The Influence Of DJ-1ON Proliferation, Apoptosis, Invasion And Migration Of Acute Lymphoblastic Leukemia Cell

BackgroundAcute lymphocytic leukemia (ALL) is the most common type of hematopoietic malignancies in children. Its immortalized malignant proliferation and invasion degree are directly and closely related to the prognosis of the children. Chemotherapy is still the main therapy at present. The5-year survival rate of ALL children given standard combined chemotherapy has reached80%. However, some children are resistant to chemotherapy, while some children accept chemotherapy of too high strength. Therefore, it is the recent focus in research on treatment of ALL children to improve and revise the prognosis assessment system, so as to strive to implement individualized treatment (including gene therapy) for children grouped according to the risk factors. Research on the molecular mechanism of ALL cells in proliferation, apoptosis and invasion not only can provide new connotation for understanding on the pathogenetic mechanism of ALL, but can also provide promising molecular targets for new individualized treatment strategies of ALL children.DJ-1gene is a kind of mitochondrial-dependent cancer gene, which is related with the occurrence, progress and prognosis, etc. of a variety of malignant tumors. As DJ-1may play an important role in the process of malignant proliferation, apoptosis and invasion, it has aroused people’s concern. However, the expression and action mechanism of DJ-1are still uncertain, such as the specific signal pathway and regulatory mechanism, etc., which need further study. DJ-1is found to be the negative regulatory factor for phosphatase and tensin homology deleted on chromosome10(PTEN), both of which are involved in cell apoptosis, differentiation and migration. It is suspected that DJ-1might participate in the malignant proliferation, apoptosis, invasion and migration of ALL by inhibiting the expression of PTEN. The present work analyzes the correlation between DJ-1and malignant proliferation, migration and invasion of ALL via ALL cell culture in vitro, thus providing new direction to clarify the molecular regulatory mechanism on ALL and providing new target for ALL gene therapy.Part I Expression of DJ-1in Jurkat cell lines and bone marrow mononuclear cells (BMMNC) of ALL children ObjectiveTo investigate the expression of DJ-1in Jurkat cells and bone marrow mononuclear cells (BMMNC) of children with acute lymphoblastic leukemia (ALL); to lay an experimental basis for further exploration on the possibility of interfering malignant biology behavior of ALL cells such as proliferation and apoptosis etc., via targeting on DJ-1.MethodsJurkat cells and BMMNCs of ALL children were selected the research objects, and BMMNCs of children with immune thrombocytopenia (ITP) were selected as the control. The Jurkat cells were cultivated in vitro; and BMMNC were isolated from new bone marrow specimens (1-1.5mL) of two groups of children in accordance to the inclusion criteria by Ficoll-Hypaque density gradient centrifugation. The mRNA and protein levels of DJ-1and PTEN were detected by RT-PCR and Western blotting respectively.ResultsAs shown in Western blot detection, the protein expressions of DJ-1were significantly up-regulated in Jurkat cells and BMMNCs of ALL children (P<0.01), while the protein expression of PTEN were significantly down-regulated in Jurkat cells and BMMNCs (P<0.01) compared to the control group. And as shown in RT-PCR, the mRNA expression of DJ-1were significantly up-regulated in Jurkat cells and BMMNCs (P<0.01), while the mRNA expression of PTEN were significantly down-regulated in Jurkat cells and BMMNC (P<0.01) compared to the control group.ConclusionDJ-1may be a new target for gene therapy of ALL, for it could affect the biological activities of ALL cells via inhibiting the expression of PTEN and regulating the signal pathway of PI3K/Akt. Part Ⅱ Influence of DJ-1on proliferation, migration and invasion of acute lymphoblastic leukemia cells ObjectiveTo investigate whether DJ-1could promote migration of ALL cells through down-regulating the expression of PTEN and up-regulating the phosphorylation of FAK; to explore a new target for gene therapy of ALL.Methods1. Jurkat cells were chosen as the experimental cells. Jurkat cells of human ALL were obtained from in vitro culture. Expression vector of DJ-1was designed and established according to the human DJ-1mRNA coding sequence. pEGFP/DJ-1vector was used to transfect into Jurkat cells. Western blot was used to determine the changes of DJ-1, PTEN, FAK and p-FAK in Jurkat cells before and after transfection. And Transwell assay was used to analyze the migration and invasion of Jurkat cells before and after transfection.2. BMMNCs were isolated from new bone marrow specimens of ALL children by Ficoll-Hypaque density gradient centrifugation, with the primary cells as the experimental cells. The small interfering RNA (siRNA) of DJ-1was constructed according to the human DJ-1mRNA coding sequence. BMMNCs of ALL children were transfected with si DJ-1, and then the changes of DJ-1, PTEN, FAK and p-FAK in the cells were determined by Western blot analysis. Flow cytometry was used to test the apoptosis rate and Transwell assay was used to detect the migration and invasion of BMMNC cells before and after transfection. Results1. The mechanism research on DJ-1inhibiting the migration of Jurkat cells through PTEN down-regulationJurkat cells were transfected by pEGFP/DJ-1for48h. Protein expressions of DJ-1and p-FAK in pEGFP/DJ-1group were significantly stronger than those in the blank control group and pEGFP-C2control group (P<0.05), while the protein expressions of PTEN were significantly decreased and obviously lower than those in blank control group and pEGFP-C2control group (P<0.05). There was no significant difference in total FAK protein expression among the three groups (P>0.05). In migration and invasion experiment, the numbers of migration and invasion cells in pEGFP/DJ-1group were significantly increased, compared with the blank control group and pEGFP-C2control group (P<0.05).2. The influence of siRNA silence DJ-1gene on migration of BMMNCsBMMNCs of ALL children were transfected by DJ-1siRNA. Then the expressions of DJ-1and p-FAK proteins were obviously decreased in DJ-1siRNA group, which were significantly weaker than those in the blank control group and nonspecific-transfection control group (P<0.05), while the expression of PTEN protein was significantly increased, which were stronger than those in blank control group and nonspecific-transfection control group (P<0.05). There was no significant difference in total FAK protein expression among three groups ((P>0.05). BMMNCs of ALL children were transfected by DJ-1siRNA. Then the apoptosis rate of DJ-1siRNA group was significantly higher than those of blank control group and nonspecific-transfection control group (P<0.05). There was no significant difference in apoptosis rate between blank control group and nonspecific-transfection control group (p>0.05).In migration and invasion experiment, the numbers of migration and invasion cells in DJ-1siRNA group were significantly decreased, compared with the blank control group and nonspecific-transfection control group (P<0.05). And there was no significant difference between blank control group and nonspecific-transfection control group (p>0.05). Conclusion1. DJ-1can promote the migration and invasion of Jurkat cells of human ALL, which may be related to the negative feedback regulation of DJ-1on PTEN expression and indirect inhibition on FAK phosphorylation. Negative regulation of DJ-1on PTEN may be the new direction for the research on gene therapy of ALL.2. Small interfering RNA down-regulating DJ-1protein expression can induce the BMMNC apoptosis, and inhibit the migration and invasion ability of cells. This function may be related to DJ-1promoting PTEN expression and improving the phosphorylation of FAK. Part Ⅲ Research on EGCG inhibiting the migration of ALL cells by down-regulating DJ-1Objective1. To study the inhibitory effect of EGCG on the migration of Jurkat cells and BMMNCs of ALL children and its mechanism.2. To explore whether EGCG could revise the proliferation of ALL cells, induce apoptosis and inhibit malignant biological behaviors of ALL cells by targeted inhibition on DJ-1.MethodsJurkat cells and BMMNCs of ALL children as the experimental cells were treated by different concentrations of EGCG. MTT assay was used to determine cell proliferation while flow cytometry was used to detect the apoptosis rates. Transwell assay was used to analyze the migration and invasion ability of Jurkat cells and BMMNCs. Meanwhile, pEGFP/DJ-1was used to block the effect of EGCG. Then Transwell assay was used to observe whether EGCG can suppress migration and invasion of ALL cells through down-regulating the expression of DJ-1.ResultsTreated with different concentrations of EGCG for different time, Jurkat cells and BMMNCs of ALL children were suppressed in proliferation to different degrees. The inhibition rate of each EGCG pretreatment group was significantly higher than that of the control group (P<0.05), and the effect of EGCG (10,25,50,80,100mol/L) was increased with the increase of the concentration and time. The inhibition rate was dose and time dependent.Treated with different concentrations of EGCG for different time, Jurkat cells and BMMNCs of ALL children were induced to apoptosis to different degrees. The apoptosis rates of EGCG pretreatment groups were significantly higher than that of the control group (P<0.05), and the effect of EGCG (10,25,50,80,100mol/L) was increased with the increase of concentration and time. The apoptosis rate was dose and time dependent.In migration and invasion experiment, Jurkat cells and BMMNCs of ALL children were treated with different concentrations of EGCG for12,24and48h. The numbers of migration and invasion cells were significantly decreased, compared to the control group (P<0.05). The numbers were dose and time dependent.Jurkat cells and BMMNCs of ALL children were treated with pEGFP/DJ-1, EGCG and pEGFP/DJ-1+EGCG for48h. The numbers of migration and invasion cells were changed differently. Treated by100μmol/L EGCG for24h, the numbers of migration and invasion cells of Jurkat cells were significantly decreased and the numbers of pEGFP/DJ-1cells were obviously increased compared to the control group. The cells transfected by pEGFP/DJ-1for24h and then treated by EGCG for24h had more migration and invasion cells than those treated by EGCG only, but less than single pEGFP/DJ-1group, namely the inhibitory effect of EGCG on migration and invasion was influenced by pEGFP/DJ-1. Conclusion DJ-1may be a target gene of EGCG. EGCG may suppress the migration and invasion of ALL cells through down-regulation of DJ-1expression.