The Role Of DJ-1 In Dedifferentiation, Migration And Invasion Of Glioma

1. BACKGROUND:Malignant gliomas are the most common type of primary brain tumor. Gliomas account for approximately 40-50% of neurological cancers. Due to the tendency of these tumors to infiltrate the surrounding brain, the cancerous area cannot be removed easily. Current understanding suggests that differentiation and invasion are the two main factors in WHO grading, thus the study of the potential disease differentiation and invasion is important to understand the pathogenesis of glioma tumors. Furthermore, it is helpful for us to identify some of the crucial biomarkers for the treatment of this cancer, providing new targets for clinical gene therapy.The present study shows that DJ-1 is over expressed in multiple cancers, thus linked to their pathogenesis. However, the role that DJ-1 plays in glioma tumors is still poorly understood. This study seeks to better understand what role DJ-1 plays in tumor pathogenesis by combining in vitro and in vivo experimentation. The present work will focus on characterizing the correlation between DJ-1, PTEN, and glioma dedifferentiation, migration and invasion.2. OBJECTIVE:This study aims to investigate the effect of DJ-1 protein on the dedifferentiation, migration, and invasion of human glioma cell, as well as explore potential mechanisms. Our work has therefore discovered and identified a biomarker for the treatment of glioma. It is significant that our study may offer a new target for inducing differentiation treatment and clinical gene therapy. 3. METHODS:1) For this study we employed three different glioma cell lines, with varying degrees of differentiation. The levels of DJ-1 and other protein expression were determined by confocal microscopy and Western blotting. These cell lines were then transfected with siDJ-1, the DJ-1 protein level of SWO-38 is the the highest in the three cell lines. To analyze the potential DJ-1 mechanism Western blotting and cell cycle test analysis were also used.2) Using immunohistochemical methods (S-P), we detected the expression of DJ-1 in specimens of 85 glioma cases. In this way, we analyzed the relationship between DJ-1 and WHO grading.3) The eukaryotic expression vector pEGFP/DJ-1 and small interfering RNA (siRNA) were constructed and transfected into human glioma SWO-38 cells. The expression of DJ-1 and PTEN, along with the phosphorylation of FAK, in SWO-38 cells was detected by Western blot. Cell migration and invasion were detected by the Transwell assay.4) Using MTT, the effect of EGCG on SWO38 apoptosis was examined, in order to select the appropriate EGCG concentration for later experiments. Cell migration and invasion were again detected using the Transwell assay. DJ-1 and PTEN expression was assayed by Western blot. Meanwhile, we used pEGFP/DJ-1 to stimulate the expression of DJ-1, then combined the EGCG and pEGFP/DJ-1. The effect of this treatment was analyzed by Western blot and the Transwell assay. With this set of experiments we analyzed the mechanism of how EGCG effects migration and invasion in SWO-38 cells.4. RESULTS:1) The expression of DJ-1 was different in three glioma cell lines.Our study found that the expression of DJ-1 in SWO-38 cells was more than the other two cell lines tested, with the lowest expression in the SWO-Z1 cells. The change in P-catenin expression was nearly the same as DJ-1. No obvious alteration in the expression of PTEN in the three cell lines was observed.2) Transfection with siD J-l in SWO-38 cells altered expression of DJ-1, PTEN, and P-catenin.si DJ-1 down-regulated the expression of DJ-1, as well as lowered P-catenin expression. In the cell cycle assay, the cell number of G2 decreased after SWO-38 was transfected with si DJ-1. Compared with control group, there was a significant difference (P<0.05).3) The expression of DJ-1 in specimens of 85 glioma cases.The expression of DJ-1 was positively correlated with the WHO grading of glioma. The expression of DJ-1 in each level of glioma was significantly different (P<0.01).4) The role of DJ-1 in the process of migration and invasion in glioma cells.After transfection of pEGFP/DJ-1 the expression of DJ-1 and focal adhesion kinase (FAK) phosphorylation were increased, whereas the expression of PTEN was suppressed. After transfection with DJ-1 siRNA, both DJ-1 and p-FAK levels were decreased while PTEN expression increased. The Transwell assay showed that transfection with pEGFP/DJ-1 promoted SWO-38 cell migration and invasion, whereas transfection with DJ-1 siRNA alone inhibited SWO-38 migration and invasion.5) The influence of EGCG on migration and invasion in glioma cells.After treated with EGCG alone for 24 h, the migration and invasion ability of SWO-38 cells decreased with a decrease in DJ-1 expression. Following this, we used pEGFP/DJ-1 to block the potential pathway of EGCG. When we combined EGCG and pEGFP/DJ-1 in SWO-38 cells the migration and invasion were not decreased by EGCG. The expression of DJ-1 and FAK phosphorylation could also not be down-regulated. 5. CONCLUSIONS:1) DJ-1 may play a role in the differentiation of glioma cells. A possible mechanism may be the up-regulation of DJ-1 andβ-catenin directly, or binding with other factors. This maybe the potential mechanism of DJ-1 stimulating glioma differentiation.2) The expression of DJ-1 is positively correlated with the WHO grading of glioma tumors.3) Over-expression of DJ-1 promotes SWO-38 cell migration and invasion, possibly through DJ-1 and PTEN/FAK pathway.4) EGCG can inhibit the migration and invasion ability of SWO-38 cell line by targeting DJ-1. There is also a correlation between EGCG and the phosphorylation of FAK.