| Backgroundesophageal cancer(EC)is a very serious one of malignant tumor patients within five years of survival rate of no more than ten percent,in the list of currently the world's ten malignant tumor in 8.About 450,000 new cases are reported globally each year,and China accounts for about half of all new cases.In China's rural,esophageal cancer is one of the highest rates of malignant tumors,serious harm to the health of our people,and because the early symptoms,typical in middle-late when the majority of patients see a doctor,so our country was increasing morbidity and mortality of patients with esophageal cancer.Conclusion on the pathological type of it,can be divided into squamous cell carcinoma and adenocarcinoma,and the highest incidence was in clinical esophageal squamous cell carcinoma.squamous cell carcinoma,ESCC),more than 90%of the total number of esophageal cancer.In recent years,with the continuous development of esophageal cancer treatment technology,radiation and chemotherapy drugs and surgical techniques have made considerable progress,but within five years of survival rate of esophageal patients is still no significant improvement.Since the early characteristics of esophageal cancer are atypical,most patients have not received sufficient attention,leading to the diagnosis of the middle and late stage,the loss of surgical opportunities,and the life expectancy is greatly shortened.Studies have shown that patients with esophageal cancer receive good treatment in the early stage,and the survival rate of patients is significantly improved.At present,the diagnosis of early esophageal cancer in clinical mainly through upper gastrointestinal barium meal and electronic digestive endoscopic for compound iodine staining esophageal mucosa,but because of the two diagnostic sensitivity and specificity is not high,so the effect is not ideal in clinical diagnosis.If you can find have stronger sensitivity and specificity of tumor markers,will provide the clinical diagnosis and treatment of esophageal cancer with powerful reference,greatly increase the clinical cure rate of esophageal cancer.Therefore,it is very important to find tumor markers for early diagnosis of esophageal cancer.At present,with the medical research and technology innovation and development of people understanding and understanding of tumor has been changing,people to the understanding of the etiology and pathogenesis of malignant tumors gradually changed,and now has discovered many in the medical community and is closely related to the tumor occurrence and prognosis judgement of oncogene and tumor suppressor genes,etc.In the human body,the gene can realize its biological function,which is realized by the protein as the coding vector.Therefore,protein is the important performer of human life activities.Because people get to know the important roles of protein,proteomics technology developed rapidly in recent years,for people to study the occurrence and development of malignant tumor,and to find effective tumor markers provide the possibility of a technical level.As a very important component of systems biology,proteomics is the study of the whole organism,tissues or cells of the whole protein of a science,all its ultimate aim is to reveal the biology protein expression level change rule and function of biological significance,the research contents include protein expression levels,protein structure,biological function and interaction,etc.By detecting different time and space,different kinds of chemical testing intervention,and different disease in the pathopHysiology of differences between protein changes,thus can get external factors affect cell metabolism and biological function such as information,by analyzing the difference of proteome in its expression change rule,in the development of these diseases will help to understand a variety of pathopHysiological process of molecular mechanism of the behind.In the field of cancer research,want to find the differentially expressed proteins,mainly in the tumor tissue protein in the application of proteomics research methods,however,these proteins could be used for early diagnosis of tumor biomarkers or screening of new targets for the drugs.In lately few years,proteomics technology platform has been in liver cancer,lung cancer and breast cancer in the early diagnosis of malignant tumor has made some research data,to find and select the function of some tumor associated protein,at the same time,also found some potential tumor molecular markers,the research for the early diagnosis of malignant tumor and pathogenesis research provides a very rich resources and valuable data.In recent years,researchers around the world to be used for early diagnosis of esophageal cancer molecular markers have done a lot of work,and successful,has been used in some markers in patients with esophageal cancer census,early diagnosis and prognosis evaluation of commonly used tumor markers are:tissue polypeptide glycogen(TPA),glycoprotein 242(CA242),neural specificity enolization enzyme(NSE)glycoprotein 199(CA199),carcinoembryonic antigen(CEA),50(CA50)glycoprotein,cell keratin(Cyfra21 1)and AFP(AFP),etc.,but the effect is not ideal,can't meet the clinical demand for early diagnosis of esophageal cancer.Past research mainly for malignant tumor markers of malignant tumor,malignant tumor and normal tissue adjacent to malignant tumour of the immortalized cell lines.However,the plasma is very important in the body tissues,collecting specimens have less pain,small trauma,collecting the advantages of the convenient,easy to preserve,study group of plasma proteins and can reflect the changes of the body's basic health.Therefore,from the plasma to find potential tumor markers can be used more effectively to the early diagnosis of malignant tumor,in view of this,many researchers have tried to from the peripHeral blood specimens to screen tumor markers,but the result is very disappointed,can't meet the clinical application.Exosomes are secreted actively by cells,with 30?150 nm of diameter,which can carry a variety of proteins and mRNA and microRNAs,and it might participate in cell communication,cell migration,angiogenesis and tumor cell growth process.It exists in a variety of bodily fluids,such as blood,urine,ascites,bronchoalveolar lavage fluid,breast milk and saliva,etc.Cancer cells can secrete exosomes to change its external environment,and exhibit some specific related proteins on the surface.Therefore,plasma might contain some specific biomarkers to be used for early diagnosis of tumors,therefore,it should be more efficient to screen specific tumor markers from plasma.In this study,we planned to screen and verify the varied proteins from plasma exosomes by using differential proteomics platform,which contains DIGE and MALDI-TOF/TOF analysis,and it would set up the experimental basis for detecting the specific biomarker for ESCC.ObjectiveTo screen and identify the specific plasma tumor biomarkers from exosomes for early diagnosis of esophageal squamous cell carcinoma,which would set the valueable experimental basis for the early diagnosis of ESCC.Materials and methods1.Clinical samples collection46 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 January 2015 to May 2015,and all the samples were collected before treatment.There were 26 male and 20 female patients in ESCC group,with 40-60 years old,T3-4N0M0 TNM stage.46 cases of normal plasma samples were collected from the department of health pHysical examination of the First Affiliated Hospital of Zhengzhou University,in April 2015,with 24 male and 22female donors,and 40-60 years old.This experiment was permitted by our hospital ethics committee,and all the subjects had signed the informed consent.The whole anti-coagulant blood was centrifuged with 4,000 g for 10 minutes at 4? to harvest plasma.The samples were stored in-80? refrigerator before use.2.Plasma exosome extraction and Western blot verificationThe plasma exosome was extract with supercentrifugation and verificated by detecting the specific exosome protein named CD9 antigen.3.Purification of plasma protein samplesWe utilized 2-D Clean-up Kit to remove salt,lipid,polysaccharides,polypHenols and other material,which may interfere the result of two-dimensional electropHoresis.4.Protein concentration measurementEttanTM 2-D Quant Kit was used to measure the protein concentrations of the plasma exosomic samples.5.CyDye labeling reaction4 exosome samples from ESCC group and control group were selected respectively,and were labeled with different CyDyes.The internal standard was protein mixture that made of all the samples with 25 ?g for each one,and 50?g of mixed protein was loaded into one gel for internal standard which was labled with Cy2.Three specfic fluorescent dyes containing Cy2,Cy3 and Cy5 were labeled respectively at the ratio of 400 pmol dyes per 50 ?g protein.6.Two dimentional electropHoresisThe labled samples were mixed firstly following the experimental plan,and there are 3 fluorescent channels for each gel.Then the rehydration process was done in the immobilized non-linear pH gradient(IPG)strips(pH 3-10 NL,24 cm),and which were incubated in the dark at RT overnight.The first dimention was then performed with EttanTM IPGpHor,finally,the gels were run in the EttanTM DALT Six.7.Image acquiring of analytic gelThe entire gels with low fluorescent glass plates were scanned with the TypHoon Imager Scanner 9400,and the scanning paremeters were set with 488/520 nm,532/580 nm and 633/670 nm to scan the channel of Cy2,Cy3 and Cy5 respectively.Afterward,the PMT value was used as the biggest grey value within 60000-90000 in the the selected region of the gel.After the pictures scanning,the blue,green,and red images,which were labeled with Cy2,Cy3 and Cy5 dyes,were observed respectively with ImageQuant TL software.8.Production of preparative gel800 ?g of unlabeled mixture proteins as internal standard was loaded into the same IPG dry stip as analytic gel,and were performed with 2DE at the same time,furthermore,the steps and parameters were also the same as the analytic gels.After post-staining with colloidal staining method,the preparative gel was matched with analytic gels to pick up the changed spots that analyzed with DeCyder 2D software.9.Varied spots analysis with software and spot pickingFirstly,co-detecting of the spots,which also was called DIG,were analyzed with DeCyder V6.5 software,and then,different images of internal standard in different gels were matched with BVA analysis,afterward,the spots with satistical differences were screened,and coordinate position of these spots were matched on the preparative gel,and finally,these spots were picked up by EttanTM Spot picker automatically.10.In-gel digestionThe frozen spots were destained and dewatered first,and then the MS grade trypsin was added into the spots,and they were incubated in 37? constant temperature in the water bath overnight;after mixing the extraction of digested peptides,the mixed solutions were concentrated to 10 ?l by vacuum freeze drying apparatus.Furthermore,ZipTip(?)C18 colum was used to purify and concentrate the peptides,finally,2?l of CHCA matrix(5 mg/ml)was added to elute the protein peptides mixture,and then they were spotted onto the MALDI sample plate directly.11.Mass spectrometry analysisAfter the crystallization and air-drying of the samples,the sample target was load into the input system of ABI 4800 MALDI-TOF/TOF mass spectrometer,after the setting of parameters and internal standard calibration,peptide mass fingerprint(PMF)of MS were harvested,and then,the parameters of tandem MS/MS were setted as follow,the strongest ten precursor ions were picked out,and then,the peptide sequence tags(PST)were got after the software analyzing.Finally,combined PMF and PST data were used to search in human SwissProt database with Mascot software,Protein score that was greater than 95%was highly confidential.12.Bioinformatics analysisBioinformatics analyses were performed for the highly confident results.GO classification of varied proteins was performed firstly,and then potential protein interaction analysis was done with the String database.13.Western blot4 plasma exosome samples of ESCC patients and 4 plasma exosome samples of healthy controls were used randomly to do Western blot validation.14.ELISA verification40 plasma exosome samples of ESCC patients and 40 of healthy controls were selected randomly to do ELISA experiment.15.Statistical analyzing methodsSPSS 17.0 was used for statistic analysis,firstly,Levene test was performed in homogeneity test of variances,and then the independent samples were analyzed with Student's t-test,and P<0.05 was the significance standard.x±SE was used to reflect the discrete tendency of the samples.Results1.Western blot verification of extracted plasma exosomesWB results showed that the specific CD9 was clearly detected in the lysis buffer of extracted plasma exosomes,and it meant our extracting method was efficient.2.Image acquiring of analytic gelsAfter the 2DE running,3 channels of protein spectra were collected with TypHoon Imager 9400 image scanning of the analytic gels in low fluorescent glass plats,after ImageQuant TL software analysis,the.samples labeled with different dyes had different colors.3.varied spots analysis with Decyder 2D software and gel pickingAccording to DIA and BVA analysis with DeCyder software,14 protein spots from plasma exsomes had significantly increased between the ESCC patients and healthy donors.4.MS identificationIn our study,14 up-regulated proteins in ESCC group were analyzed by ABI 4800 MALD-TOF/TOF mass spectrometry.Totally,8 kinds of proteins were found at last except the proteins with the same name.These identified proteins contained serum albumin alpHa-2-HS-glycoprotein(AHSG),Ig alpHa-2 chain C region,leucine-rich alpHa-2-glycoprotein(LRG),haptoglobin,CD9 antigen(CD9),CD63 antigen(CD63)and S100-A9(S100A9).5.GO classification according to molecular functions of 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 predictionProtein interaction prediction was performed with String,which was online software,for differentially expressed proteins,interestingly,other 6 kinds of proteins had the interaction relationships directly or indirectly except S100A9 and IGHA2.These proteins had close relationship with each other.7.Verification by Western blotAccording to Western blot results,AHSG and S100A9 were significantly increased in the plasma of ESCC patients compared with healthy controls.The average grey value of AHSG is about 2.9 times rising in ESCC group,and the average grey value of S100A9 is about 2.8 times rising in ESCC group.Therefore,these results of WB were more or less the same with the data of the proteomics experiments.8.Confirmation by ELISAAfter ELISA verification,AHSG and S100A9 were increased obviously between ESCC group(n=40)and control group(n=40),which were similar with the data of the proteomic experiments.The plasma AHSG concentration of control group is 70.3±2.2 ?g/ml(x±SE),and 150.1 ±4.6 ?g/ml in ESCC group.The plasma S100A9 concentration of control group is 46.9±1.4 ?g/ml,and 150.1 ±4.6 ?g/ml in ESCC group.The variances were equal after Levene testing,and then,two independent samples t test was used to perform statistical analysis,and P<0.05,which suggested that the concentrations of AHSG and S100A9 were significantly different between ESCC and controls.Conclusions1.8 up-regulated plasma proteins of exosomes were detected by differential proteomics platform,which would be potential tumor biomarkers for ESCC diagnosis;2.AlpHa-2-HS-glycoprotein and S100A9 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 S100A9 may play important roles in the pathogenesis of ESCC,but much more further study is needed to do.4.In this study,the differential proteomics technology platform was used by combining with DIGE-MALDI TOF/TOF mass spectrometry-bioinformatics analysis,which could provide an effective strategy for the screening and identification for tumor biomarkers. |
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