Discovery And Identification Of Serum Biomarkers Of Nephroblastoma

BackgroundHuman Wilms'tumor (WT) or nephroblastoma is the most frequent solid tumor of the genitourinary tract in children, occurring in every one in 10000 live births.1 WT typically occurs between 2 and 5 years of age, with 95%of the cases diagnosed before the age of 10 years.2 Rare cases can be present during adulthood3 or in utero.4 This malignancy was rated first in overall incidences among childhood abdominal malignant solid tumors. Although WT survival rates in the past four decades have risen dramatically, survivors are still at an increased risk of a broad spectrum of adverse outcomes caused by chemotherapy and radiation therapy, such as late-treatment mortality and secondary cancers.5,6 At present, diagnosis of nephroblastoma mainly relies on clinical symptoms and imaging technology, such as computed tomography. However, these diagnostic tools are used only when the disease advances to a certain extent. Early accurate diagnosis and timely treatment are critical in improving the long-term survival of WT patients. Although people do some exploration,7 the current insufficiency of effective biomarkers for WT diagnosis is disappointing. The ability to recognize the potential of serum biomarker as a non-invasive screening method for WT remains to be explored.Most types of diseases cause dynamic changes in protein during their development. Some protein levels change during various stages of tumor, even during the early stage without any clinical symptoms, and these proteins are likely to develop into early clinical diagnostic index. Proteomics study is a good prospect in offering opportunities to discover potentially new biomarkers for the early detection and diagnosis of cancer.8,9 Some proteomics technologies include surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), and high-performance liquid chromatography (HPLC). Although these methods remain to be the main technologies used in serum cancer research, other technologies such as protein arrays, isotope-coded affinity tag, isobaric tags for relative and absolute quantitation, and multi-dimensional protein identification technology also offer a great potential for future biomarker discovery in cancer. Various putative biomarkers from serum and conditioned medium have been identified using proteomics as a search tool, such as heat shock proteins 27 in breast cancer,10 dihydrodiol dehydrogenase in lung cancer,11 and cancer antigen 125 in ovarian cancer.12 We used the serum from a nude mouse WT model to search for biomarkers for diagnosing WT.We collected the serum from the nephroblastoma mouse, model that we initially constructed. We used the MALDI—TOF-MS technology to screen potential protein patterns specific to WT. We also performed joint HPLC, liquid chromatography (LC)-mass spectrometry (MS)-MS (LC-MS/MS), and a series of proteomics technology on protein separation, purification, and appraisal. Finally, the biomarkers were confirmed by Western blot using a specific antibody to provide a specific biological marker for future serological examination and diagnosis.ObjectiveTo discover and identify potential noninvasive and convenient biomarkers for the diagnosis of Nephroblastoma(wilms'tumor, WT). Materials and methods1 MiceNude mice of C57/B6 strain were obtained from Jackson Laboratory (USA). All mice were maintained in the animal breeding facility at Zhengzhou University Health Science Center under specific germ-free conditions. The experimental procedures on the use and care of animals had been approved by the Ethics Committee of Zhengzhou University Health Science Center. All animals used were 6-8 weeks old.2 Reagents and instrumentsTrifluoroacetic acid was purchased from Fluka (New Jersey, USA). ProteinChip Biosystems and WCX2 chip were purchased from Ciphergen Biosystems Inc. (Wisconsin, USA). Dithiothreitol (DTT) was acquired from BIO-RAD (Darmstadt, Germany). ZipTip C18 pipette tips were purchased from Millipore (USA). Trypsase was purchased from Promega (USA). Iodine acetamide (IAM) was acquired from AppliChem Inc. (Darmstadt, Germany). MALDI-TOF-MS was purchased from Kratos Analytical Co. (UK), whereas HPLC from Shimadzu (Japan) was employed. LC-MS/MS from Thermo Electron Corporation (USA) was also used. The antibody of apolipoprotein (APO) A-II and polyubiquitin were acquired from Sigma-Aldrich, USA.3 Cell cultureNephroblastoma cell lines were purchased from Sibio company, and cultured in RPMI 1640 medium supplemented with 10%fetal bovine serum (FBS), streptomycin, and penicillin (100 U/ml). The medium was changed once every 2 days.4 Animal model and serum samplesNephroblastoma cells were centrifiuged at 3000 rpm for 5 minutes. After the cell count,1×106 cells were injected subcutaneously into the bilateral abdominal wall of male nude mice via a 14-gauge needle. When the subcutaneous nodules had grown to 2 to 3 cm after 25 days, we conducted a follow-up experiment. Mice were sacrificed after blood samples were obtained from eyeballs, and the nodules were removed and fixed in 4%formalin. The sera were left at room temperature for 1 hour, centrifuged at 5000 rpm for 10 minutes, and then stored at-80℃.5 Western blottingSera from the WT and control groups were used to identify the target protein by Western blot. The serum sample (50μl) was separated by 10%sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to a nitrocellulose membrane. The membrane was incubated at room temperature for 1 hour in Tris-buffered saline containing 0.1%Tween 20 (TBS-T) containing 5% skimmed milk for blocking non-specific binding sites. The blocked membrane was incubated with primary antibodies at 4℃overnight. The membrane was washed 3 to 5 times for 5 minutes with the TBS-T buffer, and then incubated with 1:5000 horseradish peroxide-conjugated secondary antibody at room temperature for 1 hour. The membrane was washed as described above. Finally, the membrane was developed with ECL Reagent (Vigorous Biotechnology, Beijing) and exposed to Kodak XBT-1 film.6 Detection of the target proteinThe SELDI-TOF-MS technique allows proteins to be profiled from different biological samples on a variety of chemically or biochemically defined chromatographic surfaces. This method was performed as follows:Serum samples from-80℃were defrosted on ice, and centrifuged at 10000 r/min for 5 minutes at 4℃. The mixture of the serum sample (5μ1) and U9 buffer (10μ1,9 M urea,2% CHAPS,50 mmol/L Tris-HCL,1%DTT) added into each well of a 96-well plate was oscillated in a chromatography cabinet at 600 rev/min for 30 minutes. After adding 185μ1 of NaNC for another oscillation, the mixture was loaded onto pre-processed protein chip arrays correspondingly. After incubation in the chromatography cabinet at 600 r/min for 1 hour at 4℃, the chip bound different subsets of proteins in crude samples selectively by adsorption, partition, electrostatic interaction, or affinity chromatography according to their surface chemistries. Unbound proteins and unspecific substances were washed thrice with 200 p.1 NaAC and deionized water. Finally,1μl of 50%saturated sinapinic acid was covered on each well, and the chips were dried to prepare for analysis.The time-of-flight (ToF) reader records the ToF and calculates the accurate molecular weight of proteins in the form of a spectral map containing mass-to-charge ratios (m/z) as well as intensities corresponding to each bound protein. ProteinChip Biomarker Software version 3.1 was used to analyze the spectral map and detect differentially expressed protein with statistical significance. The instrument was calibrated using the all-in-one peptide molecular mass standard (Ciphergen Biosystems, Inc., USA).7 Purification of candidate proteinsHPLC technique was applied to separate and purify the target proteins of the mixture using SCL-10AVP with a Sunchrom C18 column and a C18 guard column. First, we mixed 100μl thawed serum samples,350μl ultrapure water, and 700μ1 pure acetonitrile, and then incubated them at-20℃for 30 minutes. The mixture was centrifuged at 13000 r/min for 10 minutes, and the supernatant was transferred into a new tube to freeze in an SPD SpeedVac. The final samples were analyzed by HPLC to obtain purified solutions of different time courses. After being freeze-dried in the SPD SpeedVac, the prepared sample was mixed with acyanoacrylate-4-hydroxy-cinnamic acid at the ratio of 1:1, and spotted onto the MALDI target plate. Finally, we employed MALDI—TOF-MS to identify the purified samples screened by SEIDI-TOF-MS. After the separation and purification of the target proteins, the structure of the proteins was identified.8 Identification of proteinsThe proteins were initially digested with enzyme before identification. The process was performed using the following standard protocol. We maintained the 40μl volume of the purified protein sample by filling with ultrapure water, and sequentially added 4μl 0.1 mol of DTT solution and 1.6μl of IAM, with separate incubation for 1 hour. Subsequently, we added DTT solution, NH4HCO3, and parenzyme into the solution, and incubated the mixture at 37℃overnight.The obtained purified protein digests were analyzed by 2-dimensional-liquid chromatography-linear trap quadrupole-mass spectrometry (2D-LC-LTQ-MS) using nitrogen laser with the following settings:laser wavelength,337 nm; pulse duration,3 ns; mass spectrum, accumulation of signal from 50 single scan; accelerating voltage, 30 kV; absorbing voltage,9.3 kV; detecting voltage,4.75 kV; vacuum degree, 1×10-6 Pa; and detecting mode, positive ion. Peptide mass fingerprints were used as searching objects in BioWorks database by SEQUEST. The biological functions and characteristics of potential biomarker proteins were analyzed using bioinformatics method. All identified proteins were confirmed by Western blotting with their specific antibodies.9 Statistical analysisData are presented as mean±standard error and were analyzed using Student's t-test. All experiments were repeated at least thrice, and the representative data are shown. P<0.05 was considered statistically significant. Statistical analysis were carried out using SPSS 11.0 software (SPSS Inc., USA).Results1 Successful construction of the WT modelIn this study,48 nude mice with wilms tumor and 50 controls were used for searching the potential protein marker. In order to make sure that the WT mice model were constructed successfully, after they were executed, we validated the subcutaneous nodules from them by H-E staining.2 Detection of the target proteinsThe serum samples were analyzed and compared by SELDI-TOF-MS. All MS data were baseline data subtracted and normalized using total ion current, and the peak clusters were generated using the Biomarker Wizard software. Twelve peaks were found with P<0.01 after conducting Wilcox rank-sum tests, and we identified two markers at positions 4509.2 and 6207.9. The 4509.2 Da protein in the WT group was remarkably elevated compared with that in the control group (P<0.05, Figure 2A). The relative molecular weights of this protein in the WT and control groups were 43.28±26.04 and 1.78±1.03, respectively. By contrast, we found that the expression of the 6270.9 Da protein in the WT group was significantly lower than that in the control group (P<0.05, Figure 2B), and their molecular weights were 2.03±1.79 and 46.73±21.53 in the WT and control groups, respectively.3 Purification and identification of candidate proteinsWe used the serum samples from the WT group for the purification of the 4509.2 and 6207.9 Da proteins. We collected purified protein solution by HPLC at a certain time according to the m/z peak value. The results of the 2D-LC-LTQ-MS analysis of the purified protein biomarker after digestion with modified trypsin are shown in Figure 3. We also found that the molecular weights of the purified proteins were identical to APO A-Ⅱand polyubiquitin with sequence coverages of 55%and 42%, respectively (Table 1). Ultimately, we identified the correction of purified protein above. The levels of 6207.9 and 4509.2 Da proteins in the sera of the WT and control groups, respectively, were observed by Western blot using specific antibody to APO A-Ⅱand polyubiquitin. The expression of APO A-Ⅱin the WT group was higher than that in the control group, in contrast to the expression of polyubiquitin (Figure 3). This result suggests that APO A-II and polyubiquitin were involved in the development of nephroblastoma.ConclusionsIn summary, we have identified a set of protein peaks that could discriminate WT from normal controls. From the protein peaks specific for WT disease, we identified apolipoprotein A-II and polyubiquitinas potential proteomic biomarkers of WT. Further studies with larger sample sizes will be needed to verify the specific protein markers. An efficient strategy, composed of SELDI-TOF-MS analysis, HPLC purification, MALDI-TOF-MS trace and LC-MS/MS identification has been proved very successful.