.Although the combined detection of multiple tumor markers can increase the sensitivity of early gastric cancer diagnosis to a certain extent and can be used to predict the progression and prognosis of patients,but the detection repeatability is not good.Combined with surface-enhanced laser desorption/ionisation time-of-flight mass spectrometry(SELDI-TOF-MS),high performance liquid chromatography(HPLC)and matrix-assisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF-MS),our research group has successfully screened out specific markers in papillary thyroid carcinoma and breast cancer in the early stage,which have reference significance for the early diagnosis and prognosis detection of related tumors.However,the previous experiments had the following problems:a)serum-specific markers were not compared with clinical tumor markers used;b)specific markers did not rule out inflammatory interference;c)Due to the deviation of various influencing factors that were not independent of each other before the serum analysis test,and affected the test efficiency.In recent years,despite the dramatic increase in the discovery of molecular biomarkers,the clinical transformation of these biomarkers remains challenging.Peptide drugs are increasingly attracting the attention of pharmaceutical companies and academia because of their advantages of non-accumulative toxicity,relatively low cost,strong biological activity,small side effects and specific target sites.This study was divided into the following two parts:Part I:Combined use of SELDI-TOF-MS,HPLC,MALDI-TOF-MS and MALDI-TOF/TOF MS technology,serum proteome in preoperative,postoperative,relapsed gastric adenocarcinoma patients,control individuals and patients with systemic inflammatory response syndrome(SIRS)were screened,isolated,purified and identified while interference of inflammatory cytokines before serum analysis was eliminated to the greatest extent and a variety of factors of uniformity was strictly controlled,and then validated by enzyme-linked immunosorbent assay(ELISA)and western blot(WB).Finally,the non-inflammatory specific markers screened were compared with three traditional tumor markers including CEA,CA19-9 and CA125 for diagnosis and prognosis by ROC curve and Kaplan-Meier survival analysis;Part II:To explore the effects of synthetic non-inflammatory specific marker on the biological behavior of gastric adenocarcinoma by in vivo and in vitro experiments and to explore the mechanism of action providing the theoretical basis for biological targeted therapy.ObjectiveTo screen and identify serum non-inflammatory specific marker for the diagnosis of gastric adenocarcinoma and explore the efficacy of non-inflammatory specific marker on the biological behavior of gastric adenocarcinoma and the mechanism of action providing the theoretical basis for biological targeted therapy.Materials and Methods1 Clinical materialsA total of 492 serum samples were included from the Division of General Surgery,the First Affiliated Hospital of Zhengzhou University(mining set)and the Affiliated Tumor Hospital of Zhengzhou University(testing set).The mining set consisted of 120 preoperative serum samples from 120 patients with gastric cancer,106 corresponding postoperative serum samples,30 corresponding relapsed serum samples,64 control individuals including 17 with gastritis and 47 healthy donors and 60 SIRS pre-treatment serum samples.The testing set consisted of 60 preoperative serum samples from 60 patients with gastric cancer and 52 individuals,including 19 with gastritis and 33 healthy donors,as the control.Meanwhile,fresh-frozen gastric cancer epithelium tissue samples from 75 patients were randomly obtained from the First Affiliated Hospital and Affiliated Tumor Hospital of Zhengzhou University,whereas samples from 15 gastritis and 60 healthy donor fresh-frozen tissue were used as control.2 Experimental methods2.1 Screening,identification,validation and diagnosis and prognostic efficacy evaluation of non-inflammatory differential biomarkers in serumRaw data of CM10 protein chips combined with sera of preoperative,postoperative,relapsed gastric adenocarcinoma patients,control individuals and SIRS patients were collected by ProteinChip Software after the dried chips into the Protein Biological System ?(PBS-?)mass spectrometer reader.Data analysis was performed using the Zhejiang University Cancer Institute-ProteinChip Data Analysis System(ZUCIPDAS).The capability of each peak in distinguishing data of different groups was estimated based on the p value of Wilcoxon t-test.The best diagnostic model was selected by genetic algorithm combined with support vector machine method.Target differential biomarkers were purified by HPLC according to the SELDI-TOF-MS screening results and each separate ingredient was dispensed.Data acquisition was performed for each fraction using MALDI-TOF-MS to identify the samples containing proteins of the same or similar mass-to-charge ratio as the non-inflammatory differential proteins screened by the previous SELDI-TOF-MS,then the corresponding peptide mass fingerprint was collected by MALDI-TOF/TOF MS and linked to the Swissprot database using Mascot search software to search for matching proteins.The expression levels of non-inflammatory biomarker in the sera of preoperative,postoperative,relapsed gastric adenocarcinoma and control serum were verified by WB and ELISA.Non-inflammatory specific biomarkers screened were compared with three traditional tumor markers including CEA,CA19-9 and CA125 for diagnosis and prognosis by ROC curve and Kaplan-Meier survival analysis.2.2 Non-inflammatory serum biomarker was synthesized and its structure and properties was predictedNon-inflammatory serum marker was synthesized by solid-phase synthesis and verified by electrospray ionization(ESI)mass spectrometry and HPLC to ensure correct synthesis and purity.Amino acid was knocked out one by one from the c-terminus to find the shortest functional fragment,at the same time performance prediction and analysis of non-inflammatory serum marker was done.N-terminal of non-inflammatory serum marker labeled FITC green fluorescence.2.3 Explore the biological function of non-inflammatory serum marker in vitroNon-inflammatory serum marker was co-cultured with gastric adenocarcinoma cells,cell growth curve and inhibition rate curve were drawn and 20%inhibitory concentration(IC20),50%inhibitory concentration(IC50)and 80%inhibitory concentration(IC80)were calculated.EdU,PI-Annexin V double staining flow cytometry analysis,scratch test,transwell transmembrane invasion assay and PI method of cell cycle analysis were used to detect the synthetic marker peptide on the proliferation,apoptosis,migration,transmembrane invasion and cell cycle distribution of gastric adenocarcinoma cells.Gastric adenocarcinoma cells were co-cultured with non-inflammatory serum marker labeled FITC green fluorescent at the N-terminal and the localization of the marker in the cell was observed by inverted fluorescent microscope and confocal laser scanning microscope.Real-time fluorescence quantitative PCR and WB were used to detect the mRNA and protein expression levels of apoptosis-related factors,cell proliferation-related factors and related signal pathway key factors to explore its mechanism of action.2.4 Explore the biological function of non-inflammatory serum marker in vivoAfter successful establishment of gastric adenocarcinoma tumor-bearing immunodeficiency mouse model,mice of the same tumor size were selected and randomly divided into four groups of three.A group of intraperitoneal injection of saline as the control group,the other three groups of intraperitoneal injection of gradient concentration markers.The dosing interval was 2 days and lasted 24 days.From the first day of manual intraperitoneal injection of non-inflammatory serum marker or saline to nude mice,the long and short diameters of the tumor were measured every 3-4 days with a caliper,the tumor volume was calculated,and the tumor growth curve was drawn.After treatment,mice were sacrificed according to the rules of euthanasia,the tumor was dissected,weighed and photographed.TUNEL method was used to analyze the apoptosis in tumor tissue of immunodeficient mice and the immunohistochemical method was used to detect protein expression levels of the apoptosis related factors,cell proliferation related factors and related signaling pathway key factors.Results1 Screening,identification,validation and diagnosis and prognostic efficacy evaluation of non-inflammatory differential biomarkers in serum of gastric adenocarcinomaSerum samples from the mining set were analysed and compared using SELDITOF-MS with the CM10 chip.All MS data were baselinesubtracted and normalised using total ion current,and peak clusters generated with Biomarker Wizard software.Wilcoxon rank sum tests to determine relative signal strength disclosed 15 differentially expressed proteins,including 9 upregulated and 6 downregulated protein peak intensities from samples of preoperative gastric cancer patients,compared with controls.Thirteen differentially expressed proteins,including 10 upregulated and 3 downregulated protein peak intensities,were observed for postoperative gastric cancer patients,compared with preoperative gastric cancer patients,and 15 differentially expressed proteins,including 9 upregulated and 6 downregulated protein peak intensities for relapsed gastric cancer patients,compared with postoperative gastric cancer patients.Nineteen differentially expressed proteins,including 9 upregulated and 10 downregulated protein peak intensities,were observed for preoperative gastric cancer patients,compared with SIRS patients,among which the protein peak of m/z 6449 was never seen and seven protein peaks of m/z 4279.326,4312.117,4968.221,5332.653,5650.360,5919.453 and 8622.667 were subsequently identified as inflammatory factors closely related to gastric cancer,intercellular adhesion molecule-1(ICAM-1),interleukin-8(IL-8),reactive oxygen species(ROS),interleukin-6(IL-6),interleukin 1 alpha(IL-1 ?),tumor necrosis factor(TNF)-a-induced protein 8-like 2(TIPE2)and cyclooxygenase-2(COX-2).From the random combination of protein peaks with remarkable variation,the SVM combined with genetic algorithm screened out the model with maximum Youden index of the predicted value,leading to the identification of a marker positioned at 6449 Da with continuous dynamic presence in the control,preoperative,postoperative and relapsed gastric cancer patient sera.Moreover,m/z 6449 Da did not appear in the 19 differentially expressed protein peaks in the serum of preoperative group and SIRS group,among which 7 were inflammatory factors closely related to gastric cancer,thus eliminating the potential interference of inflammatory factors,m/z 6449 Da was a non-inflammatory biomarker.The 6449 Da protein was remarkably elevated in preoperative gastric cancer patient sera,compared with the control,but decreased in postoperative sera and upregulated again in relapsed gastric cancer samples.In addition,the 6449 Da protein level progressively increased through clinical stages ?,?,? and ?.Using leave-one-out cross-validation SVM classifier evaluation model,the 6449 Da protein was used to effectively distinguish gastric cancer from controls with an accuracy of 85.3%(157 out of 184),sensitivity of 85.0%(102 out of 120)and specificity of 85.9%(55 out of 64)The remaining 60 gastric cancer and 52 control serum samples were analysed as a blind testing set to validate the accuracy and validity of the 6449 Da protein marker identified from the mining set.The peak intensity of the 6449 Da protein was higher than that of control,consistent with the mining group.The 6449 Da marker distinguished gastric cancer samples from controls with an accuracy of 84.8%(95 out of 112),sensitivity of 86.7%(52 out of 60)and specificity of 82.7%(43 out of 52).According to the SELDI-TOF-MS screening results,serum samples with high expression intensity of m/z 6449 Da were selected and purified by HPLC.The corresponding protein at different time points was collected respectively by EP tube and then analyzed by MALDI-TOF-MS to identify the fractionated protein with m/z 6449 Da which was enzymatically digested and then detected by the MALDI-TOF/TOF MS system.The collected data from MALDI-TOF/TOF MS were then imported into the Mascot search program and searched in the Swissprot database finding that m/z 6449 Da was polypeptide fragment of apolipoprotein C?.Expression of 6449 Da in gastric cancer serum was simultaneously confirmed by WB and ELISA,the results demonstrated that 6449 Da protein was remarkably elevated in preoperative gastric cancer patient sera,compared with the control,but decreased in postoperative sera and upregulated again in relapsed gastric cancer samples,consistent with the SELDI-TOF-MS results.Based on the ROC curve and Kaplan-Meier survival analysis,it was found that the diagnostic efficiency of 6449 Da biomarker was significantly superior to that of the three traditional tumor markers,either alone or in combination and 6449 Da candidate non-inflammatory protein biomarker represented a new and stronger potential prognostic factor of gastric adenocarcinoma.2 Synthesis of 6449 Da polypeptide and prediction of its structure and propertiesThe apoC-III precursor is 99 amino acids in length and the mature apoC-III is composed of 79 amino acids.Analysis of the signal peptide,performance,secondary structure and homology modeling of the 6449Da polypeptide were carried out by softwares such as DNAStar,PepCalc,PSIPRED and SWISS-MODEL and corresponding results showed that the first 20 amino acids of C-terminal of 6449Da polypeptide were signal peptide and the active site was predominantly located in the WSGC peptide consisting of 40 amino acids except for the signal peptide sequence,whereas the signal peptide had almost no active site combined with three parameters including antigen index,surface accessibility and hydrophilicity.Based on the above prediction results and PeptideSyn technology platform,we knocked out the amino acids one by one from the C-terminal and the remaining amino acid sequence after knockdown was synthesized.Cytotoxicity test showed that the remaining 40 amino acid residues,which was named WSGC peptide,after deletion of the signal peptide consisting of 20 amino acids at the C-terminus,were functional fragments of 6449Da polypeptide.3 In vitro experimental results of WSGC peptideAfter the intervention of WSGC peptide on gastric adenocarcinoma cells and normal gastric mucosal epithelial cells,the cell growth curve and the cytotoxicity inhibition rate curve were drawn.It was found that the WSGC peptide had stronger toxic effects on gastric adenocarcinoma cells than normal gastric mucosal epithelial cells indicating that WSGC polypeptide had relatively specific cytotoxic effect on gastric adenocarcinoma cells.Results of CCK-8,EdU,flow cytometry,scratch assay and transwell transmembrane invasion assay showed that WSGC peptides with IC50(48h)and IC80(48h)concentrations could inhibit the proliferation of gastric adenocarcinoma cells,induce cell division S phase arrest,promote tumor cell apoptosis and reduce or inhibit cell migration and transmembrane invasion.In addition,gastric adenocarcinoma cells were co-cultured with green fluorescent-labeled WSGC peptide for 48h,then observed by inverted fluorescence microscopy and laser confocal fluorescence microscopy finding that WSGC-FITC mainly distributed in the gastric adenocarcinoma cell membrane surface,rarely into the cytoplasm or cells nuclear.QRT-PCR and WB demonstrated that compared with the control group,mRNA and protein expression levels of Bax and Caspase-3 in gastric adenocarcinoma cells were significantly upregulated whereas mRNA and protein expression levels of Bcl-2,Ki67,PCNA,NOTCH1,Jagged 1,MAML3,CSL and HES1 were significantly lower after treated with IC50(48h)and IC80(48h)concentration of WSGC peptide for 48h.4 In vivo experimental results of WSGC peptideGastric adenocarcinoma tumor-bearing immunodeficiency mouse models were treated with a gradient concentration of WSGC peptide(0 nmol/g,20 nmol/g,40 nmol/g,80 nmol/g)and found that tumor weight and volume was lower than that of the control group after intervention treatment by 40 nmol/g and 80 nmol/g dose of WSGC peptide.Mice were sacrificed according to the rules of euthanasia and the tumors were dissected after the treatment.TUNEL apoptosis and immunohistochemical analysis of tumor tissue were performed respectively and the results demonstrated that compared with the control group,apoptosis rate and protein expression of Bax and Caspase-3 increased with the increase of WSGC peptide dosage whereas protein expression of Bcl-2,Ki67 and PCNA decreased with the increase of WSGC peptide dosage.Conclusions? M/z 6449 Da protein polypeptide is a specific non-inflammatory biomarker in the sera of patients with gastric adenocarcinoman and is a complement to the traditional gastric adenocarcinoma tumor marker.It is derived from apoC-III and consists of 60 amino acids and can be used for the diagnosis and prognosis assessment of gastric adenocarcinoma.? After knockdown of the signal peptide consisting of the C-terminal 20 amino acids,WSGC peptide consisting of remaining 40 amino acids is the functional fragment of the 6449 Da anti-tumor peptide.WSGC peptide can inhibit the proliferation of gastric adenocarcinoma cells,induce cell division S phase arrest,promote tumor cell apoptosis and reduce or inhibit cell migration and transmembrane invasion.? WSGC peptide exerts biological function through notch signaling pathway,which may compete with Jagged 1 ligand for NOTCH1 receptor on the surface of gastric adenocarcinoma cell membrane and on the other hand,NOTCH1 can initiate mitochondrial pathway-mediated apoptosis process through JNK signaling pathway.