Correlation Studies Of NDRG2 And Histone Acetylation In Glioma Cell Lines

Background Glioma is the most malignant tumor in the nervous system. It has high attack rate, high mortality rate and difficult treatment. At present, it has not effective therapeutic method. Although its pathogenesis has not yet entirely clear, it is considered that occurrence and development of glioma are the results from the overexpression of the oncogene and the inactivation of the anti-oncogene, one of the inactivation of the anti-oncogene plays an important role in tumorigenesis. In recent studies, the level of histone acetylation is closely related to the occurrence and proliferation of tumor and the expression level of anti-oncogene. Thus, with the study function of anti-oncogene, it makes clear the pathogenesis of glioma and in order to bring hope to treatment.NDRG2 gene was firstly discovered and cloned from normal brain tissue of human with the differential hybridization by our team numbers in 1999. Homology analysis shows, in view of the high degree of homology between NDRG1and NDRG2, it contains acyl carrier protein domain which can carrier acyl, so it called NDRG2. In previous study, it was widely distributed in human tissues. NDRG2 has the high expression in the terminally differentiated cells; NDRG2 has the low expression in high proliferation tissues; in the same tissue, NDRG2 has the low expression in embryo and neogenesis and has the high expression in adult tissues. The study also shows that NDRG2 is the differential expression gene in the tissue of different tumors and corresponding normal tissue. We discovered that the expression of NDRG2 was apparently decreased in the tissue and cell line of glioma. Simultaneously, NDRG2 is suppressor of the cell proliferation when it has overexpression in the glioma .The phrase of the cell in the glioma is apparently stopped.We firstly identified that NDRG2 is not only a candidate tumor suppressor gene, but also an important gene correlated with the cells growth and development and the regulation of cell proliferation and differentiation. In recent studies, the ways of function of anti-oncogene function are relate to apparent genetic. Now the discovery of apparent genetic is conclude with DNA methylation, histone methylation, phosphorylation, acetylation, ubiqui-tination, and non-coding RNA connected chromatin modifications. The study of histone acetylation is at most in the histone modifications. Combinated with results to bioinformatics analysis of NDRG2, its intramolecular contained acyl carrier protein domain which can carrier acyl. NDRG2 as an important gene which correlated with the cells growth and development and the regulation of cell proliferation and differentiation, may probably participate in the process of histone acetylation or decaetylaton. No person to study the relationship between with NDRG2 and histone acetylation.At present, the regulatory mechanism of histone acetylation and decaetylation on gene transcription is at the primary stage. Histone acetylation and deacetylation modification is one of the most mechanisms on regulation of gene transcription. Histone acetyltransferases and histone deacetylases which are histone acetylation key enzyme determined the level of histone acetylation and participated the expression of tumor abnormal gene. The mechanisms of histone acetylation are closely related to the tumor cell proliferation and differentiation.Trichostatin A (TSA) is a histone deacetylases inhibitor, which is reversible or in-reversible inhibitor of HDAC activity by binding to the catalytic center of HDACs. TSA induces widely chromosomal histone acetylation acting on cells, thereby which increased the level of histone acetylation, and regulates the expression of gene. Study shows that histone deacetylases inhibitors(HDACI) have regulated cell cycle, and have been shown to induce cell growth arrest, proliferation and apoptosis in many tumor cell lines.For further investigation on the function of NDRG2, to identify the relationship between NDRG2 and histone acetylation, to explore the pathogenesy and new therapy strategy of the glioma, we are going to use HDACI(Trichostatin A) to treat cells. The recombinant adenoviruses and siRNA are going to be used to detect the relationship between NDRG2 and histone acetylation in the human glioma cell lines.Methods1. Firstly, Two glioma cell lines (U251 and U87) were treated with the histone deacetylase (HDAC) inhibitor,trichostatin A (TSA). The treatment of TSA on cell growth curve and the change of cell cycle were observed. The Real-time PCR and Western blot are going to be used to detect the expression of NDRG2, Acetyl -Histone H3, H4 and HDAC1.2. The use of recombinant adenoviruses NDRG2 for the infection of U251 Cells, Real-time PCR and Western blot were applied to dectect the effects of overexpression of NDRG2 on the level of Acetyl -Histone H3, H4 and HDAC1.3. We used Real-time PCR and Western blot method to detect the interference effects of the chemical synthesis of the three pairs of NDRG2-specific siRNA and observed the influence of inhibition of NDRG2 expression on the level of three kinds of proteins.Result1. TSA can apparently inhibit the proliferation of glioma cell lines U251 and U87; caused cell cycle arrest. TSA treatment resulted in increased expression of NDRG2, Acetyl -Histone H3, H4 and in decreased expression of HDAC1.2. Overexpression of exogenous NDRG2 up-regulates acetylation of histone H3 and H4 in protein levels, and slightly decreases HDAC1 expression.3. Firstly, we have demonstrated the interference effects of NDRG2-specific siRNA by using real-time PCR and Western blot method. Chemically synthesized of the three pairs of NDRG2-specific siRNA can effectively inhibit NDRG2 expression. Inhibition of gene expression increase the HDAC1 expression at the mRNA level, and decease protein level of acetylation of histone H3 and H4, which is not increased the HDAC1 protein expression.Conclusion1. TSA can inhibit the proliferation of glioma cell lines U251 and U87, caused cell cycle arrest and not induce apoptosis.2. The expression of NDRG2 and histone acetylation was unregulated by TSA, and also downregulated HDAC1 expression.3. The level of histone acetylation and deacetylation is regulated by NDRG2, which is involved in histone acetylation and deacetylation process. Changes of the process of histone acetylation can be influenced by what means of NDRG2.That is to be studied by further experiments.