MAGE-D4 Influence On Malignant Behavior And Its Related Machanism In Glioblastoma

BackgroundGlioblastoma multiforme(GBM)is a World Health Organization grade IV malignant astrocytoma categorized clinicopathologically into primary GBM,which arises de novo,and secondary GBM,which arises from lower grade glioma(grade Ⅱ and Ⅲ glioma).Glioblastomas are highly aggressive brain tumors.The current standard of care combines maximal surgical resection,followed by radiotherapy with concomitant and adjuvant chemotherapy.Despite this multimodal approach,median survival is limited to 6 months.Up to now,it is not clear for the molecular mechanisms of the invasion and metastasis for GBM.The identification of the molecular mechanisms of the invasion and metastasis for GBM Suggest that it may be an ideal molecule to target for the treatment of GBM.Melanoma-associated antigen(MAGE)gene,is come from melanoma cell,as a antigen which can identify the tumor-specific antigen.and then,it found melanoma and glial tumors are all derived from neural ectoderm.In addition,a growing body of evidence has shown that MAGE was specially expressed in the glioma.In recent years,MAGE-D4 is a newly discovered tumor specific antigen,it plays an important role in invasion and metastasis.In previously studies,we examed the expression of MAGE-D4 at mRNA and protein levelswhich were detected by qRT-PCR and immunohistochemical techniques in glioma tissue and a varety of normal tissures thought the tissue microarray.we found that MAGE-D4 protein has relatively highly expression in glioma,but limited lower or negatively expression in normal tissue.Many studies show that MAGED4B can drive oral cance and breast cancer cell progression and metastasis,but the progression and metastasis mechanisms of MAGED4B for GBM is unclear.So,to explore the function of MAGE-D4 in invasion and metastasis for GBM,which is important that deeper our understanding of the molecular mechanisms of glioblastoma.MAGE-D4 may thus represent a valuable prognostic biomarker for GBM and an attractive target for cancer therapy.Part 1 The establishment of stably infected glioblastoma cell linesObjectives:Lentiviral vectors with knock-down MAGE-D4 and Lentiviral vectors with overexpression MAGE-D4 were estableshed.After glioblastoma cell infecting,MAGE-D4 stably silenced or overexpressed glioblastoma cell lines were established.Methods:1.The experiments of knock-down MAGE-D4:(1)Design and synthesis of MAGE-D4 shRNA,establesh the lentiviral vectors of MAGE-D4 shRNA(pGLV3-shRNA-MAGED4).(2)The high expression of MAGE-D4 glioma cell lines(U87-MG,U251-MG)were selected.After antibiotic resistant screening,MAGE-D4 stably silenced glioblastoma cell lines were established,Identified the interference efficiency of pGLV3-shRNA-MAGED4 by qRT-PCR and Western blot.According to the screening of interference efficiency,choosed the high interference efficiency sequence as the purpose sequence pGLV3 shRNA--MAGED4.2.The experiments of overexpression MAGE-D4:(1)The open reading frame of MAGE-D4 gene was amplified by RT-PCR.(2)Lentiviral vectors with overexpression MAGE-D4(LV5-MAGED4)were estableshed.(3)The low expression of MAGE-D4 glioma cell lines(SHG44 和 A172)were infected with LV5-MAGED4,After antibiotic resistant screening,MAGE-D4 stably overexpression glioblastoma cell lines were established;Identified the expression of MAGE-D4 by qRT-PCR and Western blot.Results:The down-regulation of MAGE-D4 for U87-MG and U251-MG was through pGLV3-shRNA-MAGED4.The results of qRT-PCR showed that the interference efficiency for U87-MG and U251-MG was 93.57%and 90.46%,respectively;The results of Western blot showed that MAGE-D4 protein expression level was decreased obviously,the protein expression of U87-MG and U251-MG was 69.77%and 70.11%,respectively.The low expression of MAGE-D4 glioma cell lines(SHG44 和 A172)were infected with LV5-MAGED4,The results of qRT-PCR showed that the mRNA level of MAGE-D4 was increased,U87-MG and U251-MG was 7.21 times and 5.24 times,respectively;The results of Western blot showed that MAGE-D4 protein expression level was increased obviously,the protein expression of U87-MG and U251-MG was 7.21 times and 5.24 times r,respectively.To take U87-MG,U251-MG and SHG44,A172 as parent cell lines,successful build stable silenced and overexpressed MAGE-D4 protein of glioblastoma cell lines.Conclusion:MAGE-D4 stably silenced glioblastoma cell lines were established(U87-MG 和 U251-MG),meanwhile,MAGE-D4 stably overexpressed glioblastoma cell lines were established(SHG44和A172).Part 2 The vitro experiment of influence of MAGE-D4 on malignant biological behaviors of glioblastoma multiforme cellObjectives:To observe the effect of MAGE-D4 in GBM cell proliferation,migration and invasion;To explore mechanisms of MAGE-D4 effecting on GBM cells;Preliminary understanding the role of MAGE-D4 in the development of tumor.To provide a basis for evaluating the possibility of diagnosis and targeted therapy for tumor.Methods:MAGE-D4 stably silenced and overexpressed GBM cells were used to the following experiments:(1)CCK8 assay was used to observe cell proliferation;(2)colony formation assay was used to observe situations of cell proliferation and colony formation;(3)Flow cytometry was used to test the cycle arrest and apoptosis for each type of cell;(4)Wounad-healing assay was used to observe the migration capabilities.meantime,Transwell migration and invasion models were allied to determine the migration and invasion capabilities of cells;(5)Western blot was used to detect expression of proteins related with proliferation,cycle,apoptosis,migration and invasion P53 pathways.Results:The results of knock-down MAGE-D4 experiments:(1)The CCK8 assay showed that Proliferation of MAGE-D4 silenced(U251-MG and U87-MG)cells were significantly decreased compared with control cells(p<0.001);(2)The colony formation assay showed that proliferation and colony formation ability of MAGE-D4 silenced(U251-MG and U87-MG)cells were significantly decreased compared with control cells(p<0.001);(3)The result of flow cytometry show that MAGE-D4 can arrest proliferation at S and G2M phases in MAGE-D4 silenced U251-MG cells,However,there was no significant change in U87-MG cell lines.The protein expressions of CyclinE and CDK2 of MAGE-D4 silenced cells(U251-MG)were significantly increased compared with control cells by westernblot,However,there was no significant change with the protein expressions of CyclinA and p21.The apoptosis result of MAGE-D4 stably silenced cell lines show that the apoptosis rate for U251-MG increased compared with control cells(p<0.001),Westernblot detection protein expression MAGE-D4 stably silenced cell lines U251-MG increased for Casepase-3,unchanged for Casepase-9 and increased for p53 compared with control cells;However,there was no significant change with the apoptosis of U87-MG,Westernblot detected the protein expression MAGE-D4 U87-MG cell lines decreased for Casepase-3,unchanged for Casepase-9 and unchanged for p53 compared with control cells:(4)Migration and invasion of MAGE-D4 silenced cells were significantly decreased compared with control cells(p<0.001),Westernblot detect MMP-2 and MMP-9 decreased.The results of overexpression MAGE-D4 experiments:(5)The CCK8 assay showed that Proliferation of MAGE-D4 overexpressed(SHG44 and A172)cells were significantly increased compared with control cells(p<0.001);(6)The colony formation assay showed that proliferation and colony formation ability of MAGE-D4 overexpressed(SHG44 and A172)cells were significantly increased compared with control cells(p<0.001);(7)The result of flow cytometry show that MAGE-D4 overexpression didn’t induced cell cycle arrest in SHG44 and A172,Westernblot showed that the protein expression of p21 has no obvious changed;The apoptosis result of MAGE-D4 stably overexpressed cell lines(SHG44 and A172)show that the apoptosis rate has no obvious change compared with control cells,There was no significant change with the protein expressions of Casepase-3,Casepase-9 and p53 by Westernblot.(8)Migration and invasion of MAGE-D4 overexpressed cells were significantly increased compared with control cells(p<0.001),Westernblot detect that MMP-2 and MPP-9 has no changed.Conclusion:MAGE-D4 silence can inhibite proliferation of GBM cell;MAGE-D4 silence can arrest proliferation at S and G2M phases in U251-MG cells,increase the apoptosis rate of U251-MG cells,However,there was no significant changed in the cell cycle and apoptosis of U87-MG;MAGE-D4 silence can inhibite Migration and invasion of GBM cell.MAGE-D4 overexpression can accelerate proliferation of GBM cell;However,MAGE-D4 overexpression can not affect the cell cycle and apoptosis of GBM cell;MAGE-D4 overexpression can accelerate Migration and invasion of GBM cell.Part 3 Influence on biological behaviors of Glioblastoma multiforme cell for MAGE-D4 in vivo experimentObjectives:To validate the impact of MAGE-D4 on growth of implanted GBM cell in nude mice.Methods:GBM Cells(U251-MG)were injected in the subcutaneous tissue of nude mice.Subcutaneous xenograft transplantation tumor model of GBM was established in BALB/C nude female mice.The scrambled sequence group and purposed sequence group were injected in the subcutaneous tissue,respectively.Each group with six nude mice;2.Four weeks later,the mice were sacrificed,the GBM tumor volumes of tumor were calculated,immunohistochemical staining was used to verify the result of experiment in vivo.Results:In GBM subcutaneous transplantation tumor model in nude mice,the growth of subcutaneous implanted tumors in the MAGE-D4 stably silenced group(U251-MG)was significantly decreased in contrast to the control group(p<0.001).Conclusion:MAGE-D4 silenced can inhibite the formation of subcutaneous transplantation tumor.