Proteomic Screening And Verification Of Plasma Tumor Biomarkers For Esophageal Squamous Cell Carcinoma

BackgroundEsophageal cancer(EC) is one of the most common human malignancies, which has a high malignant degree and less than 20% of 5 years survival rate, and it has become the world’s eighth largest malignant tumor. Unfortunately, EC is the most common malignant tumor in rural areas of China, among the 450,000 new detected EC patients in the world for each year, half of them are from China. But the incidence of EC is still on the rise year by year, and 90% of them are esophageal squamous cell carcinoma after pathology examination.However, with the speedy development of the new technology for cancer treatment have been made in recent years, such as adjuvant radiation, chemotherapy and surgery, the total five years survival rate of ESCC patients is still very low. The most important reason is that the majority of the ESCC patients were found to be at the late stage, so that it is unsuitable for making radical resection. Some studies have confirmed that early diagnosis and early treatment could achieve a good prognosis. But early diagnosis is usually mainly relied on endoscopic examination of esophagus mucosa staining with compound iodine solution, but the sensitivity and specificity are not ideal. More and more people are getting interested with the research and development of tumor biomarkers, who are wishing to be able to find the tumor biomarkers with a high sensitivity and a strong specificity. Therefore, looking for effective tumor biomarkers for early diagnosis for ESCC will become very urgent and significant.With the development of techniques of molecular biology, some relevant oncogenes and tumor suppressor genes about the etiology and pathogenesis of cancers have been found, and the understanding of malignant tumor were gradually deepened in recent years. However, the biological function of the gene were performed not by itself, but by proteins which encoded. Therefore, the development of proteomics technology have provided a new technology platform of looking for the new specific tumor biomarkers,as well as found technical basis for the study of malignant tumor etiology, pathogenesis, therapeutic effect and prognosis.Proteins were the performers for life activities and proteomics is a new bright spot in the life sciences. It uses many methods in fields of the clinical medicine, molecular biology, mass spectrometry analysis(MS) and bioinformatics, to explore the separation, purification and identification of protein, Proteomics is a high throughput platform for studying the expression and function of all proteins in specific tissues and cells at different time and space,It can dynamically, completely and quantitatively investigate the changes of protein species and quantity during the development of the disease and help us to find markers for the diagnosis and treatment of diseases.In 1997,the method of fluorescent dye labeling is applied to proteomics research by Unlu M. based on two-dimensional gel electrophoresis a new technique-two dimensional differential gel electrophoresis(2D-DIGE)was formed. In the two-dimensional electrophoresis(2D-DIGE)the samples were marked using different fluorescent(such as Cy2, Cy3, Cy5 mark),and then the samples were mixed together for electrophoresis in one gel. Different color fluorescence signals were obtained by using 3 different wavelengths of laser on 2D gel image, and the difference of protein between samples was judged according to the difference of these signals.The fluorescent dyes used for labeling are similar in chemical structure and molecular, and all with a positive charge, so all the samples can be moved to the same position when they react with lysine residues.In 2D-DIGE technology, every protein has its own internal standard, and the software automatically according to the internal standard of each protein point to its expression, to ensure that the detected protein concentration change is true.It avoids the difficulty in the analysis of the difference between different gels, ensures the consistency of the experimental conditions, and is conducive to the screening of the differential expression points. The 2D-DIGE technology can detect less than 10% differences between the protein expression, and the statistical reliability can reached more than 95%. At present, this technology has been widely used in the proteomics research.In recent years, a large number of useful researches on the esophageal cancer molecular markers were performed by domestic and foreign scholars, and some of them have been applied in early clinical diagnosis. The tumor biomarkers which are being used for screening, early diagnosing and prognosis forecasting mainly include: carcinoembryonic antigen(CEA), glycoprotein 50(CA50), glycoprotein 242(CA242), glycoprotein 199(CA199), tissue polypeptide glycogen(TPA), cell keratin(Cyfra21-1), neural specificity enolization enzyme(NSE) and alpha fetoprotein(AFP), etc., but the sensitivity and specificity are not very satisfied. In previous studies, cancer tissues and cancer adjacent tissues, or the immortalized cell lines of cancers were mainly used as the research objects for tumor biomarkers detecting. However, the plasma is one kind of the very important tissue fluids in the body, and the proteome changes of which can reflect the conditions of body health earlier in some diseases, in addition, this examination is without pain and trauma, and samples collection is much more convenient. Potential tumor biomarkers which are detected from plasma will be used for early diagnosis of the tumor, therefore, more and more researchers are keen on screening protein biomarkers from the peripheral blood. ObjectiveTo screen and identify the specific plasma tumor biomarkers for early diagnosis of esophageal squamous cell carcinoma, which can found the reliable experimental data for the early diagnosis for ESCC. Materials and methods 1. Clinical samples collection53 cases of plasma samples from esophageal squamous cell carcinoma patients were collected from Departments of Thoracic Surgery, the First Affiliated Hospital of Zhengzhou University from Mar 2014 to Mar 2015, and all the samples were collected before any treatment such as surgery, chemotherapy and radiotherapy treatment. There were 31 males and 22 females patients in ESCC group, with 50-65 years old, Tis-T1N0M0 TNM stage, and esophageal squamous cell carcinoma in pathologically type; At the same time, selected 53 cases of healthy group of plasma samples, these specimens were derived from the healthy physical examination department in the First Affiliated Hospital Zhengzhou University in January 2015, with 26 male and 27 female donors, and 50-65 years old. This experiment was permitted by our hospital ethics committee, and all the subjects had signed the informed consent. All the samples were stored in-80 ℃ refrigerator before using. 2. Major instruments and reagentsSIGMA Proteo Prep Blue Albumin and Ig G Depletion Kit、2-D Clean-up Kit 、EttanTM 2-D Quant Kit 、immobilized non-linear p H gradient strips、EttanTM IPGPhor apparatus,EttanTM DALT Six gel tank、Typhoon Imager Scanner 9400、De Cyder 2D software、De Cyder V6.5 software、ABI 4800 MALDI-TOF/TOFmass spectrometer。 3. Protein concentration assayThe plasma samples were treated by using the SIGMA Proteo Prep Blue Albumin and Ig G Depletion Kit, and the high abundance proteins such as albumin and immunoglobulin were removed, which enhanced the development of low abundance proteins. Other substances in plasma which may affect the results in two-dimensional differential gel electrophoresis were removed by2-D Clean-up Kit.Using EttanTM 2-D Quant Kit protein quantitation Kit determine the concentration of protein in plasma samples. 4. Production of preparative gel using Two dimentional electrophoresisSelected 50μg protein from 4 patients with ESCC and 4 healthy controls and the protein was marked with Cy3 or Cy5 fluorescent dye. The internal standard was protein mixtuer mixed with 25μg protein for each of the samples, and labeled with Cy2 dye. The plasma proteins labeled with fluorescent dyes were reacted with immobilized non-linear p H gradient strips for a rehydration process. Then the EttanTM IPGPhor apparatus and EttanTM DALT Six gel tank were used to make the first and second dimensional gel electrophoresis. 5. Production of preparative gelThe preparative gel, containing 800 μg of unlabeled proteins mixture for internal standard, was used for 2DE, the steps and parameters were the same as the analytic gels. The preparative gel was post-stained with colloid staining dyes. 6. Differentially expressed spots analysisThe gels were scanned with Typhoon Imager Scanner 9400, and the channels of Cy2, Cy3 and Cy5 were scanned respectively with 488 nm, 532 nm and 633 nm,. The blue, green, or red image, which was labeled with Cy2, Cy3 and Cy5 was observed respectively.the preparative gel was analyzed with De Cyder V6.5 software,3D images and manual proofreading to screen the differentially expressed protein spots, and then the changed spots were matched in the preparative gel, and finally, they were picked up by EttanTM Spot picker automatically. 7. Isolation and purification of differentially expressed proteinsThe picked spots were added with trypsin for In-gel digestion. And then the extraction of digested peptides were concentrated by vacuum freeze drying apparatus. After that, Zip Tip? C18 was used to purify and concentrate the peptides, 8. Mass spectrometry identificationThe peptides was spotted onto the clean and dry MALDI sample plate directly.Sample target was input into MALDI-TOF/TOF mass spectrometer, after the internal standard calibration and 3000-4000 laser bombardiment, peptide mass fingerprint(PMF) were aquired automatically, and then ten of the strongest precursor ions were selected for MS/MS mass spectrometry, and finally, the peptide sequence tags(PST) were got. PMF and PST data were used to search in NCBInr database,The differential expressed protein was identified. 9. Bioinformatics analysisBioinformatics analyses were done for the highly credible proteins. Firstly, GO classification of varied proteins was based on the molecular functions, and then, potential protein interaction analysis was performed with the String database. 10. Bioinformatics analysisBioinformatics analyses were done for the highly credible proteins. Firstly, GO classification of varied proteins was based on the molecular functions, and then, potential protein interaction analysis was performed with the String database. 11. Western blot4 plasma samples of ESCC patients and 4 plasma samples of healthy donors were selected randomly to perform the Western blot verification. 12. Statistical methodsSPSS 17.0 software was applied for statistic analysis, and independent samples Student’s t-test was used to count the mean difference between the two groups. Levene test was used in homogeneity test of variances, and P<0.05 was used as significance of differences. All the measurement data were shown with x ±SE to reflect the discrete tendency of sample means. Results 1. Protein concentration assayFirstly, 2-D Quant kit was used to determine the protein concentration, and then a random of 500μg plasma protein from different groups were loaded into SDS-PAGE gel, after the Coomassie brilliant blue staining, we can assess the accuracy of the protein assay method. The results showed that each protein bands were equal. 2. Image scanning for analytic gels4 clear protein spectrums for the gels were acquired with Typhoon Imager 9400 image scanning system. The samples labeled with different dyes showed different colors with Image Quant TL software.The blue, green, or red image, which was labeled with Cy2, Cy3 and Cy5 was observed respectively. 3. Differentially expressed spots analysisThe differentially expressed protein spotswas analyzed with De Cyder V6.5 software, 3D images and manual proofreading, In total, 31 protein spots had significant differences between the ESCC patients and the healthy controls, among those spots, 16 were up-regulated and 15 down-regulated. 4. MS identification of picked spots31 differential expressed protein spots were analyzed with MALD-TOF/TOF mass spectrometry. PMF and PST data were used to search in NCBInr database, 12 varied proteins were identified in ESCC group, in which 6 proteins were up-regulated and 6 down-regulated. Moreover, the up-regulated proteins contained alpha-1-antichymotrypsin, alpha-2-HS-glycoprotein(AHSG), leucine-rich alpha-2-glycoprotein(LRG), zinc-alpha-2-glycoprotein, complement factor I and complement C4-B, whereas the down-regulated proteins contained serum albumin, Ig alpha-2 chain C region, alpha-1-antitrypsin, fibrinogen gamma chain, haptoglobin and hemoglobin subunit alpha. 5. GO classification according to molecular functions for the differentially expressed proteinsGO classification was performed according to the molecular functions of varied proteins, and these identified proteins are functionally involved in 4 categories, in which inflammation and immune reaction proteins were appearing the biggest proportions which is 38.7% and 29% respectively, additionally, transporters and blood coagulation proteins accounts for 19.4% and 12.9% respectively. 6. Protein interaction analysis of the differentially expressed proteinsProtein interaction prediction was performed with String software for differentially expressed proteins, interestingly, other 9 kinds of proteins had the interaction relationships directly or indirectly except IGHA2, SERPINA3 and C4 B. 7. Verification results by Western blotAccording to results of Western blot, AHSG and LRG were significantly increased in the plasma of ESCC patients compared with healthy controls. The average grey value of AHSG is about 2.8 times rising in ESCC group, and the average grey value of LRG is about 1.8 times rising in ESCC group. Therefore, these results of WB verification were consistent with the data from the proteomics experiments. 8. Verification results by ELISAAccording to ELISA results, AHSG and LRG were increased obviously between ESCC patients(n=45) and healthy donors(n=45), which were consistent with the data from the proteomics experiments. The plasma AHSG concentration of control group is 140.9±27.3 μg/ml, and 308.4±62.3 μg/ml in ESCC group. The plasma LRG concentration of control group is 45.3±9.4 μg/ml, and 118.1±24.3 μg/ml in ESCC group. Levene test results showed that the variances were equal, and after independent samples t test, P < 0.05 were observed. Therefore, all the results suggested that the concentrations of AHSG and LRG had a significant difference between ESCC and healthy controls. Conclusions1. 12 differentially expressed plasma proteins were detected by differential proteomics platform, which would be potential tumor biomarkers for ESCC diagnosis; 2. Alpha-2-HS-glycoprotein and leucine-rich alpha-2-glycoprotein may become potential plasma tumor biomarkers which may provide new opportunities for the early diagnosis for ESCC, but the sensitivity and specificity of them are still needed to be verified with increasing samples; 3. Alpha-2-HS-glycoprotein and leucine-rich alpha-2-glycoprotein may play important roles in the pathogenesis of ESCC, but much more further study is needed. 4. The differential proteomics technology platform was used in this experiment by combining with DIGE, MALDI TOF/TOF MS, and bioinformatics analysis, which could provide an effective technical support for the screening and identification for the tumor biomarkers.