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Objective To investigate the effect of IKKε knock-down on the biological characteristics of U251 glioblastoma cells. Methods IKKε small interfering RNA (IKKε siRNA) mediated by lipofectamine were transfected into U251 cells while cells transfected with scrambled siRNA and control cells were prepared.RT-PCR was employed to detect expressions of IKKε in the transfected cells.The cell proliferation was determined by MTT assay.Flow cytometry was used to monitor changes in cell cycle.Cell invasion was evaluated by Transwell assay.Moreover,the proliferating cell nuclear antigen (PCNA),cyclin D 1 and matrix metalloproteinase-9 (MMP9) that regulated proliferation,invasion and cycle progression of the transfected cells and the changes of NF-κB after transfection were examined by Western blotting. Results RT-PCR revealed that the proliferation of U251 cells was inhibited,the cell cycle was arrested in G0/G1 phase and the invasive activity was attenuated in cells transfected with IKKe siRNA,with significant differences as compared with the cells transfected with scrambled siRNA and control cells (P＜0.05).The expressions ofPCNA,MMP9 and cyclin D1 were down-regulated in the IKKε knock-down cells, as compared with the other 2 groups. In addtion, transposition of NF-κB reduced from the cytoplasm to the nucleus after transfection. Conclusion As IKKε plays a vital role in proliferation and invasion of glioma cells,it may serve as a potential target ofgene therapy for glioma.
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Objective To study the inducement of U251 glioblastoma cell apoptosis in vivo through up-regulating PUMA expresion and knocking down miR-221/222, and explore its mechanism.Methods Nude mouse xenograft models were established in 5-week-old BALB/c nude mice by subcutaneous vaccination of U251 glioblastomas; 1 week later, they were treated with intratumoral injection of lipofcctamine-mediated miRNA-221/222 antisense oligonucleotides (GroupA), nonsense sequences (Group B) and controls (Group C),respectively (n=8).The tumor growth was monitored until the end of observation period (28 d after the treatment) and pathological changes of the glioblastoma tissues were observed by HE staining at the end of observation.Fluorescence in situ hybridization (FISH) and real-time PCR were employed to measure the miR-221 and miR-222 expressions. Terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end labeling (TUNEL) assay was used to detect the apoptosis of glioblastomas.Immunohistochemistry and Westem blotting were used to detect the expressions of PUMA,bax,bcl-2 and p53 in removed tumor specimens. Results The volume in Group A was significantly smaller than that of those in group B and group C 6-28 dater treatment (P=0.006). The miR-221 and miR-222 mRNA expressions in Group A were significantly decreased as compared with those of those in group B and group C.HE staining indicated that decreased heteromorphism and reduced number of new vessels in Group A were noted as compared with those in group B and group C.The cell apoptotic index in Group A was significantly higher than that in group B and group C (P＜0.05).Immunohistochemistry showed that the expression levels of PUMA and bax in Group A was significantly up-regulated as compared with those in group B and group C, while the expression of bcl-2 in Group A was significantly down-regulated as compared with that in group B and group C; and no significant changes were noted in the p53 expression. Conclusion By up-regulating PUMA expresion,knocking down miR-221/222 can induce U251 glioma apoptosis in vivo.
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objective To investigate the effects of knocking down of miR-19a and miR-19b on the biological characteristics of SNB19 glioblastoma cells. Methods Oligonucleotides inhibitor of miR-19a and miR-19b (miR-19a inhibitor or miR-19b inhibitor) mediated by lipofectamine2000 were transfected to SNB19 cells to knock down miR-19a and miR-19b; control group (without transfection),group D (performing transfection with nonsense sequence) and group E (performing transfection with both miR-19a inhibitor and miR-19b inhibitor) were established. Real time PCR was conducted to detect the expressions ofmiR-19a and miR-19b in these groups after the transfection. The cell proliferation rate and cell cycle kinetics were detected by 3-(4, 5-Dime- -thylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay and flow cytometry, respectively; the cell invasive ability was evaluated by Transwell assay.Results As compared with those in control group and group D, the expressions of miR-19a and miR-19b, proliferation activity and invasive ability of cells in the miR-19a/19b inhibitor transfected cells (group A/B) were significantly reduced (P＜0.05). The expressions of miR-19a and miR-19b and the proliferation activity and invasive ability of cells 2, 3, 4 and 5 d after the transfection in group E were significantly reduced as compared with those in group A/B (P＜0.05). Delayed cell cycle in group A/B and group E was noted as compared with that in control group and group D; and group E enjoyed more obviously delayed eell cycle than group A/B (P＜0.05). Conclusion MiR-19a and miR-19b might be oncomiRs, and may be candidate target miRNAs for gene therapy of glioma.
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Objective To investigate the suppressing effects of RNA interference (RNAi)targeting AKT1 and phosphatidylinositol 3-kinase p85 (PI3KP85) on the invasion ability of malignant glioma U251 cells in vitro.Methods Normal control group,negative control group (U251 cells being transfected by nonsense sequence of adenovirus) and gene treatment group were chosen in the experiment.A recombinant rctrovims expressing short interference RNA (siRNA) sequence targeting AKTi and PI3KP85 genes was established and transfected into the U251 cells in the gene treatment group.The silencing effect of RNAi on AKTI and PI3KP85 expressions was identified by real time PCR and Westem blotting,respectively.Western blotting was also employed to analyze the expression of some functional proteins.Enzyme-linked immuno sorbent assay (ELISA) was used to detect the changes of concentration of ectocytic matrix metalloproteinases 2 (MMP2) and MMPg.The invasion ability of U251 cells in the 3 groups was evaluated by scarification and Transwell assay.Results The expressions of AKT1 and PI3KP85 in U251 cells in the gene treatment group were dramatically down-regulated.As compared with the normal control and negative control groups,the gene treatment group showed significantly lower expression level of MMP2 and MMP9(P<0.05);meanwhile tissue inhibitor of matrix metalloproteinase-2(TIMP2)in the gene treatment group was significantly up-regulated.ELISA also indicated obvious changes of concentration of ectocytic MMP2 and MMP9.The scarification and Transwell assay showed that the invasion ability in the gene treatment group was significantly decreased as compared with that in the other 2 groups (P<0.05).Conclusion RNAi targeting AKT1 and PI3KP85 can significantly down-regulate the expressions of AKT1 and PI3KP85 and decrease the invasion ability of U251 cells in vitro.
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Objective To study the mechanism of miR-21 in regulating the invasion of human glioblastoma (GBM) cells in vitro. Methods The transfection reagent oligofectamine was mixed with antisense miRNA-21 (AS-miR-21) and nonsense oligodeoxyribonucleotides (ODN), respectively, and then, they were added into the medium of U251 GBM cell line as AS-miR-21 treatment group and nonsense ODN treatment group, respectively; control group (treated with PBS) was also established.MiR-21 luciferase reporter assay was used to detect the miR-21 knocking down effect. Matrigel cell growth assay and Transwell assay were used to determine the invasion and migration abilities of U251 cells. Western blotting was employed to test the expressions of invasion-related proteins (FAK,MMP-9/2, TIMP-1 and Tubulin-α); immunofluorescence was also employed to observe the morphology of Tubulin-α protein in GBM cells. Results Luciferase intensity in as-miR-21 treated U251 cells was significantly suppressed as compared with that in the control group and nonsense ODN treatment group (P＜0.05). The diameter of cultured clone in as-miR-21 treated U251 cells was smaller than that in the controls and nonsense ODN treatment group (F=102.819, P=0.000). Decreased cells via the transwell member in thc AS-miR-21 treatment group were detected as compared with those in the controls and cnonsensc ODN treatment group (F=243.465, P=0.000). The expressions of FAK, MMP-2/9 weredown-regulated and that of TIMP-1 was up-regulated in the AS-miR-21 treated tumor cells as compared with the other 2 groups (P＜0.05). No obvious changes were noted on the expression of Tubulin α,however, the morphology of Tubulin α protein in the AS-miR-21 treatment group changed. Conclusion High expression of miR-21 induce the ability of U251 GBM cell invasion and miR-21 can be taken as a candidate for gene therapy of human glioma.
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<p><b>OBJECTIVE</b>To study the effect of silencing Dicer by small interference RNA (siRNA) to suppress the global microRNA (miRNAs) expression on the biological characteristics of TJ905 glioblastoma cells.</p><p><b>METHODS</b>The silencing effect of RNA interference on Dicer expression was evaluated by reverse transcription-polymerase chain reaction (RT-PCR), Western blot analysis and immunofluorescence staining. The cell proliferation rate and cell cycle kinetics were detected by MTT assay and flow cytometry respectively, and the cell invasive ability was evaluated by transwell assay.</p><p><b>RESULTS</b>The siRNA targeting Dicer suppressed the expression of Dicer in TJ905 cells. Meanwhile, the proliferation activity and invasive ability were significantly enhanced in cells transfected with Dicer siRNA compared to those cells transfected with scrambled siRNA and the control cells.</p><p><b>CONCLUSION</b>Suppression of Dicer expression renders the glioma cells harboring more aggressive phenotype. This preliminary finding suggests that global lower expression of miRNAs may play an oncogenic role.</p>
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Humans , Cell Cycle , Cell Line, Tumor , Cell Proliferation , DEAD-box RNA Helicases , Genetics , Metabolism , Gene Expression Regulation, Neoplastic , Gene Silencing , Glioblastoma , Genetics , Metabolism , RNA, Small Interfering , Genetics , Metabolism , Ribonuclease III , Genetics , MetabolismРеферат
<p><b>OBJECTIVE</b>To study the inhibitory effect of knocking down microRNA(miR)-221 and miR-222 on human glioma cell growth and its possible mechanism.</p><p><b>METHODS</b>miRNA-221/222 antisense oligonucleotides (antisense miR221/222) were transfected into human glioma U251 cells by lipofectamine. Northern blot analysis was conducted to detect the mRNA expression of miR-221/222 in the control and transfected cell groups. The proliferation activity of cells was determined by MTT assay. Cell invasion ability was examined by transwell assay, and cell cycle kinetics and apoptosis were detected with flow cytometry. The expression of relevant proteins was analyzed by Western blotting. The therapeutic efficacy of antisense miR221/222 on the growth of xenograft tumors in nude mice were also observed.</p><p><b>RESULTS</b>In the antisense miR-221/222-transfected cells, the expression of miR-221/222 was significantly reduced; the cell invasion ability was suppressed, cell cycle was blocked at G(0)/G(1) phase, and apoptotic cells were increased. The growth of xenograft tumors treated with antisense miR-221/222 was also inhibited. In antisense miR-221/222 treated tumor cells, the expression of bcl-2 was down-regulated while connexin43, p27, PUMA, caspase-3, PTEN, TIMP3 and Bax up-regulated, and p53 expression not changed.</p><p><b>CONCLUSION</b>There is a significant inhibitory effect of antisense miR-221/222 on the growth of human glioma U251 cells. miR-221/222 may be considered as a candidate target for gene therapy of human gliomas.</p>
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Animals , Humans , Mice , Apoptosis , Base Sequence , Caspase 3 , Metabolism , Cell Cycle , Cell Line, Tumor , Cell Proliferation , Down-Regulation , Gene Expression Regulation, Neoplastic , Gene Knockdown Techniques , Genetic Therapy , Glioma , Metabolism , Pathology , Ki-67 Antigen , Metabolism , Mice, Inbred BALB C , Mice, Nude , MicroRNAs , Genetics , Molecular Sequence Data , Neoplasm Transplantation , Oligonucleotides, Antisense , Pharmacology , PTEN Phosphohydrolase , Metabolism , Proto-Oncogene Proteins c-bcl-2 , Metabolism , RNA, Messenger , Metabolism , Tissue Inhibitor of Metalloproteinase-3 , Metabolism , TransfectionРеферат
<p><b>OBJECTIVE</b>To study the anti-invasion effect of SEPT7 gene on U251MG glioma cells and its possible molecular mechanism.</p><p><b>METHODS</b>Recombinant adenovirus vector carrying SEPT7 gene (rAd5-SEPT7) was transduced to human glioma cell line U251MG, and empty adenovirus vector was used as control. Tumor invasion was examined by Transwell method and 3 D-Matrigel assay, and tumor cell migration by wound-healing method and 2 D-Matrigel assay. Three major molecular events associated with cell motility and migration, including changes of expression in MMP2, MMP9, MT1-MMP, TIMP1 and TIMP2, the alteration of integrin alpha(v)beta(3) expression, and the structural change of cytoskeleton protein, tubulin-alpha, in U251 cells transduced with rAd5-SEPT7 were studied by Western blotting, immunofluorescence and laser scanning confocal microscope, respectively.</p><p><b>RESULTS</b>The invasive and migratory capabilities of cells transduced with rAd5-SEPT7 were inhibited. The expression of extracellular matrix metalloproteinases MMP-2, MMP-9, MT1-MMP and integrin alpha(v)beta(3) was significantly decreased, while the expression of matrix metalloproteinase inhibitor TIMP1, TIMP2 was upregulated. Intracellular cytoskeleton protein-tubulin-alpha in U251 cells exhibited prominent morphological changes which including the appearance of distortion and aggregation resulting from redistribution of tubulin-alpha, and this feature of alteration was similar to the tubulin-alpha structure in normal non-tumor cells.</p><p><b>CONCLUSION</b>SEPT7 gene can inhibit the invasion and migration ability of U251 glioma cells. Its molecular mechanism may include that SEPT7 gene reverses the imbalanced state of MMPs/TIMPs, downregulates the expression of integrin alpha(v)beta(3) and alters the structure of tubulin-alpha of U251MG glioma cells. It is suggested that SEPT7 gene could be a good candidate for gene therapy of gliomas.</p>
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Humans , Adenoviridae , Genetics , Blotting, Western , Cell Cycle Proteins , Genetics , Physiology , Cell Line, Tumor , Cell Movement , Genetics , Genetic Vectors , Genetics , Glioma , Metabolism , Pathology , Integrin alphaVbeta3 , Metabolism , Matrix Metalloproteinase 14 , Metabolism , Matrix Metalloproteinase 2 , Metabolism , Matrix Metalloproteinase 9 , Metabolism , Microscopy, Confocal , Neoplasm Invasiveness , Genetics , Septins , Tissue Inhibitor of Metalloproteinase-1 , Metabolism , Tissue Inhibitor of Metalloproteinase-2 , MetabolismРеферат
<p><b>OBJECTIVE</b>To detect the differential expression of Notch1 and Notch2 in human astrocytoma and medulloblastoma; and to study the role of Notch1 and Notch2 in the development of both tumors.</p><p><b>METHODS</b>Immunohistochemical staining (SP method) and Western blot analysis were used to detect Notch1 and Notch2 expression in tissue arrays and freshly resected samples of normal brain tissue, astrocytoma and medulloblastoma.</p><p><b>RESULTS</b>Notch1 and Notch2 were negative in normal human brain tissue. Notch1 was highly expressed (total positive rate 80.0%, 48/60) while Notch2 was not detected in grade IV astrocytomas and sporadically observed in lower grade astrocytomas (total positive rate 10.0%, 6/60). The percentage of positive tumor cells and expression level of Notch1 increased with higher histologic grade (r = 0.859, P < 0.05). On the other hand, overexpression of Notch2 was detected in medulloblastoma (9/10) in contrast with lower expression of Notch1 (2/10).</p><p><b>CONCLUSIONS</b>Notch1 and Notch2 show differential expression in astrocytoma and medulloblastoma. This may be related to their different functional activities during the process of brain development.</p>
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Adolescent , Adult , Aged , Child , Child, Preschool , Female , Humans , Male , Middle Aged , Young Adult , Astrocytoma , Metabolism , Biomarkers, Tumor , Metabolism , Brain , Metabolism , Brain Neoplasms , Metabolism , Gene Expression Regulation, Neoplastic , Medulloblastoma , Metabolism , Receptor, Notch1 , Metabolism , Physiology , Receptor, Notch2 , Metabolism , PhysiologyРеферат
Objective To study the suppressive effect of knockdown of miR-21 on the U87 human giioma xenograft growth and the possible mechanism. Methods Nude mice bearing U87 human glioblastoma subcutaneously were treated with miRNA-21 anfisense oligonucleotides(AS-miR-21)intratumomlly every 3 d until the observation peded ended.The tumor volume of the mice treated withAS-miR-21 was measured regularly as compared with that in the control untreated mice and in the mice treated with scramble oligonucelotides(ODN).Finally,the tumors were removed from nude mice for the examination.In-sire hybridization and real-time PCR were conducted to detect the miRNA expression of miR-21.The biological charaetedsties of the tumors were evaluated by HE and immunohistochemieal staining, and the cell apoptosis was detected by TUNEL method. Resulls During the observation period,the tumor growth was delayed and the final tumor volume of AS-miR-21 heated group was smaller than that in the control and scramble ODN treatedg roup(F=6-056,P=0.007).The expression of miRNA precursor was knocked down in As-miRNA treated tunlors compared with that in untreated or scramble ODN treated tumors.Histopathological examination exhibited the appearance of degraded malignancy.The expressions of PCNA and MMP-9 were down-regulated while Septin-7 and P21 were up-regulated and apoptotic index was increased significantly (F=141.021,P=000) as well.Conclusion The suppressive effect of anti-miR-21 ODNs on the growth of U87 human glioma xenogratts is significant and miR-21 Call be taken as a candidate for gene therapy ofhuman glioma.
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<p><b>OBJECTIVE</b>To investigate the influence of SEPT7 on biological characters of gliomas cells TJ905.</p><p><b>METHODS</b>Recombinant SEPT7 constructs was transfected to human glioblastoma cell line TJ905 in which SEPT7 expression is absent. The positive clones were identified by RT-PCR and Western blot analysis. The cell proliferation was determined by MTT assay and flow cytometry, cell apoptosis was detected with Annexin V staining and cell invasion was evaluated by motility in three-dimensional culture. Moreover, the molecules regulating the cell cycle progression were examined by immunofluorescence staining and Western blot analysis.</p><p><b>RESULTS</b>When SEPT7 was successfully transfected to TJ905 cells, the cell proliferation activity of TJ905 cell was inhibited, the cell cycle was arrested in G0/G1 phase and S phase fraction (SPF) was lowered, the positive regulatory molecules for cell cycle progression including cyclin D1, CDk4, cyclin E and CDk2 were downregulated while the negative modulators including p16 and p21 were upregulated, apoptotic cells were increased and cell invasive ability was attenuated.</p><p><b>CONCLUSIONS</b>Transfection of SEPT7 construct into the glioma cells TJ905 is able to inhibit the proliferation activity and invasive ability of TJ905 cell and to induce cell apoptosis. These results revealed that SEPT7 exerted the suppressive effect on the glioma cell growth and invasion, and induced apoptosis, and suggested that SEPT7 as a gene of glioma suppressor.</p>
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Humans , Apoptosis , Blotting, Western , Brain Neoplasms , Genetics , Metabolism , Pathology , Cell Cycle , Cell Cycle Proteins , Genetics , Metabolism , Physiology , Cell Line, Tumor , Cell Proliferation , Cell Survival , Flow Cytometry , Fluorescent Antibody Technique , Glioma , Genetics , Metabolism , Pathology , Reverse Transcriptase Polymerase Chain Reaction , Septins , TransfectionРеферат
<p><b>OBJECTIVE</b>To study further the most important and frequent genetic alterations of p53 and epidermal growth factor receptor (EGFR) in astrocytic gliomas.</p><p><b>METHODS</b>(1) EGFR expression was examined in samples collected from 37 astrocytic gliomas and 6 normal brain tissue using reverse transcriptase polymerase chain reaction and immunohistochemical staining. (2) p53 gene mutation and accumulation were detected simultaneously in the same specimens using PCR-SSCP, DNA sequencing and immunohistochemical staining.</p><p><b>RESULTS</b>The frequency of p53 mutation in diffuse astrocytomas, anaplastic astrocytomas, primary glioblastomas and secondary glioblastomas was 1/10, 4/19 (21.1%), 4/6 and 2/2, respectively and the frequency of EGFR overexpression was 5/10, 10/19 (52.6%), 5/6 and 2/2, respectively. Both p53 accumulation and EGFR overexpression increased accompanied by a successive increase of degree of the glioma malignancy.</p><p><b>CONCLUSIONS</b>EGFR overexpression is not infrequently seen, however, p53 mutation is rarely seen in the low grade gliomas. Both p53 gene mutation and EGFR overexpression are often associated with primary and secondary glioblastoma. Consequently, EGFR overexpression and p53 gene mutation are not mutually exclusive in astrocytic gliomagenesis but synergistically to promote the glioma progression.</p>
Тема - темы
Adult , Aged , Female , Humans , Male , Middle Aged , Astrocytoma , Genetics , Metabolism , Pathology , Base Sequence , Brain Neoplasms , Genetics , Metabolism , Pathology , DNA Mutational Analysis , Gene Expression Regulation, Neoplastic , Glioblastoma , Genetics , Metabolism , Pathology , Immunohistochemistry , Molecular Sequence Data , Mutation , Polymerase Chain Reaction , Polymorphism, Single-Stranded Conformational , RNA, Messenger , Genetics , ErbB Receptors , Genetics , Reverse Transcriptase Polymerase Chain Reaction , Tumor Suppressor Protein p53 , GeneticsРеферат
<p><b>OBJECTIVE</b>To investigate the differential gene expression of ependymomas.</p><p><b>METHODS</b>Four fresh samples of ependymomas and 1 of normal brain tissue were collected during operation. The extracted total RNAs were converted as (32)P tagged cDNA probes, which were then hybridized with the Atlas Human Cancer Array, producing the array based hybridization maps following the protocol provided with the kit. A set of special software was applied to the analysis and RT-PCR was performed to test the result.</p><p><b>RESULT</b>In comparison with the normal brain tissue, there were 31 upregulated gene and 1 downregulated gene in ependymomas, most of which were firstly found to be differentially expressed in this kind of tumor.</p><p><b>CONCLUSION</b>The discrepancy of gene expression profiles between ependymomas and normal brain tissues is highly put through and effectively detected with cDNA array, which provides new information for the further research on the molecular mechanisms of this lesion.</p>
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Humans , Brain , Metabolism , Brain Neoplasms , Genetics , Ependymoma , Genetics , Gene Expression Profiling , Oligonucleotide Array Sequence Analysis , Reverse Transcriptase Polymerase Chain ReactionРеферат
<p><b>OBJECTIVE</b>To study the mechanism involved in the control of glioma cell proliferation with transfection of connexin (Cx) 43 gene.</p><p><b>METHODS</b>C6 rat glioma and TJ905 human glioblastoma cell lines without Cx43 gene expression were transfected with Cx43cDNA mediated by lipofectamine. Northern blot, in situ hybridization and immunohistochemical technology were used to detect the expression of Cx43mRNA and its protein with MTT assay and silver colloid stain for the detection of cell proliferation, TUNEL method for determination of cell apoptosis, scrape loading dye transfer (SLDT) for GJIC, Western blot and immunohistochemical technology for bFGF, PDGF, EGFR, IGF-I and IGFBP3 expression.</p><p><b>RESULTS</b>Cx 43 gene transfected glioma cells showed decreased proliferation, restored GJIC and decreased bFGF, PDGF, IGFBP3, except EGFR expression and cell apoptosis which showed no change.</p><p><b>CONCLUSION</b>The mechanism of Cx 43 gene inhibiting gliomas cell proliferation is the restoration of GJIC and decreased autocrine growth factors.</p>