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Objective To investigate the effect of knocking-down Yes-associated protein (YAP)on the biologi-cal characteristics of SNB19 glioblastoma cell.Methods The expression of YAB in SNB19 was knockdown by YAB small interfering RNA (YABsiRNA).The downregulation of YAP expression was identified by Western blot analysis. The proliferative ability of cell was determined by methyl thiazoyl terazolium (MTT).The invasive ability of cell was examined by Transwell assay.Flow cytometry and Annexin V staining were used to detect the cell cycle and apoptosis respectively.The results were analyzed by the statistical software SPSS18.0.Results The expression of YAP in the cells transfected with YAPsiRNA was significantly reduced.The cell proliferation activity of SNB19 cells was inhibited, which decreased from (100.00 ±0.00)%to (52.32 ±3.10)%(F=33.00,P<0.01).The cell cycle was arrested in G0-G1 phase (F=8.76,P<0.01).The cell invasive ability was attenuated apparently,which decreased from (163.20 ±10.10)to (37.71 ±2.52)(F=282.05,P<0.01).The apoptosis ratio of the tumor cell which transfected with YAPsi-RNA was increased from (3.56 ±0.35)%to (18.99 ±0.66)%,(F=931.99,P<0.01).Conclusion Knocking-down YAP expression in glioma cells could inhibit the proliferative activity and invasive ability of SNB19 cell and could induce cell apoptosis.YAP could be served as a potential target for the gene therapy of glioma.
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Objective:This study aimed to explore the effect of Yes-associated protein (YAP) on the growth of the human glioma cell line LN229. Methods:YAPsiRNA was transfected into LN229 cells to knock down the YAP expression. The downregulation of the YAP expression was identified through Western Blot analysis. Colorimetric assay using methyl-thiazolyl-tetrazolium was applied to evaluate cell proliferation ability. Cell invasive activity was examined using Transwell assay. Flow cytometry and AnnexinV were used to detect cell cycle and apoptosis, respectively. The relevant molecules regulating proliferation, invasion, cell cycle progression, and apoptosis were examined through Western Blot analysis. Results:The YAP expression was downregulated after YAPsiRNA was trans-fected into LN229 glioma cells. Reduced YAP expression could arrest the cell cycle at G0/G1 phase, inhibit cell proliferation and inva-sion, and promote apoptosis. The expression of the proliferating cell nuclear antigen (Ki-67), matrix metallopeptidase-9 (MMP-9), cy-clin D1, and Bcl-2 were downregulated. Conclusion:The downregulation of YAP in LN229 cells suppresses cell proliferation and inva-sion, as well as promotes cell apoptosis. This study provides a novel evidence for further study on Hippo-YAP signal pathway in molec-ular pathology of glioma.
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Objective To study the inhibitory effect of knocking down miR-30a-5p on the U87 human glioma xenograft growth and its possible mechanism.Methods Nude mice bearing subcutaneous U87 human glioblastoma were established and separated into three groups (eight for each group) by randomized digital table method,including control group,scr-ODN treated group and AS-miR-30a-5p treated group.After relevant subcutaneous injection treatment,tumor size was measured every other day until the observation period ended.Researchers executed the animals after the treatment,stripped tumor tissues and extracted RNA and protein.Real-time PCR was conducted to detect the expression of miR-30a-5p.The histopathological characteristics and proliferation and apoptosis biological characters (including SEPT7,PCNA,cyclin D1,MMP-2,apoptosis related factor P53,bcl-2 and caspase3) were evaluated by HE and immunohistochemical staining,Westem blot analysis respectively,and the cell apoptosis was detected by TUNEL method.Results In AS-miR-30a-5p treated group,the tumor growth was delayed and the final tumor volume was smaller than that in the control and scr-ODN treated group (F =7.167,P ＜0.05),and the expression of miR-30a-5p was knocked down.The expression of PCNA,cyclin D1 were significantly downregulated while P53,SEPT7 and caspase3 up-regulated.Apoptotic index was increased significantly.Conclusion As-miR-30a-5p suppresses the growth of U87 human gliomas xenografts significantly.Malignant phenotype of tumors are reversed to a considerable degree.Therefore,miR-30a-5p can be a candidate for targeted therapy of human glioma.
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ObjectiveTo confirm the effect of miR-93 inhibitor in glioma cell growth and invasion.MethodsMalignant glioma cells were transfected with miR-93 inhibitor by lipofectamin to downregulate their overexpression of miR-93.Real time-PCR was taken to measure miR-93 expression after transfection.The cell cycle kinetics and cell growth rate were detected by flowcytometry and MTT assay,the cell proliferative ability was evaluated by soft agar assay,and the invasive ability was detected by transwell assay.ResultsThe highlevel expression of miR-93 was downregulated effectively in glioma cells after transfecting the miR-93 inhibitor.Meanwhile,the cell cycle progress was delayed,S phase cells were reduced,the speed of growth was slowed,cloning formation ability was receded,the number of cells through the matrigel was reduced,and invasive ability was significantly repressed.ConclusionDownregulation of miR-93 expression could inhibit the proliferative ability and invasive ability of glioma cells.
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miR-106b-25 cluster is composed of miR-106b,miR-93 and miR-25,and is a paralogue of miR-17-92 cluster.Some studies have shown that the members in miR-106b-25 cluster abundantly expressed in many cancers.Over-expressions of these miRNAs promote the growth of tumor cells by negatively regulating p21 and p57,and suppress the apoptosis of tumor cells through inhibition of Bim.Moreover,high expression of miR-106b-25 cluster might endue tumor cells with resistance to inhibitory effect of cell growth induced by TGF-β signaling.
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objective To identify SEPT7as one of the target genes of miR-19a and miR-19b.MethodsmiR-19a inhibitor and miR-19b inhibitor mediated by lipofectamine2000,were transfected to SNB19 glioma cells for knocking down miR-19a/19b overexpression.Real time PCR was conducted to detect the expression of miR-19a/miR-19b in transfected cells.The expression of SEPT7was determined by Western blot analysis.RT-PCR was used to detect the mRNA expression of SEPT7; and Luciferase reporter assay was used to identify the direct regulation of miR-19a/19b on SEPT7.ResultsIn SNB19 glioma cells transfected with miR-19a/19b inhibitor,the expression of miR-19a/miR-19b was significantly reduced,whereas the protein expression of SEPT7 was upreguhtted; no significant change of SEPT7 mRNA level was found and luciferase activity became stronger as compared to control cells.ConclusionSEPT7 is the target negatively regulated by miR-19a and miR-19b.
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Objective Increasing evidence suggest that aberrant activation of PI3K/Akt is involved in many human cancers, and that inhibition of the PI3K/Akt pathway might be a promising strategy for cancer therapy. The study is to evaluate the effects of combined therapy of PI3K inhibitor (LY294002) and Ad-PTEN in athymic mice xenogeneic transplant model of human glioma and to reveal the possible mechanisms involved.Methods Twenty-four athymic mice were randomly divided into 4 groups (DMSO、Ad-vector plus DMSO、LY294002 alone and Ad-PTEN plus LY294002), and were treated, respectively. Athymic mice xenogeneic transplant model was established by inoculation (sc) with LN229 glioma cells. Body mass (BM) and diameter of tumor mass were measured. Furthermore, The protein expressions of PTEN、p-Akt、CyclinD1、Caspase-3、MMP-2、p-FAK in tumor tissues were analyzed with immunohistochemistry.Results The tumor-inhibiting rate of was significantly higher in Ad-PTEN plus LY294002 than in the LY294002 alone (92.46 vs 65.59%)( P <0.05).The protein expressions of PTEN and Caspase-3 were significantly higher, while PCNA、CyclinD1、bcl-2 and MMP-2、p-FAK was significantly lower in Ad-PTEN plus LY294002 group than in the other three groups ( P <0.05).Conclusions LY294002 plus Ad-PTEN achieve better outcome than either alone in treating glioma possibly through enhancement of the inhibitory action of PI3K/Akt pathway and Ad-PTEN pathway.
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cells, the expression of PTEN was up-regulated while pAkt down-regulated. Conclusions AS-miR-221 and AS-miR-222 may enhance the radiosensitivity of MCF-7 breast cancer cells by up-regulating the expression of PTEN.
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<p><b>OBJECTIVE</b>To study the radiation-sensitizing effect of up-regulating p27(kip1) expression by knocking down miR-221/222 in the U251 human glioblastoma cell line.</p><p><b>METHODS</b>By bioinformatic analysis, we searched the miRNA-221/222 sequence and found the relationship between p27(kip1) and miRNA-221/222. miRNA-221/222 antisense oligonucleotides were transfected into U251 human glioblastoma cells. Northern blot analysis was conducted to detect the expression of miR-221/222 in control, scrambled oligonucleotide (ODN) transfected and anti-mi-221/222ODNs transfected cell groups. The cell cycle kinetics was detected by flow cytometry. Clonogenic assay was used to measure the mitotic cell death and p27(kip1) expression was examined by Western blot analysis.</p><p><b>RESULTS</b>Based on bioinformatic analysis, we found that the seed sequences of miR-221 and miR-222 coincide with each other, and p27(kip1) is a target for miRNA-221/222. The expression level of miR-221/222 was significantly knocked down in cells transfected with antimiR-221/222 as compared to the parental cells or cells transfected with scrambled ODN. Cell cycle was arrested in G0 or G1 phase in the anti-miR-221/222 group. When combined with irradiation, S phase fraction in the anti-miR-221/222 cell group is lower than that in the other two control groups. Anti-miR-221/222 combined with irradiation could synergistically enhance mitotic cell death. The expression of p27(kip1) was up regulated in the anti-miR-221/222 group revealed by Western blot analysis.</p><p><b>CONCLUSION</b>Anti-miR-221/222 may enhance the radiosensitivity of U251 human glioblastoma through upregulation of p27(kip1).</p>
Subject(s)
Humans , Base Sequence , Cell Cycle , Radiation Effects , Cell Line, Tumor , Cyclin-Dependent Kinase Inhibitor p27 , Genetics , Metabolism , Glioblastoma , Genetics , Metabolism , MicroRNAs , Genetics , Metabolism , Molecular Sequence Data , Oligonucleotides, Antisense , Genetics , Metabolism , Radiation Tolerance , Sequence Alignment , Up-Regulation , Radiation Effects , X-RaysABSTRACT
Objective To investigate the gene expression of vascular endothelial growth factor(VEGF) and ultrastructural changes in cultured endothelial cells from human cerebral microvessels under hypoxic conditions.Methods Human cerebral microvessels were isolated from freshly obtained specimens of normal brain adherent to resected cerebral arteriovenous malformations.The expression of factor Ⅷ-relative antigen(FⅧ-RA) in cultured cells was observed with immunocytochemistry.The level of VEGFmRNA in cells and released VEGF protein in cell supernatant were determined by RT-PCR analysis and ELISA respectively when they were exposed to hypoxic conditions(95% N_2,5% CO_2;two hours,four hours,eight hours) or maintained in basal condition.Ultrastructural changes in cells were also observed by electron microscopy.Results In inverted microscope the cultured cells showed contact inhibition and a rounded cobblestone appearance.More than 90% of them were stained strongly with antibodies against FⅧ-RA.Significant VEGF mRNA and protein accumulated when these cells were exposed to hypoxia for 4 hours.However,their VEGF expression was down-regulated after hypoxia for 8 hours and a number of vesicles and swollen mitochondria were present in the cytoplasm.Conclusion The level of VEGF expression may havesignificant relationship with ultrastructural changes in human cerebral endothelial cells under hypoxic conditions.
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<p><b>OBJECTIVE</b>To construct the small interfering RNA (siRNA) expression constructs targeting epidermal growth factor receptor(EGFR) and express them in TJ905 human malignant cells.</p><p><b>METHODS</b>Two target sequences from Receptor L domain and catalytic domain were selected to create two expression constructs using psiRNA-NeoG2. Furthermore, the siRNA constructs were transfected into TJ905 cells as mediated by Lipofectamin. Meanwhile, an antisense EGFR construct p-anti-hEGFR was set as control. Immunofluorescence and Western blot were performed to detect EGFR expression.</p><p><b>RESULTS</b>With the successful construction of the two siRNA expression plasmids and the stable transfection to TJ905 cells, the expression of EGFR was down-regulated to 90% and 92% respectively, but to 82% in the anti-sense EGFR group.</p><p><b>CONCLUSION</b>The siRNA expression constructs targeting EGFR could specifically inhibit EGFR expression, and should be a new strategy in glioma gene therapy targeting EGFR.</p>
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Humans , Blotting, Western , Cell Line, Tumor , Fluorescent Antibody Technique , Models, Genetic , Plasmids , Genetics , RNA Interference , RNA, Small Interfering , Genetics , ErbB Receptors , Genetics , Metabolism , Transfection , MethodsABSTRACT
BACKGROUND: The observation of vascular endothelial growth factor gene expression of cerebrovascular diseases and ultrastructure of cells may be helpful to understand angiogenesis and its relative cellular factors involved in the pathogenesis at cellular and molecular levels. OBJECTIVE: To investigate the method of culture of human cerebral cap illary endothelial cell by separation of capillary fragment in vitro, and to ob serve vascular endothelial growth factor gene expression and ultrastructure of cells. DESIGN: A randomized controlled research on technique and method. SETTING: The neurosurgery department of a general hospital of a military area command of Chinese PLA and the neurosurgery department of a college hospital. PARTICIPANTS: Eighteen patients with arteriovenous malformation of brain(Spetzler Ⅱ-Ⅲ grade), as confirmed by aortocranial angiography before operation, in the Department of Neurosurgery, General Hospital of Shenyang Military Area Command of Chinese PLA were included. The material was obtained from fresh integrated specimen of arteriovenous malfor mation of brain with surrounding fresh brain tissues during the opera tion. Capillary endothelial cell was separated by homogenate, filtration and enzymatic digestion techniques. Cells grew well in culture flask and were divided into 4 groups(hypoxia state for 2, 4, 8 hours groups and control group). Each group contained four flasks.METHODS: Simulation of anoxia condition: volume faction 0.95 N2 and volume fraction 0.05 CO2. Expression of factor Ⅷ related antigen in cells was detected by immunohistochemistry. mRNA expression of vascular endothelial growth factor on endothelial in every group was observed by RT-PCR, protein content of vascular endothelial growth factor in supernatant detected by enzyme-linked immunoadsordent assay, and cellular ultrastructural change observed by transmission electron microscopy.MAIN OUTCOME MEASURES: mRNA expression of vascular endothelial growth factor on endothelial cell and protein content of vascular endothelial growth factor in supernatant in control group and every hypoxia groups; cellular ultrastructural changes.RESULTS: Under phase contrast microscope, cultured living cells had mono-layer pebble-like typical character. More than 90% of were factor Ⅷrelated antigen(FⅧ-RA) staining positive. mRNA and protein expression of vascular endothelial growth factor in hypoxia 4 hours group was 0.98 ±0. 19,( 180. 77 ± 20. 15) ng/L, which was significantly higher than in control group [0, (26. 20 ± 6.33) ng/L, P ＜ 0.01 ]. Eight hours later, expression decreased [(0. 35 ±0.07), (31.68 ±8.34) ng/L]; swollen mitochondrion, dilated endoplasmic reticulum, and lysosome vesiculation were found.CONCLUSION: Humane cerebral capillary endothelial cell can be cultured by separation of capillary fragment, which is easy to operate and the cellular purity is reliable. In the early stage of ischemia and hypoxia, expression of vascular endothelial growth factor is not enough to maintain cellular ultrastructure integrity. Cells may be injured along with the prolong of hypoxia.Zhao MG, Tang T, Gao YZ, Pu PY, Wei XZ. Culture of human cerebral capillary endothelial by separation of capillary fragment and the observation of vascular endothelial growth factor gene expression and cell ultrastructure. Zhongguo Linchuang Kangfu 2005; 9(21):211-3 (China) [www. zglckf. com]
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<p><b>OBJECTIVE</b>To study the role of connexin gene (Cx43) in the suppression of C6 glioma.</p><p><b>METHODS</b>Cx43 gene depleted parental C6 rats (control group) and C6 cells transfected with Cx43 cDNA (transfection group) were implanted into the right caudate nucleus of SD rats. Rats bearing cerebral C6 gliomas were treated with Cx43 cDNA (treatment group) with another group treated with empty vector (empty vector group) serving as control. The general manifestation, survival time, MRI dynamic scanning and histopathological changes in all rats were observed. Cx43 mRNA and its protein were examined by in situ hybridization and immunohistochemistry. Proliferation activity was monitored by the average number of AgNOR stain. Cell apoptosis was examined by the Tolt-mediated x-duTP nick end labeling (TUNEL) method.</p><p><b>RESULTS</b>All rats in the control and empty vector groups died of cerebral glioma within 3 weeks after implantation of C6 cells. Six in the transfection group and 8 in the treatment group were alive beyond 120 days with complete disappearance of the tumor foci, except one in this group having some residue of tumor. In the glioma of transfection and treatment groups, Cx43 gene expression was up-regulated, proliferation activity reduced while the apoptotic cells did not increase.</p><p><b>CONCLUSION</b>The development of glioma is greatly suppressed by the transfection of Cx43 gene, which has great effectiveness in rats bearing cerebral malignant gliomas. This could become a target of choice in the gene treatment of malignant gliomas.</p>
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Animals , Male , Rats , Brain Neoplasms , Mortality , Therapeutics , Connexin 43 , Genetics , Therapeutic Uses , Disease Models, Animal , Genetic Therapy , Glioma , Mortality , Therapeutics , Neoplasm Transplantation , Rats, Sprague-Dawley , Treatment OutcomeABSTRACT
<p><b>OBJECTIVE</b>To investigate telomerase activity and expression of hTR and hTERT in human neuroepithelial tumors for exploring new strategy for clinical diagnosis and treatment.</p><p><b>METHODS</b>Telomerase activity was detected by modified TRAP method and the expression of hTR and hTERT was measured by RT-PCR method in 65 human neuroepithelial tumors, respectively.</p><p><b>RESULTS</b>The positive rates of telomerase and hTERT were 61.54% and 70.77% respectively in human neuroepithelial tumors, and the positive rate and their level of expression were correlated with the degree of malignancy of tumors positively.</p><p><b>CONCLUSIONS</b>Telomerase activity and hTERT are significantly correlated with the degree of malignancyin human neuroepithelial tumors. hTERT may play a key role in the regulation of telomerase activity.</p>
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Humans , DNA-Binding Proteins , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Neoplastic , Neoplasms, Neuroepithelial , Genetics , Telomerase , Genetics , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To study the gene expression profiles of oligodendrogliomas with gene cDNA array.</p><p><b>METHODS</b>(32)P tagged cDNA probes converted from the total RNA, which had been extracted from 2 fresh samples of oligodendroglioma and 1 of normal brain tissue, were hybridized with the Atlas array. After washing the membranes, the autoradiography was performed and the autoradiograms were analyzed through the special software.</p><p><b>RESULTS</b>As compared to the normal brain tissue, there were 63 co-upregulated genes and 4 co-downregulated genes in these 2 tumor samples. However, a significant quantitative difference existed between them. The expression trend of some genes differed from the known information.</p><p><b>CONCLUSION</b>cDNA array is effective for studying the gene expression profiles of oligodendrogliomas and provides new information for the further research on their molecular mechanisms.</p>