| BackgroundThe neuroblastoma (neuroblastom NB),as the most common extracranial solid tumor, is one of the most common tumors in infants. It lists third in the tumors resulting in children’s death. NB originates from the primitive neural crest cells, may exist wherever the sympathetic nervous systems exist. Most primary tumors occur in the abdomen and sometimes get the adrenal gland involved in. The median age when the disease is mostly likely to occur is at the17th month. The prognosis of NB is rather poor, with a wide range of clinical and biological characteristics. Except for few cases may regress spontaneously, the vast majority will spread and transit wildly. Though the past few years has witnessed great progress in the diagnosis and treatment technology, there still exists a shortage of effective treatment for children with malignant NB,and children with low or moderate degree malignant NB were sometimes over-treated.The possibility for children with malignant NB to survive for5years is only40%.So it is very important to accurately define the prognosis of NB, on which the further clinical treatment is based.As NB may regress spontaneously, so accurate prognosis and appropriate treatment are very critical to patients.Signal transducer and activator of transcription sub-3(the signal transducer and activators of transcription3of STAT3), is a gene which locates at the long arm in the 17th human chromosome. Its product mediated extracellular signal transduction, transcription of downstream target genes, as well as STAT3encoding are transcription factors that are involved in various signal pathways,such as the Jak-STAT signal pathway, regulation of the growth, apoptosis and immune response reactions of neuron axon. The activated STAT3may lead to the abnornal expression of many key genes that are closely related to the cells proliferation, differentiation, survival and apoptosis.Thus, STAT3have a carcinogenic effect on cells through a variety of ways to promote cell proliferation, malignant transformation, hindering apoptosis.However the report on STAT3in neuroblastoma at home and abroad has not been found yet.Hypoxia-inducible factor1a (Hypoxia inducible factor-la, HIF-1a), a nuclear transcription protein, it will escape the polyubiquitinated constitutively activation under hypoxia environment. HIF-la can directly regulate and control the expression of VEGF at the gene level, as VEGF plays an important role in tumor angiogenesis. So it inferred that HIF-1is a major regulator factor for malignant tumor to induce the birth of new vessels.The study confirmed that in the majority of human tumors, HIF-la was over expressed and its activity is critical in maintaining normal energy metabolism of the tumor cell,neovascularity, and promoting tumor proliferation and spread.Studies on HIF-la in neuroblastoma at home are mostly conducted in vitro environment though there are differences in vitro experiments and in vivo environments.ObjectiveIn this study, the SP enzyme-linked immunosorbent assay and reverse transcriptase polymerase chain reaction (RT-PCR) technology were used for qualitative and quantitative detection of STAT3,HIF-la gene protein expression in neuroblastoma cells and normal tissue adjacent to tumor. It aims at exploring their anti-apoptotic mechanics and infinite proliferation in neuroblastoma cells under hypoxic environment, and what role they will play in the occurrence and progress of neuroblastoma, thus provides the basis for the clinical treatment of NB. Materials and Methods1.40frozen, fresh and necrosis-free intraoperative samples of NB in Pediatric General Surgery Departments, the Third Affiliated Hospital of Zhengzhou University and the First Affiliated Hospital of Zhengzhou University from July2010to February2012were collected, while the tumor adjacent tissues which are5cm away from the NB tumor tissues were collected to be a control group.General conditions of children such as gender, age at diagnosis, clinical stage, pathological type, lymph node metastasis were collected and sorted for the further statistical analysis of correlation between STAT3&HIF-la and those clinical data.2.SP enzyme-linked immunosorbent staining was used to detect the expression of STAT3and HIF-la protein in tissues, in which40sterile, fresh and necrosis-free intraoperative samples removed from NB tumor tissues act as a study group and adjacent tissues that are5cm to the NB tumor as a control group.3.Reverse transcriptase polymerase chain reaction method were adopted to detect shimada pathological stage (good prognosis, the histology group FH group and the poor prognosis group, the unfavorable histology group UFH group), and the expression of STAT3,HIF-1a mRNA of the40fresh and necrosis-free intraoperative samples and those of NB adjacent tumor issues in the control group.4.Statistical analysis:all the data was processed by means of SPSS17.0software, and rank sum test for two independent samples was adopted to explore the expression of STAT3,HIF-1a protein in both NB tumor tissues and adjacent tumor tissues in the control group; independent samples t test were used in detecting the expression of transcription factor of STAT3mRNA and HIF-lamRNA in the control group and the study group at different shimada pathological stage, Inspection level a=0.05;the correlation between STAT3mRN and HIF-lawere analyzed through correlation analysis.Results1.The expression of transcription factor STAT3and HIF-la gene protein in NB tumor tissues were significantly increased while their expression in adjacent tumor issues is scarce. And their expression in NB tumor tissues was significantly higher than that in control group, where cytoplasm is colored in STAT3,while nuclear is stained in HIF-la.(See photo) 2.As for NB tumor tissues, RT-PCR results showed that the STAT3gene protein was expressed in NB tumor FU group, UFH group and the control group of adjacent tumor tissues. Furthermore, expression of the STAT3gene protein in NB tumor tissue was higher than that in the control tumor adjacent tissues, and the expression in UFH group is higher than that of the FH group, the difference between them was statistically significant.(F=22, p=0.001)HIF-la is also expressed in both the NB tumor tissue and control tumor adjacent tissues, the expression of HIF-la in NB tumor tissue is higher than that in the control tumor adjacent tissues, while such expression in UFH group was higher than that in the FH group, and the difference was statistically significant.(F=111.820, P=0.001)3.The correlation between the expression of STAT3mRNA and HIF-1amRNA expression in NB tumor tissue was analyzed. It showed that the relative expression levels of STAT3mRNA, HIF-lamRNA in these40cases of NB tumor tissue were0.530±0.051,0.489±0.032respectively, and their expression was positively correlated (r=0.485,P<0.05).Conclusions1.STAT3gene plays an important role in promoting the occurrence and progress of neuroblastoma.2.HIF-la gene induces neuroblastoma to occur and leads to their apoptosis-free growth under low-oxygen environment.3.The interaction between STAT3and HIF-lapromotes the occurrence and progress of NB tumor. |
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