Discovery,Identification And Confirmation Of Non-inflammation Serum Biomarkers Of Wilms Tumor

1 Background and objectiveWilms tumor is the most common retroperitoneal solid cancer in pediatrics. The incidence of Wilms tumor is the highest in abdominal solid malignant tumor in children. It is constitute more than 80% of urogenital system tumor in children under 15. Normally, tumor patients are less than 5 years, the most patients between 2 years and 4 years. The incidence of Wilms tumor in left kidney is closely to right. Bilateral tumor is rare which accounting for 3%-10% of the total number. Patients are no obvious gender difference. The number of men is greater than that of women in most report.Wilms tumor patients are too young, can not clearly and accurately describe uncomfortable feeling, and the early clinical symptom is unclear. It is easy to delay the diagnosis, affects the treatment result.Imaging examinations are suitable for larger tumor, for example, Intravenous pyelography, ultrasound, computerized tomography, magnetic resonance and so on.Biomarkers which have lower sensitivity and specificity, such as serum lactate dehydrogenase and alpha-fetoprotein regard as the method to diagnose Wilms tumor. So there are limited in the diagnosis and treatment of early Wilms tumor. In the current situation, treatments for advanced Wilms tumor which consist of operation and chemotherapy are far more important than early diagnosis. But the study of National Wilms Tumor Study (NWTS) group showed the higher stage, the survival rate, whatever treatments. Thus it is most immediate problem to find early diagnosis way of Wilms tumor which is the key of raising survival rate.Development in proteomics and the relevant technique while meet needs of finding ways of early diagnosis Wilms tumor patients. In 1994, the concept of proteomics was proposed by Wilkins and Williams in Australia. Proteomics is the study of proteinic features in the level of large scale. It is consist of the expression level of protein, post-translational modification, interactions among protein and so on. Hence the study will give us a whole and overall understanding of process about occurrence of the disease and cell metabolism in level of protein. No matter what diseases, the level of serum protein is different from normal, quantitative changes or qualitative changes. Because of the above features, the diagnosis system can be established to monitoring level protein of specific disease would serve purposes of early diagnosis. It is an important research direction of early diagnosis of diseases, especially for early diagnosis of tumor. The core issue of this research direction is finding specificity protein biomarkers of the tumor. As the technology of proteomics developed, more and more biomarkers of disease were identified. Proteomics is the most effective way which screens molecule marker and target drug in disease. It can be seen that the related technical offers new facilities for finding practical way of early detecting and screening in Wilms tumor. Surfaced enhanced laser desorption/ ionization time of flight mass spectroscopy (SELDI-TOF-MS);matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy (MALDI-TOF-MS); high performance liquid chromatography (HPLC) and two dimensions-liquid chromatography-linear trap quadrupole-mass spectrometer(2D-LC-LTQ-MS)are critical technologies.The technologies analyze of quantitative and qualitative to serum of per-surgery, post-surgery and control;screen and identify specific biomarkers in serum of Wilms tumor children which is different from in serum of normal children.Western blot was used to confirm biomarkers which was screened and identified. The biomarkers could be used to early diagnosis of Wilms tumor.Inflammatory factors of non-specificity were produced by carcinoma tissue and borderline tissue. So the level of serum protein in tumor patients is similar with inflammatory diseases patients. It is possible that biomarkers which were identified in our previous work were linked to inflammatory diseases and benign diseases. Inflammatory factors interrupted the quantitative and qualitative of serum protein in tumor. The situation would reduce credibility and adaptability of biomarkers which had been identified. The underlying cause was inflammation and inflammatory factors play a key role in the course of tumor developing. Therefore, suppress interference of inflammatory factors is important work in this study which is finding specificity protein biomarkers. The purpose of the study is discovery and confirmation of non-inflammation serum biomarkers of Wilms tumor.2 Materials and methods2.1 Materials2.1.1 Clinical materials303 serum samples which were used in the study were collected from the Department of Pediatric Surgery at the First Affiliated Hospital of Zhengzhou University. Blood was taken from peripheral veins in the early morning with an empty stomach. It was incubated for 1 hour at 4℃and then centrifuged at 3,000 rpm for 15 minutes. Serum was separated in tubes for 100μL/tube. They were collected and stored at -80℃before use. The 103 serum samples were selected from primary untreated Wilms tumor (I stage 45; II stage 28; III stage 24 and IV stage 6 (The standards to divide stages from NWTS-5)); The 97 serum samples which were group of post-surgery were selected from Wilms tumor patients after surgery (The serum samples which stem from patients group of per-surgery after his operation were collected in between 7 days and 14 days after the operation (median time of 11.27 days)); The 103 serum samples were selected from healthy individuals in group of healthy controls. The cases of primary untreated Wilms tumor (57 males,46 females) which were collected in first visit had a median age of 39.12 months (ranging from 2 to 183 months).The patients were not suffered by chemotherapy, operation and interventional therapy. Patients after surgery (53 males,44 females) had a median age of 40.81 months (ranging from 2 to 183 months). Healthy individuals (57 males,46 females) had a median age of 44.31 months (ranging from 3 to 194 months). The pathology type of samples which were identified as favorable histology (FH), but cystic partially differentiated nephroblastoma (CPDN) was ruled out which was based on favourable prognosis. Pathological sections from patients were confirmed to have Wilms tumor by more than two pathologists. The study protocol was approved by the local ethics committee. Written informed consent was obtained from parents, guardians, or patients.2.1.2 Selection criteria of inflammatory factorsWe found fluctuation of level of inflammatory factors in serum of tumor patients by analyzed the correlation between inflammatory factors and tumor. All the inflammatory factors are closely related with tumor and are reported in relevant article. Compared mass spectrogram of inflammatory factors with peak values of specificity protein in Wilms tumor, and excluded the similar peaks. Interference with finding biomarkers of Wilms tumor caused by inflammatory factors and inflammation would be excluded in the process. The goal inflammatory factors include:MIP Macrophage inflammatory protein (MIP)-la, MIP-1β,MIP-3a and MIP-3βare related to Breast Adenocarcinoma, Hepatocellular Carcinoma, Pancreatic Ductal Cell Adenocarcinoma and Bone Metastases of Renal Cell Carcinoma in CC family; interleukin (IL)-10, interferon y-inducible protein (IP)-10 and monokine induced by IFN-γ(MIG) in CXC family;High expression of IP-10, monocyte interferon gamma inducing factor (MIG), MIP-la and RANTES has been reported in Renal Cell Carcinoma; IL-1,IL-6 and TNF-a are related to Colon Carcinoma and Oral Cavity Cancer; IL-10 is related to ovarian cancer; VEGF is related to Squamous Cell Carcinoma of the Oral Cavity. Interference with finding specificity protein of Wilms tumor caused by the inflammatory factors would be excluded.2.1.3 Reagents and instrumentsAcetonitrile (HPLC grade) and another mainly reagents were purchased in Sigma Inc (USA). Mouse anti-human IgG serum amyloid A1 (antiserum SAA1), rabbit anti-human IgG apolipoprotein C-III (antiserum apoC-III) and anti-mouse antiserum, anti-rabbit antiserum were acquired from santa cruz biotechnology Inc (USA). All recombinant human inflammatory factors were purchased from PeproTech Inc (USA).ProteinChip Biosystems (Ciphergen PBS II plus SELDI-TOF-MS) and WCX2 chip which were used to screen peaks of Wilms tumor and measure peaks of inflammatory factors were purchased from Ciphergen Biosystems Co (USA). HPLC which was used to purification of samples was purchased from Shimadzu (JPN) and liquid chromatographic column:C18 (250*4.6mm) was from Sunchrom Inc (GER). MALDI-TOF-MS which was used to identify to peaks value of specificity protein was purchased from Kratos Analytical Co (UK).2D-LC-LTQ-MS system was purchased from Thermo Electron Corporation (USA). Mini-PROTEAN Tetra Electrophoresis System, Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell and their accessories were purchased from Bio-Rad Laboratories Inc (USA).2.2 Methods2.2.1 Detecting the peaks value of inflammatory factors and serum protein of Wilms tumorFrozen serum samples were thawed on ice and spun; 10μl U9 was added in 5μl serum;Diluted the samples which were vibrated (600rpm,4℃,30min) with Binding Buffer to 200μl, vibrated (600rpm,4℃,2min) too. The 96-hole chip was fixed in supporting of Bio-processor, the numbers of chip are recorded; every hole was added in Binding Buffer (200μl), vibrated (600rpm, room temperature,5min) and repeated. The samples which were added in U9 were dripped into the 96-hole chip which was processed, removed samples about an hour later; every hole was added in Binding Buffer (200μl) and removed it about an hour later after vibrated (600rpm, room temperature,5min);Repeat twice. Every hole was washed by ultrapure water. Shake it dry and upside down on blotting paper. The dried chip of hole was added in 1μl 50 %saturated SPA. After drying the array surface in the air, the process was repeated. The testing process was after natural dryness. Inflammatory factors were processed in the same way.2.2.2 Analysis of data and biostatisticsAfter the raw data of mass spectrometry were filtered out the noice and with clustering analysis, the peaks of preliminary screening were analyzed by Wilcoxon test, the data of intergroup was analyzed by t-test (a=0.01).Compared pre-surgery group with post-surgery group and compared pre-surgery group with control group, the peaks of difference which were found between pre-surgery group and another two groups. Compared peaks of inflammatory factors with the peaks of difference, the same peaks and difference peaks were fallen into two groups.2.2.3 Purification of candidate non-inflammation serum biomarkersThe results of SELDI-TOF-MS show that, the serum samples of high expression on proteins of 11526 Da and 4756 Da would be purified. After samples was thawed on ice,300μl CAN and 300μl water (HPLC grade) were added in 100μl serum (stirring and standing 4℃,5min). The supernatant was taken out after centrifuged, and it was placed in SPD SpeedVac to 100μl. C18 column was washed by B fluid (95% ACN,5% ultrapure water,0.1% TFA) for 15min, and A fluid (95% ultrapure water,5% ACN,0.1% TFA) too. The HPLC separation was achieved with a linear solvent gradient:(100% A/0 min)-(20% B/15 min)-(40% B/30 min)-(70% B/80 min)-(100% B/100 min) at a flow-rate of 1 ml/min. The solution of each protein was stored in eppendorf tubes and it was placed in SPD SpeedVac to 20μl.2.2.4 Identification of candidate non-inflammation serum biomarkersThe protein samples which were freeze-dried were taken in MALDI-TOF-MS and detected. The samples which were similar with non-inflammation protein on molecular weight were found.C18 was taken in capillary column, and sample which was enzyme digested was forced into sample column. After linked sample column to gradient elution column in 2D-LC-LTQ-MS,the peptide would be detected. The result was entered into SEQUEST program and indexed protein which was similar with peptide in database of Bioworks.2.2.5 Confirmation of candidate non-inflammation serum biomarkersFrozen serum samples were thawed on ice, and centrifuged at 10,000 rpm for 5 min. The 100μl samples were separated on spacer gel and separation gel in constant voltage of 103 volts. When the Bromcresol Blue exceeded space of gel, separation of proteins was terminated. Separated gel plate from glass panel, PVDF membrane and paper of gel were sandwiched in between three-ply filtering paper, and Candidate proteins were transferred to PVDF membrane in constant voltage of 20 volts for about 5min. After antigen-antibody reaction, the image of immunocomplex was projected onto the camera film. The photographs of the Western blotting were analyzed by image pro plus 6.0 and gray values were obtained. Statistical analyses were performed using SPSS, version 13.0 and statistically significant or not were calculated.3 Result3.1 Screening serum biomarkers of Wilms tumorPeaks of protein were detected in serum samples of three groups, and the peaks of difference were screened by comparison among groups and Wilcoxon test (P<0.01). The 17 peaks were screened between group of pre-surgery and group of control,14 peaks were high expression and 1 peak was low expression in group of pre-surgery; The 14 peaks were screened between group of pre-surgery and group of post-surgery, 9 peaks were high expression and 5 peaks were low expression in group of pre-surgery. All specificity proteins were compared, the level expression of 11526 Da and 4756 Da were different in between pre-surgery and control and in between pre-surgery and post-surgery, and the proteins were high expression in group of pre-surgery. The expression of protein was different between post-surgery and control. 3.2 Detecting peaks value of inflammatory factors which were related to tumorThe inflammatory factors which are related with tumor were detected.The mass spectrogram includes inflammatory factors and their peptides.3.3 Compared peaks of biomarkers of Wilms tumor with peaks of inflammatory factorsCompared mono-charged or multiply charged of inflammatory factors and peptides with 17 peaks which were screened between group of pre-surgery and group of control and 14 peaks which were screened between group of pre-surgery and group of post-surgery, the similar peak was not found. In this process, exclude interference of inflammatory factors to a certain extent in finding specificity protein biomarkers. The proteins of 11526 Da and 4756 Da were identified as non-inflammation serum proteins of Wilms tumor.3.4 Purification and identification of non-inflammation serum biomarkersThe proteins which were purified by HPLC were stored in eppendorf tubes. The molecular weights of all the proteins were detected by MALDI-TOF-MS, and the samples of the proteins and peptides which were 11526 Da and 4756 Da in molecular weights were found. Each protein or peptides was enzyme digested and detected by 2D-LC-LTQ-MS system. The protein of 11526 Da was entered into SEQUEST program and indexed protein which was similar with peptide in database of Bioworks, and the protein was one of peptide in serum amyloid A1(SAA1).In the same detecting way, the protein of 4756 Da was one of peptide in apolipoprotein C-III (APO C-Ⅲ).3.5 Confirmation of non-inflammation serum biomarkersThe method of measure the contents of SAA1 and APO C-Ⅲ:First of all, gray values which came from bands of interest protein were measured of three groups (the group of per-surgery, post-surgery and control) in the same gel plate. In the second place all the gray values were measured of all gel plates. Finally, the gray values of three groups were used to statistical analysis. The results showed that there was statistic significance between the gray values of SAA1 and APO C-Ⅲin group of pre-surgery and in group of post-surgery, and the statistic significance also existed between in group of pre-surgery and in group of control (p<0.05).4 ConclusionsThe study confirmed the protein of 11526 Da was SAA1 and the protein of 4756 Da was APO C-Ⅲ. The expression of level of the two proteins was statistic significance between group of pre-surgery and post-surgery, between group of pre-surgery and control. Interference of inflammatory factors which were related to tumor was excluded in the process of finding biomarkers of Wilms tumor. With the above experiment process, SAA1 and APO C-Ⅲwould become more meaningful biomarkers. From what has been studied above, not only the biomarkers of Wilms tumor with more specificity were identified and confirmed,but also the experimental considerations and technique flow is practicable and it offers new facilities for finding biomarkers of another diseases.