| BackgroundGlioma in the adult central nervous system tumors account for about 40-50% of intracranial tumors, gliomas showed diffuse, invasive growth and poor prognosis, with an average survival time is short. Current treatment programs include surgery, radiation therapy, chemotherapy, and other comprehensive treatment. But the treatment is not satisfactory. In particular, the average survival time of glioblastoma (WHOâ…£) patients were approximately 1 year.Chemotherapy is an important adjunct to the treatment of glioma, but a considerable part of glioma which easily develop resistance to chemotherapy can not achieve the desired effect, chemotherapeutic drug resistance of glioma has become the major obstacles . We Often see the prognosis for patients with the same clinical pathological grade, extent of surgical resection, and underwent radiation therapy and chemotherapy is not the same. This difference in survival time with the individual's probably depended on sensitivity to chemotherapy. But molecular mechanisms of glioma chemotherapy resistance and related target have not yet been fully elucidated. At this stage, the mechanisms of resistance to chemotherapy of malignant glioma are as follows: ( 1 ) O~6-Methylguanine Methyltransferase(MGMT);(2) Mismatch Repair;(3) Poly(ADP-ribose)polymerase;(4)Apoptosis-Regulating Genes;(5)Transporter Proteins.Our previous work carried out some genes which were associated with chemotherapy resistance by gene chip detection of glioma cases and clinical follow-up, MDM4 (Mouse double minute 4) is one of chemotherapy-related genes which were selected from the gene chip analysis of biological information.MDM4 is a key regulator of p53, which was affected on apoptosis through regulation of p53 transcriptional function or the mitochondrial pathway. USP2a (Ubiquitin-specifc protease 2a) is one of Deubiquitinating enzymes(DUBs), MDM4 is a substrate of USP2a. the protein expressions of MDM4 and USP2a in the clinical specimens from longer survival patients were higher than that in clinical specimens from shorter survival patients. In clinical glioma specimens and cell lines, MDM4 and USP2a can happen immunoprecipitation and form complex. MDM4 and p53Ser46~P were co-localized in the mitochondria. In the artificial induction of apoptosis conditions, MDM4 promote mitochondrial pathway of apoptosis depends on p53, but not p21. USP2a can promote stability of MDM4.Part I: Study on the expression and significance of MDM4 in human glioblastomaObjective: To investigate the expression and significance of MDM4 mRNA and protein in brain glioblastoma.Methods: The mRNA expression of MDM4 in 8 cases of human glioblastoma tissues were detected with quantitative real-time PCR. The protein expression in these samples were detected using Western blot. The protein location and relationship of MDM4 and prognosis of patients were detected by IHC.Results: The expression of MDM4 mRNA was no difference in patient of poor prognosis and in patient of good prognosis. The expression of MDM4 protein is higher in patient of good prognosis than in patient of poor prognosis. Survival in patients with MDM4 high expression were longer than in patients with MDM4 low expression. The positive expression of MDM4 was associated with USP2a,but not p53.Conclusion: In human glioblastoma of brain, USP2a and MDM4 may take effect with each other in the cytoplasm; MDM4 and p53Ser46~P may take effect with each other in the mitochondria. The elevated levels of MDM4 protein expression may be due to post-transcriptional regulation by USP2a. MDM4 was an independent predictor of prognosis of glioblastoma patients.Partâ…¡: Study on the expression of MDM4 and relationship with USP2a and p53 in human glioblastoma cell line U87MGObjective: To detect the expression of MDM4 and relationship with USP2a and p53.Methods: By immunoprecipitation and confocal immunofluorescence was used to detect the expression of MDM4 and relationship with USP2a and p53.Results: In the U87MG cell line, MDM4 could combined with USP2a in the cytoplasm, MDM4 combined with p53Ser46~P in the mitochondria; MDM4 was co-localized with p53Ser46~P, USP2a. Conclusion: USP2a and MDM4 could interact with each other, and MDM4 and p53Ser46~P could also interact with each other in the U87MG cell line.Part III Study on effects of MDM4 on apoptosis of U87MG and mechanism of itObjective: To investigate effects of MDM4 on apoptosis of U87MG and to explore the mechanism of regulating tumors.Methods: In vitro cell lines, to observe the relationship between MDM4 and the various proteins on the apoptosis pathway using lentiviral interference and overexpression of relevant target.Results: 1. Under the conditions of UV induced U87MG cells apoptosis, after overexpression of USP2a, apoptosis increased significantly; after USP2a knocked down, apoptosis decreased; p53 knocked down can block the effect of it. 2. Under condition of normal growth of U87MG cells, after overexpression of USP2a, the expression of MDM4 protein increased significantly; after USP2a knocked down, the expression of MDM4 protein decreased. 3. Under condition of normal growth of U87MG cells, after overexpression of USP2a , the expression of p53 and p53Ser46~P proteins in the mitochondria increased; MDM4 knocked down can block the effect of it,but not p21. 4. Under the condition of UV induced U87MG cells apoptosis, after USP2a knocked down, the expression of Bcl-2 protein increased and the expression of Caspase3 protein decreased; Overexpression of MDM4 can block the effect of it. 5. Under the condition of UV induced U87MG cells apoptosis, after overexpression of MDM4, apoptosis increased significantly; after MDM4 knocked down, apoptosis decreased; p53 knocked down can block the effect of it. 6. Under the condition of UV induced U87MG cells apoptosis, after USP2a knocked down, the expression of p53, p53Ser46~P and Cytochrome C proteins in the mitochondria increased and the expression of Cytochrome C protein which was outside of mitochondria decreased.7. Under the condition of UV induced U87MG cells apoptosis, after p21 knocked down, apoptosis did not change significantly. 8. Under the condition of UV induced U87MG cells apoptosis, after overexpression of MDM4 , the expression of Bcl-2 protein decreased and the expression of Caspase3 protein increased; p53 knocked down can block the effect of it,but not p21. After Overexpression of MDM2, the expression of Bcl-2 protein and Caspase3 protein did not change significantly.Conclusion: Under the condition of UV induced U87MG cells apoptosis, MDM4 could promote apoptosis by its effects on p53 mitochondrial pathway; USP2a could promote stability of MDM4. |
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