| Objectives and SignificanceLaryngeal and hypopharyngeal cancers were common malignant tumors of head and neck which threaten people’s lives and healths seriously, and the most common pathological type was squamous cell carcinoma. In recent years, due to the interaction of different carcinogenic factors such as environment, laryngeal and hypopharyngeal cancer incidences have been on a gradual upward trend. While the progresses of combination treatment which based on surgical measures mainly have been made, the5-year survival rate of laryngeal carcinoma patients was still no noticeable improvement after multimodality treatment. However, clinical data suggested that different stages had significant influences on prognosis of patients with treatment. According to statistics data, the results of treatments on the early-stage of laryngeal and hypopharyngeal cancers were better, and the laryngeal functions were better reservated commonly; however, because of the local infiltration and metastasis of lymph nodes for advanced laryngeal and hypopharyngeal carcinomas, the treatment results were disappointed and the5-year survival rate was decreased. Thus, to improve the consequences of laryngeal and hypopharyngeal carcinoma, early diagnosis and early treatment was important. However, since there were no reliable and specific indicators for early diagnosis of the tumors currently, early diagnosis of laryngeal and hypopharyngeal carcinoma were difficult, therefore, it was urgently to look for specific tumor markers of laryngeal and hypopharyngeal cancer for early clinical diagnosis.With the development of techniques of molecular biology and the further research on cancers, the understanding of etiology and pathogenesis of cancers were gradually deepened, and some relevant oncogenes and tumor suppressor genes about tumorigenesis, development and prognostic have been found. Functions of gene were performed by proteins which encoded, and proteins were the true performers for lives. The development of proteomics technology have provided a new technology platform for overall study on tumor development and malignant transformation process, as well as found technical basis of looking for the new specific tumor markers.Proteomics is a scientific study of proteom compositions and their variation rules of cells, tissues or organism, and the aims of which is to clarify the mode of protein expression patterns and functional patterns of all living organisms, including proteins expression, existences, structures, functions and interactions and so on. We can understand the effects of external factors to cell metabolism, the functions of changed proteins in various disease, and molecular mechanisms of all the pathophysiological processes by detecting the level of proteins in different environments, different time, drugs treatment, diseases and changes in physiological processes. In cancer research, advanced studies have been performed by proteomic approach, which were mainly on protein quantities, structures, characters, relationships and biological functions of tumor tissues, to further clarify the mechanism of tumor development, to look for all kinds of abnormal expression of proteins which can be used as markers for cancer diagnosis and screening of drugs target for cancer treatment. In recent years, the proteomics techniques were well progressed in early diagnosis of lung cancer, liver cancer, stomach cancer, renal cell carcinoma, and cervical cancer, and a series of potential cancer-related proteins and tumor markers were discovered, which may provide wealthy resources for cancer-related research. Both domestic and overseas studies have observed that there were differential expressioned proteins between head and neck cancers and adjacent normal, primary cancers and lymph node cells, and in salivary proteins, additionly, the results were confirmed by experimental data. At present, there were few plasma proteomics reports on laryngeal and hypopharyngeal carcinoma, and there were no available tumor markers for early diagnosis, treatment and prognosis for clinical monitoring.According to the study situation of plasma proteomics and its significance of tumor markers for laryngeal carcinoma and hypopharyngeal carcinoma, the purpose of this study is to utilize proteomics technology to screen differential expressed plasma proteome to look for potential serum tumor markers for patients with laryngeal and hypopharyngeal cancers, therefore, our study will provide reliable experimental data for early diagnosis of laryngeal and hypopharyngeal carcinoma.Materials and methods1. Clinical samples collectionThe plasma samples from20cases of laryngeal cancer patients and20cases of hypopharyngeal cancer patients were collected in Otolaryngology Head and Neck Surgery of Nanfang Hospital from January2010to June2012, and all the samples were collected before treatment. There were16male and4female patients in laryngeal cancer group, with53-60years old, T1-4N1-3M0TNM stage, and laryngeal squamous cell carcinoma (LSCC) of pathologically type; there were18male and2female patients in hypopharyngeal cancer group, with50-60years old, T1-4N1-3M0TNM stage, and hypopharyngeal squamous cell carcinoma (HSCC) of pathologically type.20cases of normal plasma samples were collected from healthy physical examination department of Nanfang Hospital in June2012, with15male and5female donors, and50-60years old. The samples were deposited in-80℃refrigerator.2. High abundance proteins removement of plasma samplesTo remove high abundance proteins such as albumin and IgG to strengthen the development of low abundance proteins, a SIGMA ProteoPrep Blue Albumin and IgG Depletion Kit was used.3. Purification of plasma protein samples2-D Clean-up Kit was used to remove salt, fat, polysaccharides and other material which interfere with two-dimensional electrophoresis.4. Protein concentration determinationThe protein concentration of the samples were determined by EttanTM2-D Quant Kit.5. CyDye labelingInternal standard was protein mixture of all the samples with25μg each, and50μg total protein for internal standard in one gel, which was marked with Cy2dye. Three special fluorescent dyes named Cy2, Cy3and Cy5were labeled with50μg protein:400pmol dyes.6. Two dimentional electrophoresisThe rehydration process was performed with immobilized non-linear pH gradient (IPG) strips (pH3-10NL,24cm) which were later rehydrated by CyDye-labeled samples in the dark at room temperature overnight. Isoelectric focusing was then performed using a IPGphor apparatus. The gels were then run in an Ettan DALT Six gel tank.7. Image scanning of analytic gelThe gels after electrophoresis were scanned with Typhoon Imager Scanner9400, and the channels of Cy2, Cy3and Cy5were respectively scanned by488/520nm,532/580nm and633/670nm, and the PMT value was set as the biggest grey value within the range60000-90000in the whole gel or the interested region. The blue, green, and red images which were labeled with Cy2, Cy3and Cy5dyes were observed respectively.8. Production of preparative gelA preparative gel, containing600-800μg of unlabeled internal standard mixture proteins, was prepared for2DE, the steps and parameters were the same as the analytic gels. After post-stainning with colloid staining dyes, the preparative gel was matched with analytic gels to pick the differential spots analyzed with DeCyder 2D software.9. Differential spots analysis with software and spot pickingDifferential in-gel analysis and biological variation analysis were analyzed with DeCyder V6.5software to look for the differential expressed protein spots, and then the varied spots were matched on the preparative gel, finally, they were picked by EttanTM Spot picker automatically.10. In-gel digestion of differential expressed spotsThe picked spots, which were added trypsin, were placed in37℃constant temperature water bath for14-16hr; After the extraction of digested peptides were concentrated to about10μl by vacuum freeze drying apparatus. ZipTip (?) C18were used to purify and concentrate samples, and finally,2μl CHCA matrix (5mg/ml) was used to elute the protein mixture, and then placed to the clean and dry MALDI sample plate directly.11. Mass spectrometry identificationSample target was input to ABI4800MALDI-TOF/TOF mass spectrometer, after internal standard calibration, the peptide mass fingerprint (PMF) were aquired automatically, and then the strongest tenth precursor ions were selected for MS/MS mass spectrometry, and finally, the peptide sequence tags (PST) were got after amino acid sequencing. PMF and PST data were used to search in database with Mascot software, and MS combined MS/MS pattern was matched to NCBInr database, protein score which was greater than95%was highly credible. Bioinformatics analysis were performed for those highly credible proteins.12. Western blot4plasma samples of laryngeal cancer,8of hypopharyngeal cancer and8of healthy persons were selected randomly to perform Western blot verification.13. Enzyme-linked immuno sorbent assay20plasma samples of laryngeal cancer, hypopharyngeal cancer and healthy persons were respectively selected randomly to perform ELISA verification.14. Statistical methodsFor ELISA analysis, independent samples Student’s t-test was used to determine mean differences between two groups, and Levene test was used in homogeneity test of variances, and a P<0.05was used to assess significance of differences using SPSS17.0software. All measurement data was shown with x±SE to reflect the discrete tendency of sample mean.Results1. Results of protein concentration assay2-D Quant kit was used to assay protein concentration firstly, and then a stained pictures of SDS-PAGE was to verify the accuracy of protein concentration. The results showed that each protein bands were equal.2. Image scanning of analytic gelClear protein expression spectrums were got after two-dimensional gel electrophoresis and Typhoon Imager9400image scanning. Samples labeled with different dyes showed different color maps by ImageQuant TL software.3. Differential expressed spots analyzed by Decyder2D softwareAccording to the DeCyder software analysis,28protein spots had significant differences occurring between the LSCC patients and the healthy honors, among those spots,9were up-regulated and19down-regulated. Addtionally,36protein spots had significant differences occurring between the HSCC patients and the healthy honors, among those spots,16were up-regulated and20down-regulated.4. MS identification of picked spotsThe differential expressed protein spots were analyzed by ABI4800MALD-TOF/TOF mass spectrometry. Mascot software was used to search the proteins in Swiss-Prot database, except proteins with the same name,16proteins were identified in LSCC and18proteins in HSCC. 5. Verification results of Western blotAccording to Western blot results, AGP1was significantly increased in the plasma of LSCC patients, and AHSG was significantly increased in the plasma of HSCC patients. These results were consistent with the data from the proteomics experiments.6. Verification results of ELISAAccording to ELISA results, AGP1was increased between LSCC patients (n=20) and healthy donors (n=20), which was consistent with the data from the proteomics experiments. The plasma AGP1concentration of control group is965.53±49.24μg/ml (x±SE), and LSCC group is2109.13±99.94μg/ml (x±SE). Levene test results showed that the variances was equal, and after independent samples t test, t=10.282, P<0.05(0.000) was observed, therefore, the results suggested that AGP1concentration had a significant difference between LSCC and healthy controls. AHSG was increased between HSCC patients (n=20) and healthy donors (n=20), which was consistent with the data from the proteomics experiments. The plasma AHSG concentration of control group is129.36+4.04μg/ml (x±SE), and HSCC group is261.61±8.39μg/ml (x±SE). Levene test results showed that the variances was equal, and after independent samples t test, t=-14.197, P<0.05(0.000) was observed, therefore, the results suggested that AHSG concentration had a significant difference between HSCC and healthy controls.ConclusionsThis study identified differentially-expressed plasma proteomes of LSCC and HSCC by DIGE and MALDI-TOF/TOF platform, and two of the interested proteins were consistent with the results of functional experiments. Through this study,4conclusions could be drawn as follows:1. The differentially expressed DIGE maps of plasma were established successfully in LSCC patients and healthy controls, and28varied spots were picked by an spot picker automaticly. These28protein were identified by mass spectrometer successfully, except for proteins with the same name,16kinds of proteins were got at last.2. The differentially expressed DIGE maps of plasma were established successfully in HSCC patients and healthy controls, and36varied spots were picked by an spot picker automaticly. These36protein were identified by mass spectrometer successfully, except for proteins with the same name,18kinds of proteins were got at last.3. Among the candidates proteins, AGP1and AHSG were verificated by Western blot and ELISA, and the results were consistent with the data from the proteomics experiments.4. Both AGP1and AHSG were upregulated in plasma of LSCC and HSCC patients, the differences were significant by statistical tests, therefore, they were expected to be specific candidate serum tumor biomarkers for the early diagnosis of LSCC and HSCC.According to the results above, the candidate differential proteins should play an important role in the process of development of LSCC and HSCC, and these proteins should be as the specific serum tumor markers for these cancers. Proteomics technology could provide a new way for look for tumor related biomarkers in blood to help diagnosis of head and neck cancers. |
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