| Background and objective:Esophageal carcinoma (EC) is one of the sixth most common malignant tumours in the world. Multiple factors and pathways cause the carcinogenesis and development of the esophageal carcinoma. In recent years. with the development of molecular biology. the molecular mechanism of the esophageal carcinoma has been known deeply. but its true pathogenesis is not figured out.Recently,It has been found that the occurrence of esophageal carcinoma has a relationship with the higher levels of proliferation. Nuclear transicription factor kappaB(NF-κB) was a type of important nuclear transicription factor.The main form of NF-κB was dimmer composed of p50 and p65. It play a vital role in regulating the cell proliferation. NF-κB was a mediator of Ras and cyclin D1 in Ras signaling pathway. NF-κB gene was activated by Ras links growth factor signaling from cytoplasmic into the nucleus.that resulted in NF-κB binded cyclinD1,then up-regulation the expression of cyclinD1,that accelerate the transition from G0/G1 to S phase,which resulted in abnormality cell proliferation induces tumorigenesis.Cyclin dependent kinases (CDKs) regulate cell cycle by Cyclin-CDK- CDI complexes. CDK4 protein was an important member of CDKs, the over expression of CDK4 can induce uncontrolled cell proliferation and eventual malignant transformation.The over expression of cyclooxygenase-2 (COX-2) protein promote cell proliferation, and depress cell apoptosis. which result in dysequilibrium of cell proliferation and apoptosis and eventual malignant transformation. There is no report about the the expression of NF-κBp50,p65 mRNA by in situ hybridization in esophageal squamous cell carcinoma.There is no report about correlation between the expression of NF-κB p50,p65, CDK4 ,C0X-2 and the carcinogenesis,invasion and metastasis of esophageal squamous cell carcinoma, too. In order to further explore the relation between the expression of NF-κB p50,p65 and the carcinogenesis,invasion and metastasis of esophageal squamous cell carcinoma, to find early markers of invasion and metastasis of esophageal carcinoma and to find out useful ways to suppress the invasion and metastasis of esophageal carcinoma,this article detected the expression of NF-κBp50,p65 mRNA in 49 cases of esophageal squamous cell carcinoma and adjacent-carcinoma epithelium by in situ hybridization.And detcted the expression of NF-κBp50,p65 protein,CDK4 and COX-2 proteins by immunohistochemistry, and explored the correlation between NF-κBp50%,p65,CDK4 and COX-2.Materials and methods1.Sample:49 cases of esophageal squamous cell carcinoma were collected formthe First Affiliated Hospital,Zhengzhou University in 2002.All cases has no the history of the chemotherapy,radiotherapy and immunotherapy.All cases were identified by pathologist,it is entirely esophageal squamous cell carcinoma.24 cases are grade I ,16 cases are grade II ,9 cases are grade III.23 example appeared the lymph node metastasis,26 cases are non-lymph node metastasis. 17 cases are on the lower layer invaded and 32 cases on the deeper layer invaded.Each all in site of tumor and adjacent-carcinoma mucoma cut a piece of tissue involve of 33 cases proliferention ,19 cases mild dysplasia,14 cases severe dysplasia,12 cases carcinoma in situ and 32 cases normal esophageal epithelium.2.Methods:Using streptomycete avidin peroxides(SP) immunohistochemistry and in situ hybridization(ISH) to examine the expression of NF-κBp50,p65, CDK4 and COX-2 in 49 cases esophageal squamous cell carcinoma.3.A11 the data were analyzed by SPSS10.0 software,the comparision of positive rate used the Chi-square.The relation of two variable are analyzed by correlationanalysis.The level of significant difference wasα=0.05.Results1. The positive rate of NF-κBp50 protein in normal epithelirm, simplehyperplasia epithelium,mild atypical hyperplasia epithelium,severe atypical hyperplasia epithelium,carcinoma in situ and invasive carcinoma were 28.1% (9/32) ,30.3% (10/33) ,47.4% (9/19) ,50.0% (7/14) ,33.3% (4/12)和55.1% (27/49) respectively, there were significant difference between the normal epithelium, the simple hyperplasia epithelium and the invasive carcinoma (P<0.05). The positive rate of NF-κBp65 protein in normal epithelirm, simple hyperplasia epithelium,mild atypical hyperplasia epithelium,severe atypical hyperplasia epithelium,carcinoma in situ and invasive carcinoma were 18.8% (6/32) ,18.2% (6/33) ,36.8% (7/19) ,42.9% (6/14) ,50.0% (6/12)和57.1 (28/49) respectively, there were significant difference between the normal epithelium, simple hyperplasia epithelium and the invasive carcinoma (P<0.05). The positive rates of NF-κBp50 protein in esophageal squamous cell carcinoma of grade I,II,III were 37.5%,62.5%,88.9% recpectively, there was a significant difference among them(P<0.05). the positive rates of NF-κBp50 protein on lower layer invade of esophageal squamous cell carcinoma group and deeplayer invade of esophageal squamous cell carcinoma group were 15.4% and 69.4% respectively, a significant difference between them(P<0.05).The positive rates of NF-κBp50 protein in the lymph node metastasis group and nonlymph node metastasis group were 60.9% and 50.0% respectively, a significant difference between them(P<0.05). The positive rates of NF-κBp65 protein in esophageal squamous cell carcinoma of grade I,II,III were 41.7%,68.8% and 77.8% recpectively,although they were increased gradually, there was no significant difference among them(P>0.05). the positive rates of NF-κBp65 protein on lower layer invade of esophageal squamous cell carcinoma group and deeper layer invade of esophageal squamous cell carcinoma group were 7.7% and 75.0% respectively, a significant difference between them(P<0.05).The positive rates of NF-κBp65 protein in the lymph node metastasis group and nonlymph node metastasis group were 87.0% and 30.8% respectively, a significant difference between them(P<0.05).2. The positive rate of NF-κBp50 mRNA in normal epithelirm, simple hyperplasia epithelium, mild atypical hyperplasia epithelium,severe atypical hyperplasia epithelium,carcinoma in situ and invasive carcinoma were 21.9%(7/32) ,24.2% (8/33) ,31.6% (6/19) ,42.9% (6/14) ,41.7% (5/12)和153.1 % (26/49) respectively, there were significant difference between the normal epithelium, simple hyperplasia epithelium and the invasive carcinoma (P<0.05). The positive rate of NF-κBp65 mRNA in normal epithelirm, simple hyperplasia epithelium, mild atypical hyperplasia epithelium,severe atypical hyperplasia epithelium,carcinoma in situ and invasive carcinoma were 18.8% (6/32) ,18.2% (6/33) ,31.6% (6/19) ,35.7%(5/14) ,33.3% (4/12) ,55.1% (27/49) respectively. there were significant difference between the normal epithelium, simple hyperplasia epithelium and the invasive carcinoma (P<0.05). The positive rates of NF-κBp50 mRNA in esophageal squamous cell carcinoma of grade I,II,III were 37.5%,62.5% and 77.8% respectively,although they were increased gradually, there was no significant difference among them(P>0.05). the positive rates of NF-κBp50 mRNA on lower layer invade of esophageal squamous cell carcinoma group and deeper layer invade of esophageal squamous cell carcinoma group were 15.4% and 66.7% respectively, a significant difference between them(P<0.05). The positive rates of NF-κBp50 mRNA in the lymph node metastasis group and nonlymph node metastasis group were 78.3% and 30.8% respectively, a significant difference between them(P<0.05). The positive rates of NF-κBp65 mRNA in esophageal squamous cell carcinoma of grade I,II,III were 45.8%,50.0% and 88.9% respectively, although they were increased gradually, there was no significant difference among them(P>0.05). the positive rates of NF-κBp65 mRNA on lower layer invade of esophageal squamous cell carcinoma group and deeper layer invade of esophageal squamous cell carcinoma group were 15.4% and 69.4% respectively, a significant difference between them(P< 0.05).The positive rates of NF-κBp65 mRNA in the lymph node metastasis group and nonlymph node metastasis group were 73.9% and 38.5% respectively, a significant difference between them(P<0.05).3. The positive rate of CDK4 protein in normal epithelium, simple hyperplasia epithelium, mild atypical hyperplasia epithelium,severe atypical hyperplasia epithelium,carcinoma in situ and invasive carcinoma were 9.4%(3/32),24.2%(8/33), 42.1% (8/19), 64.3% (9/14), 66.7% (8/12), 77.6% (38/49) respectively. There was significant difference between mild atypical hyperplasia epithelium,severe atypical hyperplasia epithelium,carcinoma in situ ,invasive carcinoma and normal epithelium (P< 0.05).The positive rate of CDK4 protein in esophageal squamous cell carcinoma of grade I,II,III were 66.7%, 87.5% and 88.9 % respectively, there was no significant difference among them (P>0.05),although they were increased gradually. The positive rate of CDK4 protein on lower layer invade of esophageal squamous cell carcinoma group and deeper layer invade of esophageal squamous cell carcinoma group were 46.2% and 88.9% respectively,there was a significant difference between them(P< 0.05). The positive rate of CDK4 protein in the lymph node metastasis group and nonlymph node metastasis group were 95.7% and 61.5% respectively, there was a significant difference between them(P< 0.05).4. The positive rate of COX-2 protein in adjacent normal epithelium, simple hyperplasia epithelium, mild atypical hyperplasia epithelium,severe atypical hyperplasia epithelium,carcinoma in situ and invasive carcinoma were 0.0% (0/32),6.1%(2/33), 31.6%(6/19), 46.2%(6/14), 58.3%(7/12), 73.5%(36/49), respectively. There was significant difference between mild atypical hyperplasia epithelium,severe atypical hyperplasia epithelium,carcinoma in situ ,invasive carcinoma and normal epithelium (P<0.05). The positive rate of COX-2 protein in esophageal squamous cell carcinoma of grade I, II ,III were 62.5%, 81.3% and 88.9 % respectively,they are increased gradually,while there was no significant difference among them (P>0.05). The positive rate of COX-2 protein on lower layer invade of esophageal squamous cell carcinoma group and deeper layer invade of esophageal squamous cell carcinoma group were 38.5% and 90.6% respectively,there was a significant difference between them(P< 0.05). The positive rate of COX-2 protein in the lymph node metastasis group and nonlymph node metastasis group were 91.3% and 57.7% respectively, there was a significant difference between them(P< 0.05).5.There were positive correlations between the positive expression rate of NF-κBp50 protein and that of CDK4 protein, COX-2 protein in esophageal squamous cell carcinoma(P<0.05). There were positive correlations between the positive expression rate of NF-κBp50 mRNA and that of CDK4 protein, COX-2 protein in esophageal squamous cell carcinoma(P<0.05). There were positive correlations between the positive expression rate of NF-κBp65 protein and that of CDK4 protein, COX-2 protein in esophageal squamous cell carcinoma(P<0.05). There were positive correlations between the positive expression rate of NF-κBp65 mRNA and that of CDK4 protein, COX-2 protein in esophageal squamous cell carcinoma(P< 0.05).6. There was a positive correlation between the positive expression rate of CDK4 protein and that of COX-2 protein in esophageal squamous cell carcinoma(P< 0.05). there were 33 cases positive expression of COX-2 protein in 38 cases positive expression of CDK4 protein,there were 8 cases negative expression of COX-2 protein in 11 cases negative expression of CDK4 protein.7. There was a positive correlation between the positive expression rate of NF-κBp50 protein and that of p65 protein in esophageal squamous cell carcinoma(P< 0.05). There was a positive correlation between the positive expression rate of NF-κBp50 mRNA and that of p65mRNA in esophageal squamous cell carcinoma(P<0.05). There was a positive correlation between the positive expression rate of NF-κBp50 protein and that of p50 mRNA in esophageal squamous cell carcinoma(P<0.05). There was a positive correlation between the positive expression rate of NF-κBp65protein and that of p65 mRNA in esophageal squamous cell carcinoma(P<0.05).Conclution1.The positive expression of NF-κBp50,p65 protein and mRNA in esophagealsquamous cell carcinoma were both higher than that of NF-κB p50,p65 protein and mRNA in normal esophagus epithelium,and the positive expression of NF-κB p50, p65 protein and mRNA in tumor-adjacent atypical hyperplasia epithelium had a trend of increasing gradually.It hints that over expression of NF-κB p50,p65 maybe play an important role in the tumorigenesis and development of esophageal squamous cell carcinoma.2. The positive expression of NF-κB p50,p65 protein and mRNA were related to invasion and lymph node metastasis. It was indicated that NF-κB p50,p65 was correlated with invasion and lymph node metastasis of esophageal squamous cell carcinoma.3.The CDK4 protein and COX-2 protein might contribute to occurrence,invasion and metastasis of esophageal squamous cell carcinoma, From the higher to the lower differented carcinoma tissues,the positive rate of CDK4 protein had a trend of increasing gradually, it hints that CDK4 protein maybe correlated with the development of esophageal squamous cell carcinoma.4.There was a positive correlation between the expression of NF-κB p50,p65 protein and NF-κB p50,p65mRNA,it implied that the expression of NF-κB p50,p65 protein and mRNA had consistency, the positive expression of NF-κB p50,p65 were correlated with CDK4 protein and COX-2 protein, it implied that the positive expression of NF-κB can ascending the expression of CDK4 protein and COX-2 protein.
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