| 1 Background and objectiveAt present, radical nephrectomy is the primary intervention for WT; adjunct chemotherapy and/or radiotherapy are also beneficial, increasing the long-term survival rate to 90% in children with stage I or II WT and 70–80% in children with stage III or IV WT. However, these therapies have complications that can be serious, including heart failure, reproductive dysfunction, and kidney dysfunction; some patients develop severe complications in adulthood. Thus, developing new therapeutic strategies for WT is imperative for decreasing medical costs and improving patient outcomes. Wilms’ tumor is the most common children kidney malignant tumor in children, and early diagnosis and early therapy are effective in increasing survival. Color Doppler ultrasound, CT, and MRI are routinely used to diagnose and prognose Wilms’ tumor, but these modalities are less effective for sensibility and early diagnosis. The treatment of Wilms’ tumor is given priority to the comprehensive treatment with surgery, chemotherapy, radiation therapy, and according to the situation of stages with different treatments. The treatment situation of Stage I is relatively optimistic, and 5 years of survival rate after surgery can reach more than 90%. But the treatment situation of Stage I and above staging is not optimistic. In view of the fact that early diagnosis, clinical pathologic staging and prognosis monitoring is vital for the treatment of Wilms’ tumor, a more sensitive, more accurate methods of early diagnosis and prognosis monitoring is required.This project consists of two parts content. The first part, relying on the proteomics technology and method, we found out the Wilms’ tumor specific protein marker protein polypeptide m/z 6455.5 Da, and established the diagnosis staging model and the prognosis monitoring model of Wilms’ tumor. The second part, we studied the impact of the protein polypeptide m/z 6455.5 Da on Wilms’ tumor biological behavior in vivo and in vitro experiments. And we explored the mechanism of action of the protein polypeptide m/z 6455.5 Da in action on Wilms’ tumor at the genetic level. 2 Materials and methods 2.1 Experimental material 2.1.1 Material of clinical casesScreening of Wilms’ tumor serum markers, protein and polypeptide. 54 serum samples extracted from normal healthy children during physical examinations were treated as a normal group; and 48 serum samples extracted from children with Wilms’ tumor during first visit examinations were treated as a tumor group. All children with Wilms’ tumor received no treatment before.Staging diagnosis model establishment of Wilms’ tumor serum markers. 41 serum samples extracted from normal healthy children during physical examinations were treated as a normal group; and 94 serum samples extracted from children with Wilms’ tumor during first visit examinations were treated as a tumor group. All children with Wilms’ tumor received no treatment before. According to NWTS-5 staging, the tumor group included 23 stage I cases, 30 stage II cases, 26 stage III cases, and 15 stage IV cases. The tumor group was further divided into a WT-I group, a WT-II group, a WT-III group and a WT-IV group.Prognosis monitoring model establishment of Wilms’ tumor serum markers. 30 serum samples extracted from normal healthy children during physical examinations were treated as a normal group. There are 40 children with Wilms’ tumor. Among them, 27 children had a radical operation of Wilms’ tumor for complete removal of tumors and were completely cured and 13 children could not have an operation for complete excision or had varying degrees of recurrence or metastasis or died after operation. 160 serum samples of children with Wilms’ tumor were extracted by four times. The extraction time was before operation, 2 weeks, 2 months and 6 months after operation. According to treatment conditions, serum samples of children with Wilms’ tumor were further divided into a preoperative group with 40 cases, a healed group（2 weeks after operation） with 27 cases, a healed group（2 months after operation） with 27 cases, a healed group（6 months after operation） with 27 cases, an unhealed group（2 weeks after operation） with 13 cases, an unhealed group（2 months after operation） with 13 cases, an unhealed group（6 months after operation） with 13 cases.All serum samples came from Department of Pediatric Surgery, the First Affiliated Hospital of Zhengzhou University and the collection time was from January, 2007 to December, 2013. All children with Wilms’ tumor mentioned above had no preoperative chemotherapy and all pathological diagnostic results were favorable histology. The diagnosis of all pathological samples was demonstrated by more than two pathologists after testing pathological sections. The study was authenticated and approved by the Ethics Committee of Zhengzhou University and the Ethics Committee of the First Affiliated Hospital of Zhengzhou University. Legal guardians of all subjects signed informed consent. 2.1.2 Artificially synthesized protein polypeptide m/z 6455.5 DaOn the platform of Life Tein’s Peptide Syn TM technology, protein polypeptide m/z 6455.5 Da was synthesized by FOMC-protected amino acid solid-phase synthesis. The specific industrial production process was finished by Life Tein LLC. After synthesis, protein polypeptide m/z 6455.5 Da was a single chain compound with 55 amino acids and the purity of the sample was 96.57%. At a normal temperature, protein polypeptide m/z 6455.5 Da was dissolved in normal saline to prepare 1μmol /ml medicinal stoste. Then the stoste was stored at-20℃ and kept out of the sun. 2.1.3 Establishment of the Wilms’ tumor cell line and the tumor-bearing mouse modelThe Wilms’ tumor cell line SK-NEP-1 was selected as a study object and purchased from ATCC. The established cell line SK-NEP-1 was a suspending and half-anchorage-dependent cell line and the solution was changed every three days and the passage interval was 9 days. 4-week-old female BALB/c Nude inbred line athymic mice were chosen as study objects and fed in a standardizing SPF animal laboratory and established a stable environment of a 12 h night and 12 h daytime cycle. All operations were conducted a sterile environment.In culture of SK-NEP-1 cells, cells were extracted during the exponential growth phase and resuspended in serum free medium and BD Matrigel in a 1:1 ratio. The concentration of SK-NEP-1 cells was adjusted to 1×107/0.2ml. After sterilizing the skin of nude mice, 0.2ml cell mixture was subcutaneously injected in the armpit of left forelimb. Then they were observed every 3 days in two weeks and a Wilms’ tumor tumor-bearing mouse model was established. After model establishment, mice were divided into an experimental group and a control group. In the experimental group, 4mg/kg of protein polypeptide m/z 6455.5 Da diluent was injected in the abdominal cavity of the tumor-bearing mouse model at 08:00AM every day; and in the control group, a corresponding volume of normal saline（NS） was injected in the abdominal cavity of the tumor-bearing mouse model at 08:00AM every day.The study was authenticated and approved by the Ethics Committee of Zhengzhou University and the Ethics Committee of the First Affiliated Hospital of Zhengzhou University. 2.1.4 The main instruments and reagentsProfiling Kit 100 MB-WCX and MALDI-TOF-MS were purchased in Bruker Inc.（Germany）; Protein Chip, PBS II+ SELDI-TOF-MS, Bio-processor and WCX2 Protein Chip were purchased in Ciphergen Biosystems Inc.（U.S.A.）; SPD Speed Vac; 2D-LC-LTQ-MS and Thermo Scientific Varioskan Flash were purchased in Thermo Inc.（U.S.A.）; HPLC was purchased in Shimadzu Corporation（Japan）; Applied Biosystems 7500 Fast Real-Time PCR System was purchased in ABI Inc.（U.S.A.）; BD Influx was purchased in Becton, Dickinson and Company（U.S.A.）.CAN, CHAPS, Urea, et al.were purchased in Sigma Inc.（U.S.A.）; Reagent of vitro experiment was purchased in Gibco Inc.（U.S.A.）; Reagent of PCR and Western blot experiment was purchased in Thermo Inc.（U.S.A.）. 2.2 Experimental methods 2.2.1 Proteomics screening serum difference proteinThe supernatant was obtained by centrifugation after unfreezing serum samples. A weak cation kit was used and serum samples were pretreated in line with instructions and requirements. After treatment, they stood still for mass spectrometry. WCX2 Protein Chip was pretreated. After pretreated serum samples and U9 diluent were mixed well, the mixture was added into the hole of WCX2 Protein Chip. Shake WCX2 Protein Chip for sufficient reaction between samples to be tested and WCX2 Protein Chip. Then the liquid was disposed. The chip was cleaned with ultrapure water and SPA solution. After drying, it was inserted in Bio-processor and chip coordinates were recorded for computer use. Parameters of PBS II+ SELDI-TOF-MS were set and Bio-processor was installed in SELDI-TOF-MS for detection and screening of serum difference protein.Calibration was conducted by the original data of Ciphergen Biosystems software Version 3.1 each m/z and corresponding protein peak data were gained. ZUCIPDAS network service software was applied to analyze data of the protein chip. Wilcoxon rank sum tests were carried out on data of preliminary screening and the test standard was P<0.01. T-tests were done on spectral counts of different groups and the test standard was α=0.01. Different difference proteins of peak values with same or corresponding m/z were obtained by analyzing values of different groups. Then, by SVM, a combined model with the highest index was screened.According to SELDI-TOF-MS screening results, serum samples with higher target protein expression were selected as separating and purifying objects. After pretreatment of serum samples, they were separated by C18 HPLC Columns. Each peak component was collected, shifted to SPD Speed Vac and stored in a vacuum concentration volume. After extraction and purification, each components sample and WCX2 Protein Chip were pretreated and put into MALDI-TOF-MS for mass-to-charge ratio testing of protein. Enzymatic hydrolysis was conducted on purified target protein. Then its volume was vacuum concentrated, added in gradient elution columns of C18 protein sample detection and connected in series with the spray system of the 2D-LC-LTQ-MS system. Testing of peptide mass-to-charge ratio was conducted to obtain PMFs of target protein. Finally, results were introduced in the SEQUEST sequence and the amino acid sequence of target protein was obtained by searching in Bioworks database.2.2.2 In vitro cytology experimentsSK-NEP-1 cells during the logarithmic phase were selected to be inoculated in a 24-well culture plate. After inoculation for 1 day, cell counting was conducted to get the initial cell number. The medicinal stoste of protein polypeptide m/z 6455.5 Da was added in the 24-well culture plate. The final concentration of each well was 0, 1×10-6, 1×10-5, 1×10-4, 1×10-3, 1×10-2μmol/ml. Cell counting was conducted on the observation group and the control group every 24 h. The average value of 3 complex holes was calculated every day of 7 days. A cell growth curve was drawn and the cell doubling time was calculated. SK-NEP-1 cells during the logarithmic phase were inoculated in a 96-well culture plate. After inoculation for 1 day, the medicinal stoste of protein polypeptide m/z 6455.5 Da was added in the 96-well culture plate and the final concentration of each well was 0, 5, 10, 15, 20, 25, 30×10-3μmol/m L. After 24 h, 48 h and 72 h, a microplate reader was used to detect absorbance OD value. The cell viability was computed and a calibration curve of cytotoxic effects was drawn. The median inhibitory concentration（IC50） and the 80% inhibitory concentration（IC80） of protein polypeptide m/z 6455.5 Da in the SK-NEP-1 cell line after 24 h, 48 h and 72 h were calculated.SK-NEP-1 cells during the logarithmic phase were selected to be inoculated in 2 25cm2 culture bottles with a cell concentration of 1×106/m L. After inoculation for 1 day, the protein polypeptide m/z 6455.5 Da was added in a culture bottle with a final concentration of IC50. This was the experimental group. Another bottle was as a blank group. After 48 h, cells in the experimental group and the blank group were collected and the total RNA was extracted. Reverse transcription of c DNA was conducted by a kit. During Real-time PCR, amplification of genes to be tested in the experimental group and the blank group was detected and software was applied to analyze PCR data.SK-NEP-1 cells during the logarithmic phase were selected to be inoculated in 2 75cm2 culture bottles with a cell concentration of 1×106/m L. After inoculation for 1 day, the protein polypeptide m/z 6455.5 Da was added in a culture bottle with a final concentration of IC50. This was the experimental group. Another bottle was as a blank group. After 48 h, cells in the experimental group and the blank group were collected and the total protein was extracted. The protein concentration was tested. The SDS-PAGE running gel of protein was prepared. Ionophortic separation of protein sample loading was carried out. Then membrane transfer was conducted by Western blot. Antibodies were incubated on the PVDF membrane. The PVDF membrane was detected and results were analyzed by photo taking. 2.2.3 In vivo experiment of the nude mouse modelSK-NEP-1 cells during the logarithmic phase were inoculated in nude mice with an adjusted concentration of 1×107/0.2m L and a Wilms’ tumor tumor-bearing mouse model was established. In the experimental group, 4mg/kg of protein polypeptide m/z 6455.5 Da diluent was injected in the abdominal cavity of nude mice every day for 6 weeks. In the control group, a corresponding volume of normal saline（NS） was injected. Tumor growth of nude mice was recorded. A vernier caliper was used to measure diameters and volumes of transplantation tumors every third day. After 6 weeks, they were killed by cervical dislocation. Tumors were dissected, measured and photographed. After anatomy of nude mice in the experimental group and the control group, paraffin embedding of tumor tissue was conducted. 4um sections were used to carry out immunohistochemical analysis and TUNEL apoptosis detection. 3 Result 3.1 Proteomics experiment resultsThere were 3 high-expression protein peak values of serum of children with Wilms’ tumor and 9 low-expression protein peak values of serum of children with Wilms’ tumor. By SVM, a combined model with the highest index was screened and protein markers of protein polypeptide m/z 6455.5 Da were obtained. The protein marker was of low expression in the tumor group with expression strength of 881.68 ± 484.86; and it was of high expression in the normal group with expression strength of 3503.27 ± 780.49 with statistically significant differences.In the experiment of the Wilms’ tumor staging diagnosis model, expressions of protein markers in the normal group, the tumor group, the WT-I group, the WT-II group, the WT-III group and the WT-IV group were analyzed and were 3305.50 ± 821.90, 1279.50 ± 919.17, 2579.95 ± 644.57, 1116.63 ± 504.98, 911.70 ± 294.15, 248.76 ± 228.21 respectively. Statistical comparison of the normal group, the WT-I group, the WT-II group, the WT-III group and the WT-IV group were conducted and there were statistically significant differences between any two groups. It was confirmed that protein polypeptide m/z 6455.5 Da could be a standard of the Wilms’ tumor staging diagnosis model.In the experiment of the Wilms’ tumor prognosis monitoring model, expressions of m/z 6455.5 Da protein markers were analyzed. The expression strength of the normal group was 3619.27 ± 760.79 and that of the preoperative group was 881.36 ± 355.29 with statistically significant differences. The expression strength of the postoperatively healed group was 863.05 ± 250.79（2 weeks after operation）, 743.47 ± 375.99（2 months after operation）, 480.72 ± 250.04（6 months after operation）. There were statistically significant differences between the expression of m/z 6455.5 Da protein markers of different phases of the postoperatively healed groups and the expression of the preoperative group and there was no statistically significant difference between the expression of m/z 6455.5 Da protein markers of different phases of the postoperatively unhealed group and that of the normal group. After children with Wilms’ tumor were completed cured by a complete tumors resection and corresponding postoperative chemotherapy, the expression of the protein marker increased again. What’s more, with postoperative recovery, it gradually increased to a level of normal healthy children. In cases of palliative resection of Wilms’ tumor or complete resection with recurrence, the protein marker expression was continuously low.After separation and purification of protein polypeptide m/z 6455.5 Da were proved to be correct by MALDI-TOF-MS, the target protein was detected by the 2D-LC-LTQ-MS system and amino acid sequences of various protein fragments were obtained. Amino acid sequences of these proteins were introduced in SEQUEST and were searched in Bioworks database for matching and restructuring to gain a complete amino acid sequence.Serum samples, imageological examination results and prognosis survival data of normal healthy children, children with Wilms’ tumor and children with other abdominal solid tumors were gathered. Sensitivity and specificity of protein polypeptide m/z 6455.5 Da were analyzed. Sensitivity of protein polypeptide m/z 6455.5 Da has obvious advantages on staging diagnosis compared with other diagnostic methods. Sensitivity and specificity of protein polypeptide m/z 6455.5 Da have obvious advantages on prognosis monitoring compared with other diagnostic methods. 3.2 Results of the in vitro cell experimentIn the observation group, the growth trend of SK-NEP-1 cells intervened by protein polypeptide m/z 6455.5 Da was worse than that without drug intervention in the normal group with a characteristic of dose-dependence. According to the cell growth curve, the cell doubling time of SK-NEP-1 cells was obtained by the Patterson function. 24 h after entering into the logarithmic phase, the number of SK-NEP-1 cells reached 7.551×105 in the control group and the cell doubling time was 21.88 h. When the concentration of protein polypeptide m/z 6455.5 Da was 1×10-2μmol/ml, 24 h after entering into the logarithmic phase, the number of SK-NEP-1 cells intervened by the protein polypeptide m/z 6455.5 Da was 1.434×105 with cell doubling time of 67.40 h. According to absorbance OD values of the observation group and the control group, the cytotoxicity inhibition rate curve was drawn and toxic effects of protein polypeptide m/z 6455.5 Da on SK-NEP-1 cells were confirmed. The data showed that protein polypeptide m/z 6455.5 Da inhibited growth of SK-NEP-1 cells with characteristics of dose-dependence and timedependence.A flow cytometry was used to analyze the proportion of cell apoptosis. The results showed that after protein polypeptide m/z 6455.5 Da intervened SK-NEP-1 cells for 48 h, the proportion of cell apoptosis in the blank group, the IC50 group and the IC80 group were 8.4%, 22.3% and 69.6% respectively. In the study, we found that protein polypeptide m/z 6455.5 Da could inhibit effectively SK-NEP-1 cell proliferation. By analyzing cell growth cycles with a flow cytometry, the results showed that protein polypeptide m/z 6455.5 Da intervened SK-NEP-1 cells; in the cell growth cycle, cell elongation in the G2/M phase was greater than that in the blank group; and protein polypeptide m/z 6455.5 Da induced blockage of SK-NEP-1 cells in the G2/M phase.In Real-time PCR, amplification expression of PCNA, Bcl-2 and Bax was detected. The results showed: after protein polypeptide m/z 6455.5 Da intervened SK-NEP-1 cells, expression of PCNA and Bcl-2 was lowered and expression of Bax was increased, verifying that inhibiting effects of protein polypeptide m/z 6455.5 Da on SK-NEP-1 cell proliferation. At the same time, key proteins of four signaling pathways, Wnt Pathway, Hedgehog Pathway, TGF-β Pathway and Growth factor recepator Pathway, were tested. The results showed that after protein polypeptide m/z 6455.5 Da intervened SK-NEP-1 cells, expression of β-catenin was lowered. When protein polypeptide m/z 6455.5 Da inhibited SK-NEP-1 cell proliferation, β-catenin/Wnt Pathway played an important role. The Western blotting trial confirmed that expression of PCNA, Bcl-2, Bax and β-catenin, which was consistent with the result of PCR.3.3 In vivo experimental results of the nude mice modelCompared with the control group, volumes and diameters of tumors in the tumor group intervened by protein polypeptide m/z 6455.5 Da were smaller. Protein polypeptide m/z 6455.5 Da had obvious anti-Wilms’ tumor effects in nude mice. HE-staining sections showed that the growth of tumor tissue was good in the control group, while massive necrosis of tumor tissue occurred in the observation group. The structure of tumor tissue was damaged. After TUNEL staining, under a fluorescence microscope, large apoptosis of tumor cells was observed in the treatment group. Results of immunohistochemistry tests indicated that after treating with protein polypeptide m/z 6455.5 Da, expression of PCNA, β-catenin, Bcl-2 and Bax was low in tumor tissue. 4 ConclusionsProtein polypeptide m/z 6455.5 Da refers to a specific marker of Wilms’ tumor separated from human serum and a long peptide consisting of 50 amino acids which can be a biological marker of the Wilms’ tumor staging diagnosis model and the prognosis monitoring model. At the same time, sensitivity of protein polypeptide m/z 6455.5 Da has obvious advantages on staging diagnosis compared with other diagnostic methods. Sensitivity and specificity of protein polypeptide m/z 6455.5 Da have obvious advantages on prognosis monitoring compared with other diagnostic methods. Protein polypeptide m/z 6455.5 Da could be a new laboratory diagnosis method for staging diagnosis and prognosis monitoring of Wilms’ tumor.Protein polypeptide m/z 6455.5 Da is a anti-tumor peptide which can induce cell apoptosis of Wilms’ tumor and inhibit tissue growth of Wilms’ tumor effectively. By preliminary exploration, the action mechanism of protein polypeptide m/z 6455.5 Da against Wilms’ tumor might be that protein polypeptide m/z 6455.5 Da regulates the Wnt signaling pathway of Wilms’ tumor resulting in low expression of the key protein, β-catenin, leading to a series of complex changes of cytobiology. This new finding has great potential significance in researching new treatment methods and means of Wilms’ tumor and maybe generate a new protein polypeptide drug against Wilms’ tumor. |
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