Expression Of Cyclooxygenase-2 In Astrocytic Tumors And Its Clinical Significance

IntroductionAstrocytic tumor is common neuroepithelium tumor that incidence rate is 13.0% ~26.0% of intracranial tumor and 21. 2% ~51.6% of glioma. Due to the character of infiltrative growth, operative risk of astrocytic tumor is bigger; operation is difficult to cure radically; it is easy to recrudesce after operation; survival rate and quality both are low. So it is the hot topic that further study molecular biology mechanism of astrocytic tumors occurrence and development, search and find the method of early diagnosis and effective treatment. Cyclooxy-genase (COX) is a key enzyme during the change of arachidonic acid to prosta-glandin. Recent studies showed that over expression of COX-2, the induced iso - enzyme of COX, was related to occurrence and development of tumors closely , but reports about the relationship of COX-2 and astrocytic tumours are rare yet. We detected the COX-2 protein and mRNA levels in astrocytic tumours tissues and non - tumor tissues by immunohistochemistry and RT-PCR technique to explore the relationship between COX-2 expression and occurrence, develoption of astrocytic tumours , and give evidences for finding gene markers of early diagnosis and gene therapy.Materials And Methods1. Specimens selectionSpecimens of astrocytic tumours ( n = 45) were obtained from patients ofbrain tumors in the second affiliated hospital of China Medical University between Aug, 1999 to Aug, 2002. All tumours were graded according to World Health Organization ( WHO) Classification of Tumours of the Nervous System (2000) , pilocytic astrocytoma ( grade I ) : n = 10; diffuse astrocytoma ( grade II ) : n=13; anaplastic astrocytoma ( grade HI) : n = 10; glioblastoma ( grade IV): n = 12. And collection 43 fresh tissues specimens including 36 astrocytic tumours and 7 non - tumor tissues, low grade astrocytoma ( grade I ~ II ) : n = 11; anaplastic astrocytoma (grade HI) : n = 15; glioblastoma ( grade IV) : n = 10. All the specimens were giagnosed by pathological examination.2. Methods(1) Immunohistochemistry technique :(2) S-P methods were used. <2)RT-PCR technique:raction of total RNA: Extract with TRI-zol reagent in only ane step. Reverse transcription to synthesize cDNA; The operation was taken according to the directions of test kit.<3)PCR amplification; 3jjl1 cDNA product, 0. l|xlprimers respectively, 2. 5yJ Bca Buffer( x 10) , 2jxl dNTPs( lOmmol/L) , 0. 2jxl Taq enzyme, 16. 3|xl ddH2O, pre - degeneration for 90s at 90tI, degeneration for 45s at 941, annealing for 45s at 55*0 , then extension for 90s at 72*C , after 35 cycles extension for 10 minutes at 721.@ Products analysis; The amplification products were taken agarose gel eletroresis, take j3-actin as internal contral, stain with EB, observe under ultraviolet lamp and take photos, detect content of products in each amplification zone with automatic analysis system of electrophoresis gel imaging.3. Result determination;(1} Immunohistochemistry result:The immunohistochemistry positive matter of COX-2 assumes brown -yellow particles in cytoplasm or nucler membrane.(2) RT-PCR result:After EB staining, it was posivise result if with both 305bp zone and |3-ac-tin 690 bp zone and it was negative result if only with p-actin 690 bp zone com-pared with Marker.Relative content of COX-2 mRNA was obtained by standardizing absorbency of COX-2 by the absorbency of internal control.4. Statiscal analysis;Take statistical with SPSS 11.5. The data were expressed as average ± standard difference, t-test was used in compare of mean value of two samples. 5(2-test and fisher' s exact probability were used to rates of samples, a =0.05.ResultsThe expression of COX-2 protein in each group by immunohistochemical staining ( IODA ) was; non -tumor tissues(2. 18 ±0.94) , astrocytic tumours (6. 38 ± 3. 68) , pilocytic astrocytoma (3. 54 ± 1. 29) , diffuse astrocytoma (5. 58 ± 2. 78) , anaplastic astrocytoma (6. 88 ± 2. 70) , glioblastoma (9. 19 ± 4. 62) ; The rate of posivise expression of COX-2 mRNA was: non - tumor tissues (2/7) , astrocytic tumours (83. 33% ); The relative content of COX-2 mRNA was: non - tumor tissues(43. 14 ± 19. 26) , astrocytic tumours(119. 67 ±46. 56) ; The expression of COX-2 in astrocytic tumours was significantly highter than in non - tumor tissues (P < 0.05).Discussion1. Structure, function and carcinogenesis mechanism of COX-2 COX-2 is a key enzyme in arachidonic acid metabolism. The main products are prostaglandins(PGs) . The human COX-2 gene is 8. 3 kb in size. It is composed of 10 exons. Fluorescence in situ hybridization localized human COX-2 to chromosome Iq25.2 -25. 3. The transcription of COX-2 yields mRNA is 4.5kb in size. After the stimution of mitogen COX-2 mRNA rapidly increased within thirty minutes and keep high level for six to eight hours, It majority cells and tissues , the expression of COX-2 is low level and even undertecable but COX-2 expression in inducible by a wide range of extracellular and intracellular stimuli, incuding cellular factor, growth factors, Its expression increase ten to eighttimes, but COX-2 protein is induced by epidermal growth factor ( EGF) and stimulate the activation of COX-2 promoter. It is reveals that COX-2 also expressed in normal tissue.2. Expression of COX-2 protein and mRNA in astrocytic tumor tissues and non-tumor tissues and significanceThe result of study shows: COX-2 both has the expression in tumor and non-tumor tissues, the expression in tumor tissues is higher than that in non-tumor tissues, the expression in low-grade tumor tissues are also higher and the difference has statistics significance. The result shows the over-expression of COX-2 has close relative with astrocytic tumorigenesis and important action to come into being early. COX-2 acts to early canceration of astrocyte. Examination of COX-2 may be the index of early gene diagnosis for astrocytic tumor.Expression of little COX-2 exists in non - tumor tissues. So presume that COX-2 has complicated action in the course of organ growth and maintaining normal physiological function besides COX-2 joins the pathological courses of tu-mogenesis and inflammatory occurrence etc.COX-2 expression strengthens along with tumor pathological level in astrocytic tumor tissue and it is strongest in glioblastoma that show COX-2 over - expression has close relation with not only astrocytic tumorfc occurrence, but only tumors development. The result presumes that COX-2 over - expression has relation with higher malignant phenotypic development of low grade astrocytoma and the end is highest malignant phenotypic glioblastoma. COX-2 high expression may be important reason of tumor evolution from low malignant phenotypic to high malignant phenotype. It may reflect malignant potential of astrocytic tumor in certain significance.The result of this study also shows: COX-2 expression is low in pilocytic astrocytoma and the difference is significant with other grade tumor. It shows that genetic bases of pilocytic astrocytoma and diffuse astrocytoma are different. Pilocytic astrocytoma is a kind of genetic change in biology and morphology that is different from other astrocytic tumours.Pearson correlation analysis shows; COX-2 expression in translation has good correlation with that in transcription. It shows regulation of COX-2 genetic...