| Backgroud and ObjectiveBrain glioma is the most common one of primary intracranial tumor.Despite the significant advances in neurosurgical techniques and oncology treatment regimens,the prognosis of patients with brain malignancies remains dismal.It is urgent to develop a new treatment for such malignancy.As the rapid progresses occur in the fields of molecular biology,immunology and cellular biology,the biological treatment,mainly including gene therapy and induced-differentiation,has become a brand new method of the treatment of brain glioma.The dysregulation of signaling pathway is associated with glioma tumorigenesis. As an important signaling transducer and activator of transcription, STAT3 and its constitutive activation (pSTAT3) expression were observed in a variety of human glioma. pSTAT3 is required for maintenance of malignancy to human glioma and result in oncogenic transformation of cell line and induces expression of genes against apoptosis and promotes proliferation of cells. GRIM-19 (Genes associated with Retinoid–IFN-induced Mortality-19) was originally identified as an interferon-βand retinoic acid (RA)-inducible gene with apoptotic effects in human cancer cell lines. Most recently, as a tumor suppressor to inhibit cell transformation induced by constitutively activated Stat3, It was found that GRIM-19 was up-regulated in induced-differentiation of glioma cells.In this study, a recombinant adenovirus carrying GRIM19 gene Ad-GRIM19 was constructed, packaged and purified. To investigate effects of GRIM19 espression on induced differentiation of human glioma CHG-5 cells, the changes of cell morpH2Ology were observed,and apoptosis and proliferation, cell adhesion, invasion after infection of Ad-GRIM19 were investigated,and its mechanism were also explored.Methods1. AdEasy-1 System was used to generate the adenoviral pAd-GRIM19 plasmid.The human GRIM19 gene was subcloned to adenovial shuttle vector pAdTrack-CMV from pCDNA3.1 (+)-GRIM19 plasmid .Then the resultant pAdTrack-CMV-GRIM19 was transformed into BJ5183 baeteria with the Plasmid AdEasy-1. The adenoviral Plasmid pAd-GRIM19 was obtained with H2Omologous recombinant in E.coli BJ5183.2. The recombinant replication-deficient adenovirus Ad-GRIM19 and Ad-GFP control were packaged in 293T cells by transfection with lipofectamin 2000, further confirmed by PCR and purified and isolated using CsCl2.The infective efficiency and virus titer were test with GFP expression and TCID50 metH2Od. its MOI to CHG-5 cells were detected by GFP expression. Western Blot was used to analyze the GRIM19 expression.3. The glioma CHG-5 cells were infected with Ad-GRIM19 and Ad-GFP control respectively. Experimental glioma cells were devided into three groups including non-infected, Ad-GFP-infected and Ad-GRIM19- infected cells. Cell MorpH2Ology were observed by fluorescence microscope after infection at 24h,48h and 72h. Cell Viability was measured Typan Blue Staining.The proliferation were surveyed by cell counting kit and soft agar colony formation assay. The adhesion assay was used to detect cell adhesion ability,and the cell invasion to ECM was assessed by Transwell chambers. Cell apoptosis was detected by TUNEL assay and H2Ochest 33342 stainning.4. Expression of pSTAT3, GFAP and Vimentin in CHG-5 cells were first detected by Immnol Fluorence staining before adenovirus infection. The expression of Bcl-2, Bax, PCNA, C-myc,CCND1,GFAP or STAT3 genes were measured by Real-Time PCR with 2-△△C T metH2Od, and further confirmed by Western blot. CD133 expression was detected by cell Immunohistochemistry. ROS production were examined using DHE Fluorescent Probe.Results1. pAdTrack-CMV-GRIM19 and pAd-GRIM19 are constructed. The recombinant adenovirus carrying human GRIM19 gene and Ad-GFP gene are constructed and its titer was 3.5×1010 PFU/ml and 3.5×1010 PFU/m1. Ad-GRIM19 increases expression of GRIM19 in CHG-5 cells.2. pSTAT3,GFAP and Vimentin proteins were detected in CHG-5 cells. Marked cell morpH2Ological changes are observed in Ad-GRIM19 infected CHG-5 cells. Adenovirus-mediated GRIM19 expression significantly inhibits cell proliferation and colony formation (P<0.01),and reduce abilities of cell adhesion and invision(P<0.01), compared with its controls. Infection of Ad-GRIM19 down-regulates expression of PCNA and cyclin D1 at mRNA and protein, Up-regulation of GRIM19 significantly induces cell apoptosis after Ad-GRIM19 infection.3. Expression of Bcl2 and GFAP protein are increased, and PCNA and cyclin D1 expression are reduced in CHG-5 cells infected with Ad-GRIM19. ROS productions are also up-regulated .of and increase of Bax.Adenovirus-mediated GRIM19 expression significantly. via down-regulation of Real-Time PCR and Western blot resultsConclusions1. pAdTrack-CMV-GRIM19 and pAd-GRIM19 are constructed. The recombinant adenovirus carrying human GRIM19 gene and Ad-GFP gene can infect CHG-5 cells efficiently and effectively.2. Marked changes of cell morphology maybe associated with up-regulation of GFAP. significantly change Cell morpH2Ology, reduce abilities of cell adhesion and invision. Increase of GRIM19 expression Inhibits cell proliferation and colony formation via down-regulation of STAT3,PCNA and cyclin D1, and promotes cell apoptosis via decrease of Bcl2 and increase of Bax.3. Adenovirus-mediated GRIM19 expression increase GFAP expression and promote differentiation of glioma, It maybe associate with ROS production of mitochondria and decrease of pSTAT3 expression in glioma CHG-5 cells.
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