Expression Of CDKN2A And EGFR In Gliomas And Their Effection On Chemosensitivity

Abstract:Objective1.To explore the expression of CDKN2A in glioma and to evaluate its mechanism in the generation and development process; The effect of EGFR expression on the growth and chemosensitivity of glioma derived cell lines;3.The relationship between CDKN2A and EGFR on the growth and chemosensitivity of U251cell line.Method1.(1) A total of61patients with glioma were included in this study. All patients underwent surgery at Xiangya Secondary Hospital during the period2007-2011. None of the patient had received adjuvant treatments such as radiotherapy or chemotherapy treatment prior to surgery in order to eliminate their effects on gene expression;(2) The expression of CDKN2A in61glioma tissues and glioma derived cell lines U251-MG, T98G, U87-MG, A172, SW1783, U118-MG, U138-MG, H4and HS-683were detected by immunohistochemistry and Western blot, respectively; The correlation between CDKN2A expression and the pathological characteristics was analyzed by statistics;(3) To determine the effect of CDKN2A on the growth and survival of glioma cells, a recombinant plasmid carrying the CDKN2A cDNA transfected into glioma derived cell lines. Then transfected cells were selected by G418. The analysis of CDKN2A expression after transfection was by immunohistochemistry and Western blot; The morphology detection of the glioma cell lines after CDKN2A transfection by microscope. The growth curve was detected by viable cell counts;(4) Synthesis of siRNA targete CDKN2A and non-silencing control siRNA. Transfected HS-683cells and H4cells with siRNA, then examined the expression of CDKN2A, Cyclin D and pRb by Western blot after48h. The morphology detection of the glioma cell lines after siRNA transfection by microscope. The growth curve was detected by viable cell counts;(5) Flavopiridola, a cyclin D1inhibitor, added to the glioma cell lines after siRNA transfection, then examined the expression of Cyclin D and pRb by Western blot and the growth curve was detected by viable cell counts;2.(1) The expression of EGFR in the above61glioma tissues and detected by immunohistochemistry and Western blot; The correlation between EGFR expression and the pathological characteristics was analyzed by statistics;(2) The U251glioma cell line was infected with the EGFR-siRNA; The morphology detection of the glioma cell lines after konckdown of EGFR by microscope. The growth curve was detected by viable cell counts;(3) The U251cell proliferation of untransfected group, EGFR RNAi group, BCNU group and EGFR RNAi+BCNU group were detected by the MTT method.3.(1) The U251 glioma cell line was infected with EGFR-siRNA and pcDNA3.1-CDKN2A by the techniques of genetic modification. The morphology detection of the U251glioma cell line after transfection was observed by microscope. The growth curve was detected by viable cell counts;(2) The U251cell proliferation of untransfected group, EGFR-RNAi group, CDKN2A group and EGFR-RNAi+CDKN2A group undergo the the same concentration of BCNU respectively, were detected by the MTT method, and the data had been analysed in statistics.Result1.(1) Expression levels of CDKN2A in high-grade glioma tissues were significant lower than that in lowgrade glioma tissues. Decreased CDKN2A in high-grade, and the high grade glioma cells have a lower levels of CDKN2A than that of low-grade glioma cells, which in consistent with glioma tissues from patients;(2) Transfection of CDKN2A into glioma cells resulted in a reduction in the rate of cell growth (3) siRNA knockdown was performed in some lowgrade glioma cell lines (H4and HS-683). When the expression of CDKN2A interfered effectively, the cell growth accelerates, and also increased the phosphorylation of pRb and cyclin D1. However, flavopiridola, a cyclin D1inhibitor, can reverse the accelerated cell growth both of HS-683and H4cell lines and the expression of pRb and cyclin D1;(4) Increased cyclin D1also detected in high-grade gliomas tissues comparing low-grade gliomas tissues.2.(1) The expression levels of EGFR in high-grade glioma tissues were significant higher than that in lowgrade glioma tissues;(2) EGFR interference’ significantly inhibited glioma cell line proliferation and increased the chemotherapy sensitivity of BCNU;3. Compared to the other three groups, glioma U251cell proliferation, with the treatment of EGFR knockdown and CDKN2A overexpression, significantly decreased.Conclusion1.There is a negative positive correlation between the expression of CDKN2A and degree of malignancy. In contrast, it’s a positive correlation between the expression of EGFR and degree of malignancy.2. The level of CDKN2A expression may present the feedback mechanisms of the cell cycle in the malignant cell populations, and antitumour effect of CDKN2A is Cyclin D1-dependent. CDKN2A aplays a key role on malignant gliomas formation and that therapeutic targeting of this gene locus may be useful in malignant gliomas treatment;3. CDKN2A overexpression and siRNA knockdown EGFR enhance the chemotherapy sensitivity of BCNU, has a better synergistic antitumour effect. It is confirmed that the combined gene therapy can improve the effectiveness of gliomas treatment, which provide a reliable theoretical basis for the research and development of new drugs and the new treatment options.