The Study Of Downregulating DNMT1 Gene To The Proliferation, Cloning And Cycle Of Human Brain Glioma Cells

Objective The purpose of the present study was to investigate the method and basis of clinical treatment of glioma cells in vitro. Further, this effects of targeting the DNA methyltransferase 1(DNMT1) gene was evaluated in relation to gloma cell proliferation, cloning and the cell cycle of human glioblastoma cells. Whether targeting DNMT1 gene could expose us with a clinical therapeutic target was the sole aim of the study.Methods The expression of DNMT1 gene in 62 human glioma tissue specimens were analysed by immunohistochemistry(IHC) and different levels of DNMT1 gene expression was found in the organization for human brain glioma in correlation to the pathological grading. Similarly, the DNMT1 si RNA transfection in vitro cultivation were targeted against two kinds of human brain glioma cells(A172 and U87). The cells that were knocked out of DNMT1 gene were set as the experimental group which were compared with the groups that had only been transfected by si RNA(negative control sequence) of glioma cells. The expression of DNMT1 gene was confirmed by q RT-PCR. The expression of DNMT1, PCNA and Cyclin D1, markers of glioma cell proliferation were analyzed by western blot. The degree of cell survival, cell proliferation rate and cellular clonality were determined by MTT and colony formation assay. The cell cycle distribution was analyzed by flow cytometry.Results IHC results showed that the expression of DNMT1 gene were significantly up-regulated and positively correlated with pathological grading of glioma betweenⅠ~Ⅱ and Ⅲ~ Ⅳ(P<0.05) as compared to the control group. Similarly, q RT-PCR results revealed that DNMT1 m RNA levels were significantly decreased in DNMT1 si RNA transfected cells(P<0.01), as compared to the control groups. On the other hand, the western blot revealed that the levels of protein expression of DNMT1, PCNA and Cyclin D1 were dramatically reduced in the experimental groups( P<0.01). MTT and colony formation assay results demonstrated that the experimental group had a much lower cell survival(P<0.05)along with lower levels of proliferation rate(P<0.01). Moreover, the cycle G0/G1 phase of glioma cell in experimental group were significantly longer(P < 0.01). All the observation observed in this study were statistically significant.Conclusion In summary, The in-vitro study of the effect of DNMT1 gene on glioma cell proliferation, colony formation and cycles have a very important influence. This study has exposed DNMT1 gene as a potential therapeutic target for future clinical application.