| 一、Research backgroundWith the development of economy and society, the etiology, diagnosis and treatment of various diseases of children have been improved. The incidence and mortality rate of infectious diseases and congenital diseases decreased significantly. In the era of molecular biology and precise medical mode, compared with adults, the etiology and the basic research of early diagnosis and treatment of malignant tumor in children have lagged behind, and influence the cancer in children accurate diagnosis and treatment. So, making clear the pathogenesis of malignant tumor in children the differentially expressed genes, finding effective tumor markers, improving the level of diagnosis and treatment, increasing the survival rate of the patients, reduce the impact of short-term and long-term side effects in the treatment, become the pressing concerns and problem of society.Wilms Tumor is a kind of embryonal malignant tumor of the kidney. It is also the most common tumor of the urinary and reproductive system in children. Its incidence accounts for about 14% of children with solid tumors, the peak ages rangedfrom 1 to 5 years old. Although the constant improvement of surgery, chemotherapy, radiotherapy and other comprehensive treatment programs in nearly 50 years, Wilms Tumor survival rate is also significantly improved after treatment and the five year survival rate reach 87%, a certain number of cases with tumor cell changes, late clinical stage and tumor rupture show a high recurrence rate and distant metastasis rate, result in a poor prognosis. In addition, the lack of effective tumor markers may lead to severe complications and adverse consequences because of the excessive treatment of low risk cases and the lack of adequate treatment in high risk cases. Therefore, there is the key to search for closely related biological markers and study the pathogenesis of progression for improving the survival rate and quality of life. There is also important step to refine the treatment plan and reduce the adverse reactions.The regulation mechanism of tumor is a complex network, which involves many regulatory signaling pathways. In recent years, the basic research of tumor has been developed, and it has found a lot of regulatory signaling pathways that are related to the occurrence, development, invasion and metastasis of tumor. And these signaling regulatory pathways provide some more accurate markers for the early diagnosis, the staging and grading of tumors, the development of therapeutic drugs, and the prognosis. This is not only a more deep understanding of the mechanisms in tumor development, better assessment of tumor grading, providing accurate treatment options, increased treatment, reducing complications. At present, there is little research on the signaling pathways in Wilms tumor, especially the important regulatory genes and components in the downstream of the signaling pathway, so it is helpful to understand the mechanism of Wilms tumor pathogenesis and the accurate judgment of markers in the downstream of the more signaling pathways.Transforming growth factor beta (TGF-β) is a kind of active cytokine, which is known to have a variety of functions, and it is also the initial factor of regulating the signialing pathway and is involved in the formation of tumor. The mechanism of activity includes:promoting angiogenesis in tumor angiogenesis by the enhancement of VEGF expression; the induction of immune suppression and immune escape in tumor cells; In the process of tumor metastasis, epithelial mesenchymal transition is induced, which improves the ability of invasion and metastasis of tumor cells. TGF-β signaling pathway superfamily is one of the most important signal pathways involved in many biological processes. Its function is very extensive, including cell proliferation, differentiation, migration and apoptosis. In some tumor studies, the various biological mechanisms of the TGF-β signaling pathway are needed for the complex signal transduction process in the downstream, which requires the coordination of various genes and molecules. So it is more accurate to understand the mechanism of the occurrence and development of tumors by studying the changes and interactions of downstream signaling pathways.There is a general condition of hypoxia in solid tumor tissue. Due to excessive growth of tumor blood vessels and oxygen supply capacity is relatively insufficient, resulting in hypoxia. However, during the growth of malignant tumor, it can increase the new blood vessel, expand the blood vessel, increase the cell fermentation, and overcome the deficiency of hypoxia and nutrition. Therefore, the growth, proliferation and metastasis of tumor are bound to be able to overcome the state of hypoxia and the environment by a series of cytokines. Wilms Tumor in the peritoneum, the location is more insidious and there is no specific symptoms and signs in the early stage,and growing relatively rapidly, which is often greater than 10 cm and relatively large diameter after finging it. Furthermore, more cases of tumor tissue showed cystic and solid phase. So in the internal part of tumor has obvious hypoxic microenvironment for liquefaction and part necrosis. In recent years, some studies at home and abroad began to study the mechanism of HIF-1 alpha in Wilms Tumor.. The results showed that the expression of HIF-1 in Wilms tumor tissue was significantly increased, which may be a positive regulator in Wilms tumor, and has a close relationship with clinical stage and prognosis. It is helpful to guide clinical treatment and prognosis. HIF-1 in Wilms Tumor tissue may stimulate the expression of vascular epithelial growth factor (VEGF), to ensure the blood supply to the tumor, which may also be a target for future treatment. Hypoxia inducible factor 1 alpha (HIF-1α) is one of the key transcription factors for the mediated cell hypoxia response in the Wilms Tumor. HIF-1 alpha is an important transcription factor that mediates hypoxia effect under hypoxic conditions. It is through the combination of hypoxia response factor activation downstream of a large number of target genes involved in tumor invasion, metastasis, angiogenesis, energy metabolism, and put chemotherapy resistance, and many other aspects. The occurrence of epithelial mesenchymal transition (EMT) is a key mechanism in the development of embryo, the invasion and metastasis of tumor cells. HIF-1 alpha mediates the initiation of hypoxia signaling and the occurrence of EMT in tumor hypoxia microenvironment. Multiple mechanisms involved, the known signaling pathways related with Notch signaling pathway, Wnt signaling pathway, stem cell growth factor/stem cell growth factor receptor signaling pathway HGF/ Met and hedgehog signal pathway. But in the Wilms Tumor of HIF-1 alpha specific regulatory mechanisms how remains unclear are involved in the invasive and metastatic ability of Wilms Tumor cells increased is not clear.二、Research objectiveThe purpose of this study was to collect the clinical and pathological data of Wilms Tumor patients, and to study the pathological tissue samples and follow up. Molecular biological methods were used to detect the mutations of 84 important genes in the TGF-beta signaling pathway in the patients with Wilms Tumor. And the differentially expressed genes were used for the correlation analysis with the clinical pathological indexes in order to establish a more accurate diagnosis, prediction of prognosis of patients with Wilms Tumor. In addition, the SK-NEP-1 human Wilms Tumor cell line was cultured in the hypoxic environment, and the model was established in vitro, By comparing the results of normal oxygen concentration in the environment, the morphological changes and growth conditions of SK-NEP-1 human renal cell tumor cells were analyzed. The expression of HIF-1 was detected, In order to show the changes of the expression level of SK-NEP-1 human Wilms Tumor cells in two environments. Another sign of epithelial cells detected nephroblastoma cells of the SK-NEP-1 protein, namely epithelial cadherin (E-cadherin), Interstitial cell marker proteins, namely the expression of vimentin (Vimentin), So as to analyze whether the role of HIF-1 alpha in Wilms Tumor through EMT transformation increased tumor invasion and migration. Finally, we speculate on possible target and reverse pathway.三、Materials and methods1. Clinical WT tissue specimen collection and groupingThis experiment was approved by the ethics committee of Guangzhou women and children’s medical center. Clinical cases of 28 cases with unilateral Wilms Tumor pathology were collected from January 2010 to December 2012, respectively All preoperative chemotherapy and bilateral Wilms Tumor cases were excluded from this study. In the operation,28 patients were taken as the tumor group (28 cases) and the adjacent renal tissue as the adjacent cancer group (28 cases).2. Tissue treatment and RNA extraction and detection of clinical tumor specimensWilms Tumor tissue was used as the tumor group, and the adjacent renal tissues were extracted as the side groups, respectively, and the total RNA. NanoDrop UV-Vis (2000/2000 ultra-micro spectrophotometer) was used to detect OD260/OD280, to determine the concentration of extracted RNA; In order to eliminate the degradation of RNA or genomic DNA contamination, a good RNA solution was taken to be placed on the agarose gel electrophoresis of 1%, with RTN (Integrity Number RNA) value,28S and 18S ratio and the degree of clarity, to observe the total RNA of the sample purity and integrity.3. Preliminary screening of differentially expressed genes in the TGF-beta/ BMP signaling pathwayPhase Ⅰ, phase Ⅱ and phase Ⅲ Wilms Tumor tissue samples were randomly selected from each of the 2 patients with Ⅲ stage, to be mixed with RNA, for initial screening, the use of TGF-Human beta/Signaling Pathway PCR Array BMP standard test kit to detect the difference of the 84 genes in the signal pathway.4. Using RT-PCR method in 28 cases of tissue specimens to verify the TGF-beta signaling pathway in the superfamily of mutations in the gene Based on the detection results of TGF-BMP Signaling Array PCR Pathway Human, the possible differences of the different genes were analyzed and screened,7 genes in the 84 genes showed more than 10 times the difference in the mutation gene, respectively, AMH (12 times), BMPR1A (27 times), CHRD (43 times), GDF2 (10 times), IGF (27 times), COL1A2 (14 times), NROB1 (11 times). TGF-beta signaling pathway is considered to be in the 7 genes, and the RT-PCR method is used to detect and verify the tumor group and the adjacent group of 28 cases in all cases.5. Correlation analysis between Wilms Tumor and clinical pathological factors in the super family of TGF-beta signaling pathwayVarious factors, including gender, age, stage, pathological type and invasion/ metastasis, were studied in this study, the patients were followed up for 3 to 5 years. The patients had no recurrence and the indexes were compared with those of BMPR1A mRNA expression.The difference expression of gene expression and clinical pathological factors were compared using. P<0.05 test Chi-Square was considered the difference was statistically significant.6. Morphological evaluation of SK-NEP-1 human Wilms Tumor cells cultured under different oxygen concentrationsSK-NEP-1 human Wilms Tumor cell line (Shanghai Institute of life sciences, Chinese Academy of Sciences) in two kinds of oxygen concentrations in the culture. The experimental group, the hypoxia inducible group 20, cultured liquid oxygen concentration was adjusted to 1%,37 degrees Celsius under 6 hours of training. Control group, normal group 20, for normal oxygen concentration 37,21% degrees Celsius for 6 hours under the conditions of the growth of tumor cells were observed under the microscope.7. RNA extraction and RT-PCR detection of tumor cells in normal group and hypoxia inducible group were detected by HIF-1 mRNA8. Transwell invasion experiment and Transwell Migration ExperimentTwo groups of cultured cells were implanted into Transwell plate in normal group and hypoxia inducible group, respectively, the tumor cells were stained on the plastic film, and the paired T test was carried out under the inverted microscope. The statistical differences were compared.9. Western Blotting in SK-NEP-1 method was used to detect the expression of HIF-1, Vimentin, E-cadherin, β-actin and in normal and hypoxia induced group四、Research results1. Screening and validation of the TGF-beta signaling pathway superfamily geneThe total RNA quantity, quality and integrity of the clinical renal cell tumor tissue samples were in line with the further testing standards.TGF-BMP Signaling Pathway PCR Array Human standard test kit for detection of mutations in the 84 genes in the TGF-beta signaling pathway superfamily, Preliminary screening of the 7 tumor groups and the comparison of the adjacent cancer group, the expression of mRNA is more than 10 times the difference between the possible mutations, Are AMH (anti Mullerian duct hormone) (12.07 times), BMPR1A (bone morphogenetic protein 1A receptor) (27.15 times), CHRD (tenascin) (4.34 times), COL1A2 (type I collagen) (10.45), GDF2 (growth differentiation factor 2) (27.84 times), IGF (insulin-like growth factor 1) (14.54 times), NROB1 (nuclear receptor subfamily) (11.18 times).Results (p=0.036<0.05) and NROB1 (p=0.039<0.05) are the differentially expressed genes in (WT) and (BMPR1A), which are the results of the RT-PCR method in the tumor tissues and adjacent renal tissues of 28 patients.2. Correlation analysis between BMPR1A gene expression and clinical pathological data of patients with Wilms TumorBMPR1A gene expression in the clinical pathological data of various factors (gender, age, stage, invasion and metastasis, recurrence, pathological type), P were greater than 0.05, there is no significant difference between the groups.3. Morphological evaluation of SK-NEP-1 human Wilms Tumor cells cultured under different oxygen concentrations In the normal group, the tumor cells showed polygonal and arranged rules closely. In the hypoxia group, the tumor cells showed oval or fusiform, arranged loosely and irregularly.4. Comparison of HIF-1 alpha mRNA levels in SK-NEP-1 human Wilms Tumor cells in hypoxia-induced group and normal groupSK-NEP-1 human Wilms Tumor tumor cell lines in the normal group and the hypoxia induced group were extracted RNA quantity, quality and integrity in line with the further testing standards.RT-PCR test results showed that the expression level of HIF-1 in hypoxia induced group (6.36±0.76) was significantly higher than that in normal group (2.69±0.86) (p<0.05).5. Comparison of the expression of epithelial mesenchymal transition related protein in hypoxia-induced and normal cellsWestern Blotting method was used to detect the expression of HIF-1, Vimentin, E-cadherin and β-actin protein, respectively, The results showed that the protein levels of HIF-1 and Vimentin were significantly higher in the hypoxia induced group than in the normal group (0.96±0.51 vs 0.15±0.06; 1.01±0.13 vs 0.81±0.06, p<0.05), The protein level of E-cadherin decreased in the hypoxia induced group and was significantly lower than that in the normal group (1.03±0.13 vs 1.15±0.11, p<0.05).6. Invasion and mobility of SK-NEP-1 human Wilms Tumor cells in hypoxia-induced and normal controlsDetection results using Transwell migration assay and invasive test method:The number of Wilms Tumor cells in the two groups was detected and the number of tumor cells in the two kinds of plastic film was significantly higher than that in the normal group. (P<0.05)五、Discuss1. Using the method of PCR chip in the clinical Wilms Tumor tumor tissue samples screened TGF-beta signal transduction pathway in the super family of mutations in the gene for BMPR1A and NROB1TGF-/BMP signal transduction pathway PCR chip testing and validation of the large sample of the mutant gene for BMPR1A and NR0B1.BMPR1A (bone morphogenetic protein type 1A receptor) gene is located in human chromosome 10q23, also known as ALK3. It belongs to the category of TGF-beta cell factor superfamily receptor in the superfamily of TGF-beta signaling pathway, The lack of BMPR1A not only affects the initiation of tumor cell proliferation, but also affects the epithelial mesenchymal transition of tumor cells, and its high expression can significantly increase the invasiveness of breast cancer. The use of mouse knockout BMPR1A model to be able to work in the pre BMP receptor can be better for the treatment of high stage glioma help. MiR-656 was able to inhibit the growth and progression of glioma, which was inhibited by BMPR1A expression. In the present study, the mRNA gene BMPR1A was significantly elevated in the Wilms Tumor tumor tissue. It can be explained that it plays an important role in the occurrence and development of Wilms Tumor, and then affect the role of TGF-beta signaling pathway superfamily in cancer, which may promote the growth of tumor. But the specific mechanism is not clear, whether the intervention can delay the growth of tumors and so on have yet to be confirmed in vitro and in vivo experiments.NROB1 is currently associated with tumor research is less, so this article temporarily not the expression of NROB1 and Wilms Tumor relationship is discussed.2. There was no significant correlation between the TGF-beta/BMP signaling pathway and the clinical pathological characteristics of the mutant gene BMPR1AWe have preoperative information about the patient, postoperative tumor stage, invasion and metastasis (lymph nodes, ureter, renal pelvis, renal and external vessels), recurrence and pathologic type (good prognosis and poor prognosis). Respectively, the amount of mRNA of BMPR1A and the factors are done statistical correlation analysis. The results showed that there was no significant difference in mRNA of BMPR1A elevation in these clinical factors.This may be related to the number of cases of this study, it should be more than the number of centers, to expand the number of cases. Increased comparison of factors such as survival and disease-free survival, and so on, may be able to find valuable clinical implications.3. SK-NEP-1 cell growth model is a good in vitro model for the study of HIF-1 alpha in Wilms Tumor tumor cells under hypoxic condition4. HIF-1 alpha in human Wilms Tumor tumor SK-NEP-1 cell hypoxia induced model is through the role of epithelial mesenchymal transition mechanism, play an important role in the invasion and metastasis of tumorHypoxic microenvironment can increase the expression of HIF-1 in human renal cell carcinoma SK-NEP-1 cells, promote the morphological changes of the tumor cells, and improve the ability of tissue invasion and migration. The upregulation of Vimentin protein expression and down-regulation of E-cadherin protein also suggested that EMT could promote the metastasis of tumor cells. This may be an effective way to inhibit tumor invasion and distant metastasis, if it is possible to improve the microenvironment of the tumor by blocking the conduction of the downstream signal pathway of HIF-1α. Although this need to be clear about the mechanism and further research, but this can give us the rapid growth and metastasis of the tumor to provide a way of thinking, to some of the surgical treatment of some patients to bring more hope.六、Conclusion1. In the super family of TGF-P beta signaling pathway in Wilms Tumor, the differential expressed genes are BMPR1A and NROB1. There was no significant correlation between the increase of BMPR1A expression and clinical pathological factors.2. SK-NEP-1 cell growth model is a good in vitro model to study the growth of HIF-la cells under hypoxic conditions.3. HIF-la plays an important role in the invasion and metastasis of tumor by inducing epithelial mesenchymal transition in the hypoxia inducible model of human Wilms Tumor SK-NEP-1 cells. |
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