Discovery, Identification And Confirmation Of Biomarkers Of Wilms Tumor After Eliminating Inflammation Factors Interference

1 Background and objectiveWilms’ tumor(WT), also known as nephroblastoma or embryonic renal tumor, is a rare solid tumor that is embryo-derived and mostly occurs in children. In the developing kidney of affected patients, gene regulation becomes abnormal in certain renal embryonic cells, and these cells develop disordered differentiation and maturation, along with uncontrolled proliferation and division.In Wilms’ tumor cells, can often see the original tubules and glomeruli. 75% of WT is most common in the first 5 years of life, and is especially common in patients aged 2–3 years. The incidence of WT is comparable for the right and left kidneys, and 10% of patients develop bilateral WT either simultaneously or sequentially. The prevalence of WT is similar for males and females, but most studies report a slightly higher incidence in males. Several case studies have reported WT in adults.WT usually appears as solitary with a large volume and clear borderline, and is covered bya pseudomembrane in some cases. Bilateral and multifocal tumors occur in a small number of patients. The tumor is usually soft, flesh-like in cross-section, and grey in color. There may be hemorrhage and necrosis, and bone-like tissues may be present.The major factors influencing the prognosis of children with Wilms’ tumor include early diagnosis, pathological classification, and appropriate treatment, including surgery or chemotherapy for children with stage I and II tumors and tumor resection combined with adjuvant chemotherapy or even radiotherapy for high-stage tumors. Diagnosis of Wilms’ tumor is dependent on clinical manifestation, ultrasonography, intravenous urography, and computed tomography. Although most patients are ultimately diagnosed, late diagnosis delays treatment and may affect prognosis.Thus, recent studies have investigated markers for the early diagnosis of WT, and proteomics studies have examined the role of specific proteins in the pathogenesis of WT. Surface-enhanced laser desorption/ionization time of flight mass spectrometry(SELDI-TOF-MS) is the most widely used technique in proteomics, and is often coupled with protein microarray methods. Protein microarrays can be classified as analytical or functional microarrays. Analytical microarrays selectively identify proteins based on their chemical characteristics(hydrophobicity, hydrophily, and charge). Thus, SELDI-TOF-MS can be used with protein analytstical microarrays to screen differentially expressed genes at different stages of a disease. Previous studies of tissues and body fluids reported that some proteins had altered abundance in patients with certain tumors, and may therefore be useful for tumor screening.Tumor markers can enable early diagnosis and permit monitoring therapeutic outcomes. Although we previously screened and identified specific protein markers for Wilms’ tumor using proteomics analyses of patient sera, the analysis was confounded by the presence of inflammatory factors, which may affect their efficacy for monitoring and diagnosis of Wilms’ tumor. Therefore, it is necessary to exclude inflammatory factors during screening and identification of tumor markers.Exclusion of inflammation-associated factors cannot be performed by only excluding thetumor-associated inflammatory factors. In the present study, serum samples from systemic inflammatory response syndrome(SIRS) patients were used as a control group, thus extending the range of inflammatory factors to be excluded. SIRS is characterized by the uncontrollable self-amplification and self-destruction of the body due to an excessive response to either infectious or noninfectious origin. Many inflammatory factors and cytokines, including interleukin-1(IL-1) and tumor necrosis factor-α(TNF-α), in addition to neutrophil degranulation products, complement fragments, arachidonic acid derivatives, and a variety of chemotactic factors are detected in the blood of SIRS patients.Surface-enhanced laser desorption/ionization-time of flight-mass spectrometry(SELDI–TOF–MS) was employed to analyze the sera of patients with Wilms’ tumor, healthy children, and patients with SIRS. After exclusion of inflammatory factors, protein markers were screened and identified using mass spectrometry.In hope of being consistent with previous findings, proteins were purified with high-pressure liquid chromatography(HPLC) as well as matrix-assisted laser desorption ionizationtime of flight-mass spectrometry(MALDI-TOF-MS), and the screened specific protein markers were identified by two-dimensional liquid chromatography- linearion trap mass spectrometry(2D-LC-LTQ-MS). Meanwhile, in order to verify whether there is similar non-inflammatory intratumor protein marker in Wilms’ tumor, Surface-enhanced laser desorption/ionization-time of flight-mass spectrometry(SELDI–TOF–MS) was employed to check proteins expressed in tumor tissue and adjacent normal kidney tissue and compare them with the sera of patients with SIRS for identification of potential tumor-specific markers by excluding inflammatory factors. TRICINE-SDS-PAGE was performed to purify and collect the target proteins, followed by identification by MALDI-TOF/TOF.Therefore, the objective of the thesis is to identify and screen out specific protein markers for Wilms’ tumors after exclusion of factors associated with inflammation. 2 Materials and Methods: 2.1 Materials 2.1.1 Patients and SamplesSerum samples were obtained from 50 preoperative patients with Wilms’ tumor(28 males and 22 females, mean age of 2.9±0.1 years), 60 healthy children(34 males and 26 females, mean age of 3.2±0.1 years), 60 patients with SIRS(37 males and 23 females, mean age of 3.1±0.1 years) from the First Affiliated Hospital. There were no significant differences in age and gender among the patient groups. Peripheral venous blood samples were harvested in the morning prior to their first meal, placed at room temperature for 1 h, centrifuged at 3000 g for 10 min, and stored at-80 °C.WT tissues were collected from 45 children(23 boys and 22 girls, mean age of 2.9 ± 0.1 years) who received surgery in the First Affiliated Hospital of Zhengzhou University from 2011 to 2013. Adjacent normal kidney tissues(controls) were collected from the same kidneys of 40 children(22 boys and 18 girls, mean age of 2.8 ± 0.1 years) who received radical surgery(n = 31) or palliative surgery(n = 7). WT was pathologically confirmed after biopsy or surgery by more than 2 pathologists. Age and gender were comparable in the control and WT groups. All samples were stored at-80°C before use.All experiments approved by the ethics committee. Written informed consent was obtained from each patient or their guardian. 2.1.2 Reagents and InstrumentsAcetonitrile(HPLC grade) and other major laboratory reagents were purchased from Sigma(USA); Na AC、SPA、DTT、CHAPS were purchased Promega(USA);all inflammatory factors used to compare with the peak value of specific proteins were purchased from Pepro Tech(USA). RNeasy Plus Micro Kit and RNAprep pure Tiss ue Kit were from Qiagen Inc.(Bei Jing).As for SELDI-TOF-MS/PBS II+and Bio-processor: WCX2 microarrays were purchased from Ciphergen(USA); HPLC(used to purify proteins from serum samples) from Shimadzu(Japan); chromatographic column C18(250*4.6mm) from Sunchrom(Germany); MALDI-TOF-MS(used to identify non-inflammatory protein markers for Wilms’ tumor) from Kratos Analytical(UK); 2D-LC-LTQ-MS from Thermo Electron(USA); Mini-PROTEAN Tetra Electrophoresis System and Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell from Bio-Rad Laboratories(USA). 2.2 Methods 2.2.1 Extraction of total proteinsProcedures were performed on ice. Tissues were weighed and mixed with lysis buffer(100 mg tissue/500 μL buffer). Then, 2 μL of protease inhibitors were added, and homogenization was performed until there were no evident macroscopic tissues. The homogenate was transferred into a pre-chilled tube, and then centrifuged at 14000 g /min for 25-30 min at 4°C. The resulting supernatant was transferred to another pre-chilled centrifuge tube and served as the total protein solution. The supernatant was stored at-80°C before analysis. 2.2.2 Screening for intra-tumor proteinsChip pretreatment was carried out at the same time that the samples were prepared. After the chip was placed into the Bioprocessor and the chip number was recorded, 200 μL Na AC(100 mmol/L, p H4) was added to each well. After the chip was shaken at 600 g for 2 min, the procedure was repeated once more.Simultaneously, serum samples were thawed on ice and then centrifuged at 10,000 g at 4 °C for 2 min. A 96-well plate was placed on ice, and 10 μL U9(9M urea, 2% CHAPS, 1% Bar) and 5 μL serum were added to each well. The plate was shaken at 600 g for 30 min at 4 °C. After it was placed on ice, 185 μL Na AC was added, and the plate was shaken at 600 g for 2 min. After 100 μL of the sample was added to the pretreated chip, it was then shaken at 600 g for 60 min at 4 °C. The remaining supernatant was removed from the plate, which was quickly dried. After 200 μL Na AC was added to the plate, it was shaken at 600 g for 5 min, and the supernatant was removed. This procedure was repeated three times. Each well was washed twice with 200 μL deionized water. After the chip was dried, 1 μL 50% saturated SPA was added to each well. The chip was then dried and preserved.The SELDI–TOF–MS system was corrected to an error of less than 0.1% in molecular weight using a protein chip of a known molecular weight. After the WCX2 protein-binding chip was analyzed using mass spectrometry, the original data were filtered for noise, and cluster analysis was performed. The Wilcoxon rank-sum test was carried out for the mass-to-charge ratio of the peak value obtained by the initial screening. A 0.01 significance level was used. 2.2.3 Data Analysis and Statistical TreatmentNoise should be filtered first, and then cluster analysis was performed. In the next place, Wilcoxon rank-sum test was carried out to analyze m/z peaks, and last mass spectrometric data from different groups should be analyzed through t testwith thetest criterionα=0.01. The differential protein peak values of patients group(Wilms’ tumor patients) could be identified through comparison with normal control groups. Compare peak values of inflammatory factors with the screened differential ones and classify similar ones and differential ones into groups. 2.2.4 Purification of non-inflammatory serum protein markersSerum samples were thawed on ice, and 100 μL was added to 300 μL deionized water and 600 μL CAN. After mixing, the samples were placed at 4 °C to precipitate the serum proteins. After 30 min, the samples were centrifuged at 10,000 g for 30 min at 4 °C. The supernatant was collected and dried under vacuum to a volume less than 50 μL. After 450 μL H2O/0.1% trifluoroacetic acid(TFA) was added, desalination was carried out using solid-phase extraction columns packed with C18.Separation of the treated sample was carried out using C18 reverse phase high pressure liquid chromatography column. The mobile phase A consisted of H2O/0.1%, and the mobile phase B was ACN/0.09% TFA. The samples were eluted as follows: 100% mobile phase A for 15 min, 20%–40% mobile phase B for 15 min, 40%–70% mobile phase B for 50 min, 100% mobile phase B for 10 min. The flow rate for the whole process was 0.5 m L/min, and the detected wavelengths were 214, 254 and 280 nm. The components at their peak values were collected and concentrated in a vacuum to a volume of less than 20 μL. The samples obtained by HPLC separation underwent MALDI–TOF–MS analysis in linear mode. 2.2.5 Purification of non-inflammatory tissue protein markersFor SDS-PAGE, 0.75-mm thick glass was used. Both glasses were aligned at the bottom, clamped closely and placed in the bracket. Their bottom was closely pressed on the gel mat to prevent the leakage of gel. The separating gel was added to 2/3 height of the glasses and allowed to stay at room temperature for 1.0 h. When the gel became solidified, the stacking gel was added, and an appropriate comb was placed in the stacking gel, which was allowed to stay at room temperature for 1.0-1.5 h. When the stacking gel became solidified, the instrument was placed in the chamber, and buffer for electrophoresis was added, and the comb was taken out. Samples were located onto lanes. The concentrated samples were mixed with an indicator(5u L), boiled for 15-20 min, centrifuged at 5000 rpm for 5 min, and then subjected to SDS PAGE with Coomassie Brilliant Blue. Marker was added to the first lane and used to define the molecular weight, and samples in other lanes should be recorded in detail. The instrument was connected to the power supply, and the voltage was set at 30 V. When the samples reached the borderline between the separating gel and stacking gel, the voltage was set at 100 V. When the marker reached the bottom of the gel 4-6 hours later, the instrument was powered off, the glasses were taken out, the stacking gel was gently removed from the separating gel. The stacking gel was discarded, and the separating gel was carefully separated and placed in a dish, followed by incubation with Coomassie Brilliant Blue for 6-8 h under vortexing. Then, the gel was washed with deionized water, and de-coloring solution was added, followed by incubation for 4-6 h under vortexing. When the color was absent, the gel was washed with deionized water, and target bands were present in the gel. The target protein bands were excised and transferred into different Eppendorf tubes on ice for identification by MALDI-TOF-MS. 2.2.6 Identification of non-inflammatory serum protein markersAfter isolation, 20 μL of the target protein was incubated with 60 μL of 8 M urea to give a final concentration of 6 M. After shaking at room temperature for 20 min, 0.8 μL of 1 M DTT was added, and the sample was then placed at room temperature. After 1 h, 3.2 μL of 1 M IAM was added, which was placed in the dark for 45 min after which 3.2 μL 1 M DTT was added and incubated at room temperature for 30 min. Following addition of 400 μL 50 m M NH4HCO3, the urea concentration was reduced to 1 M with a p H of approximately 8.0. After addition of 0.08 μg trypsin to each sample, they were incubated overnight at 37 °C. The enzymatic digestion was stopped by the addition of 0.2% TFA. After centrifugation at 12,000 g for 10 min, the supernatant was collected and concentrated to a volume of less than 10 μL. The sample was then loaded onto the C18 column, which was connected to the spray system within the 2D-LC-LTQ-MS system for detecting the m/z spectrum of the peptides. The results were imported into the SEQUEST search program and the related proteins were identified in the Bioworks database. 2.2.7 Identification of non-inflammatory tissue protein markersFor protein identification, 80 μL of washing buffer was added to each Eppendorf tube, the tube was vortexed for 20 min at 37°C, and the medium was refreshed with buffer. These procedures were repeated three times until the gel color was absent. The temperature of bath water was set at 90°C and the gel was dried for 15 min. Then, 20 μL of digest Buffer and 2 μL of reducing reagent were mixed and added, followed by incubation at 37°C for 10 min. The mixture was allowed to cool to room temperature, and 20 μL of blocking agent was added, followed by incubation at room temperature for 10 min. Following addition of 0.5 μL of diluted trypsin(final concentration: 8 ng/μL), centrifugation was performed at 5000 rpm for 5 min. The gel was immersed in the liquid, and vortexed at 37°C for 10-12 h. After centrifugation at 10000 rpm for 10-15 min, the supernatant was transferred into a new Eppendorf tube.Then, 1 μL of sample was added to a protein microarray chip which was then dried. Following addition of 1 μL of matrix, the microarray chip was subjected to MALDI-TOF/TOF. After adjustment and laser desorption ionization, the different peptides were harvested. The Swissprot database and Mascot software were used for protein identification. 2.2.8 Validation of targeted tissue protein expression by RT-PCRThe RNAprep pure Tissue Kit was used to extract total RNA from tumor and normal tissues. RNA was used as a template and transcribed into c DNA with the Revert Aid First Strand c DNA Synthesis Kit. Scientific Maxima SYBR Green q PCR Master Mixes were employed to detect the expression of c DNA in tumor tissues and cancer tissues by real time PCR. The primers used for PCR were as follows:Profilin-1: 5′-ACGCCTACATCGACAACCTC-3′(Forward), 5′-TGATGTTGACGAACGTTTTCC-3′(Reverse); Ubiquitin: 5’GTCACTAAGCCATCGGTCGT3’(Forward), 5’ACACGGACACAACCAGTTCA3’(Reverse).The following two-step method was employed for PCR: UDG pre-treatment at 50°C for 2 min, pre-denaturation at 95°C for 10 min and 40 cycles of denaturation at 95°C for 15 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s. Molecular Analyst software was used to analyze the expression of targeted genes, which were normalized to that of β-actin. 2.2.9 Validation of targeted serum protein expression by ELISAAn enzyme linked-immuno-sorbent assay(ELISA) was used to measure the expression of ubiquitin and profilin-1 in tumor and normal tissues. Standard ubiquitin and profilin-1 solutions(0 pg/m L, 62.5 pg/m L, 125 pg/m L, 250 pg/m L, 500 pg/m L, 1000 pg/m L, 2000 pg/m L, and 4000 pg/m L) were prepared. Then, 100 μL of each standard was added to a 96-well plate pre-coated with human ubiquitin and profilin-1 antibodies(3 wells per group). 10 samples each were collected from the tumor group and normal group and diluted 100-fold. Then 100 μL samples were added to a 96-well plate pre-coated with human ubiquitin and profilin-1 antibodies(3 wells per group), followed by incubation at temperature for time. The solution was removed, and 100 μL of biotin-conjugated human ubiquitin and profilin-1 antibodies were added, followed by incubation. After washing the plates, ABC working solution was added, followed by incubation at temperature for time. The plate was washed, and 90 μL of TMB was added for visualization, followed by incubation at temperature for time. Then, TMB stopping solution(100 μL) was added to each well. The optical density(OD) was measured at 450 nm for calculation of concentration. 2.2.10 Validation of targeted tissue protein expression by ELISAAn enzyme linked-immuno-sorbent assay(ELISA) was used to measure the expression of ubiquitin and profilin-1 in tumor and normal tissues. Standard ubiquitin and profilin-1 solutions(0 pg/m L, 62.5 pg/m L, 125 pg/m L, 250 pg/m L, 500 pg/m L, 1000 pg/m L, 2000 pg/m L, and 4000 pg/m L) were prepared. Then, 100 μL of each standard was added to a 96-well plate pre-coated with human ubiquitin and profilin-1 antibodies(3 wells per group). 10 samples each were collected from the tumor group and normal group and diluted 100-fold. Then 100 μL samples were added to a 96-well plate pre-coated with human ubiquitin and profilin-1 antibodies(3 wells per group), followed by incubation at temperature for time. The solution was removed, and 100 μL of biotin-conjugated human ubiquitin and profilin-1 antibodies were added, followed by incubation. After washing the plates, ABC working solution was added, followed by incubation at temperature for time. The plate was washed, and 90 μL of TMB was added for visualization, followed by incubation at temperature for time. Then, TMB stopping solution(100 μL) was added to each well. The optical density(OD) was measured at 450 nm for calculation of concentration. 3 Rusults 3.1 Determination of the target serum proteinsThe pretreated sera from patients with Wilms’ tumor, healthy children and children with SIRS were screened with SELDI–TOF–MS to obtain the peak value of related proteins, peak value of inflammatory factors, and their decomposed peptides. Wilcoxon rank-sum test was carried out to compare the peak values of related proteins in each group(p < 0.01), and the differential protein peak values. Of the proteins analyzed, 37 differential protein peak values were obtained by screening sera in the Wilms’ tumor and normal control groups. Two protein peak values were highly expressed in the Wilms’ tumor group while 35 protein peak values were highly expressed in the normal control group. Twenty-seven differential protein peak values were obtained by screening sera in the Wilms’ tumor and SIRS groups. The differential proteins were compared, and similar protein peak values were identified(there was 0.3% of deviation in the results). The proteins or the peptides with m/z of 6438 Da were screened out, which had the same specificity with the normal control and SIRS groups. After 15 tumor-related inflammatory factors were identified to eliminate the effect of inflammatory factors on screening specific Wilms’ tumorrelated protein markers, the m/z of the non-inflammatory, Wilms’ tumor-related protein marker was 6438 Da. 3.2 Determination of the target tissue proteinsWe used SELDI-TOF-MS to analyze proteins from the tumor tissues of 40 children with WT and adjacent normal kidney tissues from 38 of these 40 children who received radical or palliative surgery. The resulting m/z peaks in these samples, which correspond to proteins(peptides) were compared by the Wilcoxon rank sum test. The results indicate that there were 50 differentially expressed proteins in WT and normal tissues; 29 proteins had higher expression in the tumor and 21 proteins had higher expression in the normal tissue. Fifty differential protein peak values were obtained by screening sera in the Wilms’ tumor and SIRS groups. The differential proteins were compared, and similar protein peak values were identified(there was 0.3% of deviation in the results). The proteins or the peptides with m/z of 8350 Da and 5363 Da were screened out, which had the same specificity with the normal control and SIRS groups. After 15 tumor-related inflammatory factors were identified to eliminate the effect of inflammatory factors on screening specific Wilms’ tumorrelated protein markers, the m/z of the non-inflammatory, Wilms’ tumor-related protein marker was with an m/z of 8350 Da and the other with an m/z of 5363 Da. 3.3 Purification and Identification of the Target Serum ProteinsHPLC was used to isolate and purify the specific marker proteins with m/z of 6438 Da in the serum samples of patients with Wilms’ tumors. After isolation of the proteins and peptide segments with different peak values by HPLC, MALDI–TOF– MS analysis was undertaken to identify the proteins and peptide segments with m/z of 6438Da(SELDI–TOF–MS results had 0.3% of deviation).Enzymatic digestion of the proteins and peptide segments with m/z of 6438 Da was carried out and the peptide segments were detected using 2D-LC-LTQ-MS. The sequence of proteins and peptide segments with m/z of 6438 Da was E.LKEFGNTLE DKARELISRIKQSELSAKMREWFSETFQKVKEK.G. Subsequent analysis of the peptide segment using the SEQUEST search program and the Bioworks database identified this peptide segment as that of apolipoprotein C-I(APO C-I). The coverage rate of two peptide segments in APO C-I was analyzed using the SEQUEST search program. 3.4 Purification and Identification of the Target Tissue ProteinsTRICINE-SDS-PAGE was used to determine the molecular weight, separate, and purify the target proteins. There was a modest difference(~0.3%) in the molecular weights based on SDS-PAGE and MALDI-TOF-MS.Based on the molecular weights of the markers, the targeted bands were excised and digested, followed centrifugation. The supernatant was collected and subjected to MALDI-TOF/TOF. Searched from Swissprot database and Mascot software, this peptide segments was identified as one of Ubiquitin’s. The sequence of peptide segments with m/z of 5363 Da was K.IQDKEGIPPDQQRLIFAGKQLEDGRTL SDYNIQKESTLHLVLRLR.G. Searched from Swissprot database and Mascot software, this peptide segments was identified as one of Profilin-1’s. The sequence of peptide segments with m/z of 8350 Da was K.DSPSVWAAVPGKTFVNITPAEVG VLVGKDRSSFYVNGLTLGGQKCSVIRDSLLQDGEFSMDLRTKSTGGAPTFNV TVK.T. 3.5 Validation of non-inflammatory serum protein marker expression by ELISAELISA was adopted to test APO C-I expression in both WT and normal serum. According to standard curves drawn based on absorption values of reference standards, Protein concentration of APO C-I in WT and normal serum could be obtained by bringing absorption values of samples into equation. The results indicated that APO C-I expression was lower in WT than normal. 3.6 Validation of non-inflammatory tissue protein marker expression by RT-PCRFor confirmation of proteinidentity based on MALDI-TOF-TOF, we performed real time fluorescence quantitative PCR to measure the expression of ubiquitin and profilin-1. The results showed that ubiquitin expression was lower in WT tissues than normal tissues, but that profilin-1 expression was higher in WT tissues than normal tissues. 3.7 Validation of non-inflammatory tissue protein marker expression by ELISAWe confirmed these results by performance of ELISA. In agreement with the RT-PCR results, expression of ubiquitin protein was lower in WT tissues than normal tissues, but expression of profilin-1 protein was higher in WT tissues than normal tissues. 4 ConclusionExpression index of the proteinwith m/z of 6438Da(or peptide fragment of APO C-I) was confirmed to be different in WT and normal serum,which was in accordance with previous research results. Expression index of theproteins with m/z of 5363 Da and 8350Da(or peptide fragment ofubiquitin andprofilin-1) was also verified to be different in WT and normal serum, and there were no same or similar protein markers in WT serum or tissue. In addition, inflammatory factors were excluded to make the three targeted WT protein markers be more specific. This research suggested not only a harvest of more specific non-inflammatory WT protein markers, but also an experimental consideration and technique process to indentify tumor protein markers. It laid the foundation for the identification of more specific protein markers for other tumors.