ABSTRACT
<p><b>OBJECTIVE</b>To study the expression of RAS protein in human glioma tissues and its influence on tumor growth.</p><p><b>METHODS</b>RAS protein expression in glioma tissues was determined by immunohistochemical (IHC) staining. Subsequently, MTT cell proliferation assay, flow cytometry and Western blotting were used to assay U251 cells with reduced RAS expression.</p><p><b>RESULTS</b>The expression of RAS in glioma was increased and strongly correlated with pathological grade. Downregulation of RAS resulted in glioma cells growth suppression and increased apoptosis.</p><p><b>CONCLUSION</b>The expression level of RAS protein in human glioma was increased. Downregulation of RAS can inhibit glioblastoma cell growth through the RAS signal pathway.</p>
Subject(s)
Humans , Brain Neoplasms , Genetics , Metabolism , Pathology , Cell Growth Processes , Genetics , Cell Line, Tumor , Down-Regulation , Glioma , Genetics , Pathology , Immunohistochemistry , ras Proteins , GeneticsABSTRACT
<p><b>BACKGROUND</b>Invasion growth is the most characteristic biological phenotype of glioblastoma, but the molecular mechanism in glioma cell invasion is poorly understood. Recent data have showed that microRNA plays an essential role in tumor invasion. Our study aimed to explore the mechanism of miR-7 involved in the control of glioblastoma cell invasion.</p><p><b>METHODS</b>Glioma cell invasion was evaluated by transwell and scratch assays after up-regulation of miR-7 using miR-7 mimics in U87 and U251 cells. Luciferase reporter assay was used to determine focal adhesion kinase (FAK) as a target of miR-7. The levels of miR-7, matrix metalloproteinases (MMP)-2 and MMP-9 mRNA were detected by PCR assay, and the levels of FAK, MMP-2, MMP-9, total and phosphorylation serine/threonine kinase (AKT), and extracellular signal-regulated kinase (ERK) 1/2 were measured by Western blotting analysis.</p><p><b>RESULTS</b>Over-expression of miR-7 inhibited the invasion and migration activity of U87 and U251 cells. And up-regulation of miR-7 reduced FAK protein expression, Further, luciferase reporter assay showed that miR-7 modulated FAK expression directly by binding 3'UTR of FAK mRNA. In addition, miR-7 repressed p-ERK1/2 and p-AKT level, MMP-2 and MMP-9 expression. Finally, the inverse relationship between FAK and miR-7 expression was certificated in human glioma tissues.</p><p><b>CONCLUSION</b>To our knowledge, these data indicate for the first time that miR-7 directly regulates cell invasion by targeting FAK in glioblastoma and that miR-7 could be a potential therapeutic target for glioblastoma intervention.</p>
Subject(s)
Humans , Blotting, Western , Cell Line, Tumor , Focal Adhesion Protein-Tyrosine Kinases , Genetics , Metabolism , Glioblastoma , Genetics , In Vitro Techniques , Matrix Metalloproteinase 2 , Genetics , Metabolism , Matrix Metalloproteinase 9 , Genetics , Metabolism , MicroRNAs , Genetics , Metabolism , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
<p><b>OBJECTIVE</b>To investigate the role of bone morphogenetic protein 4 (BMP4) on the proliferation and apoptosis in glioma stem cells.</p><p><b>METHODS</b>Stem cells were isolated from a human glioma cell line U87 by using vincristine and characterized by immunofluorescence assay. Proliferation and apoptosis were determined by soft agar colony assay and flow cytometry; Cyclin D1, Bcl-2 and Bax were detected by Western blot analysis.</p><p><b>RESULTS</b>BMP4 inhibited cell proliferation and promoted apoptosis in U87 glioma stem cells. Moreover, Bcl-2 and Cyclin D1 expression were decreased by BMP4, while Bax level was elevated.</p><p><b>CONCLUSION</b>BMP4 can inhibit U87 glioma stem cells proliferation through downregulating Cyclin D1 level, and promote apoptosis through induction of Bax expression and inhibition of Bcl-2 level. It suggests that BMP4 plays an important role in human glioma stem cell biology.</p>
Subject(s)
Humans , Apoptosis , Bone Morphogenetic Protein 4 , Genetics , Metabolism , Cell Line, Tumor , Cell Proliferation , Cyclin D1 , Genetics , Metabolism , Gene Expression Regulation, Neoplastic , Glioma , Genetics , Metabolism , Neoplastic Stem Cells , Cell Biology , Metabolism , Proto-Oncogene Proteins c-bcl-2 , Genetics , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To investigate the potentiality of herpes simplex virus thymidine kinase transduced endothelial progenitor cells (EPC-TK) as angiogenesis-targeting vector in the glioma treatment in vitro and in vivo.</p><p><b>METHODS</b>EPC-TK were mixed with human umbilical vein endothelial cells (HUVECs), U87 or U251 cells at various ratios for ganciclovir (GCV) treatment. The bystander effect was observed by counting the survival cells using MTT assay, and the apoptotic cells were determined by annexin-V and propidium iodide (PI) staining. EPC-TK, EPCs, or phosphate buffered saline (PBS) were injected into the nude mice model of glioma xenograft by tail vein, for the EPC-TK group, EPC group, and PBS group, respectively. And then the changes of tumor volume and tumor vasculature were observed.</p><p><b>RESULTS</b>GCV killed most EPC-TK and reduced the number of other viable cells in a cell:cell ratio-dependent and time-dependent manner. EPC-TK obviously inhibited tumor growth. The tumor volumes on day 21 were 1741.20+/- 576.10 mm(3), 3275.52 +/- 710.86 mm(3) and 3033.09+/-1134.86 mm(3) in the EPC-TK, EPC and PBS group, respectively. EPC-TK also displayed a significant effect on the inhibition of tumor angiogenesis.</p><p><b>CONCLUSION</b>EPC-TK can exert a potent antiglioma effect via inhibiting angiogenesis.</p>
Subject(s)
Animals , Humans , Mice , Angiogenesis Inhibitors , Pharmacology , Antiviral Agents , Pharmacology , Bystander Effect , Cell Transformation, Viral , Physiology , Endothelial Cells , Virology , Endothelium , Genetic Vectors , Glioma , Therapeutics , Mice, Nude , Simplexvirus , Genetics , Thymidine Kinase , Genetics , Transduction, Genetic , Transfection , Xenograft Model Antitumor AssaysABSTRACT
<p><b>OBJECTIVE</b>To investigate the effect of osteopontin silencing on the invasion and apoptosis of U251 cells.</p><p><b>METHODS</b>The invasion, apoptosis and levels of uPA, MMP-2 and MMP-9 were determined by invasion assay, flow cytometry, Western blot and real-time fluorescence quantitative PCR respectively.</p><p><b>RESULTS</b>Osteopontin small interference RNA (siRNA) inhibited osteopontin expression and cell invasion, promoted apoptosis in U251 cells. In addition, the expression of Bcl-2, uPA, MMP-2 and MMP-9 was decreased, while Bax level was elevated.</p><p><b>CONCLUSION</b>Osteopontin siRNA can inhibit U251 cells invasion via the down-regulation of uPA, MMP-2 and MMP-9 levels, and promote apoptosis through induction of Bax expression and inhibition of Bcl 2 level. It suggests that osteopontin plays an important role in human glioma progression.</p>
Subject(s)
Humans , Apoptosis , Cell Line, Tumor , Gene Expression Regulation, Neoplastic , Gene Silencing , Gene Transfer Techniques , Genetic Vectors , Genetics , Metabolism , Glioma , Genetics , Metabolism , Pathology , Lentivirus , Genetics , Metabolism , Neoplasm Invasiveness , Osteopontin , Genetics , Metabolism , RNA, Small Interfering , GeneticsABSTRACT
<p><b>OBJECTIVE</b>To construct a recombinant lentivirus RNA interference (RNAi) vector carrying hTERT gene, and to obtain the titer of the lentiviral stock for investigating the expression in the eukaryotic cells and the effect on the hTERT gene silencing in the eukaryotic cells.</p><p><b>METHODS</b>Two complimentary oligos of small interference RNA (siRNA) with hairpin structures targeting the hTERT gene and a negative control were synthesized, then ligated with pLVTHM vector and sequenced. The recombinant vectors were then transfected with viral packaging mix into T293 cells, viral supernatant was harvested to determine the titer. U87 cells infected by virus were harvested and the expression of hTERT, telomerase activity and apoptosis were detected by reverse transcription-PCR(RT-PCR), TRAP assay and flow cytometry separately.</p><p><b>RESULTS</b>Sequencing data showed that the constructed plasmids contained the correct sequences of hTERT siRNA transcript templates. A vector producing cell line T293 was established, and the titer for transfection was obtained. RT-PCR and TRAP flow cytometry analyses demonstrated that hTERT shRNA expression construct could suppress the expression of hTERT and telomerase activity and induce apoptosis.</p><p><b>CONCLUSION</b>A lentivirus RNAi vector targeting hTERT gene was successfully constructed, which decreased the expression of hTERT and telomerase activity effectively and induced apoptosis. It has set up a research platform for the gene therapy of tumors which take hTERT as the target.</p>
Subject(s)
Humans , Base Sequence , Cell Line , Flow Cytometry , Gene Knockdown Techniques , Methods , Genetic Vectors , Genetics , Lentivirus , Genetics , Molecular Sequence Data , Oligodeoxyribonucleotides , Genetics , Metabolism , Plasmids , Genetics , Metabolism , RNA Interference , RNA, Small Interfering , Genetics , Metabolism , Reverse Transcriptase Polymerase Chain Reaction , Telomerase , GeneticsABSTRACT
<p><b>OBJECTIVE</b>To study inhibitory efficacy of combined gene therapy for malignant gliomas transfected with antisense human telomerase reverse transcriptase (hTERT)/PTEN in vitro and in vivo.</p><p><b>METHODS</b>To construct two adenovirus recons which contained antisense hTERT and wild-type PTEN respectively with high performance homologous recombination system in bacteria. The two adenovirus recons were transfected into U251 human malignant glioma cells combinedly or respectively in vitro and in vivo. U251 cell proliferation in vitro was determined by MTT assay and flow cytometry, tumor growth in vivo was measured by the volume of glioma in nude mice. Telomerase activity was detected by telomeric repeat amplification protocol (TRAP) assay. Expression of hTERT and PTEN protein was detected by Western blotting methods.</p><p><b>RESULTS</b>After transfection in vitro, the growth of U251 cells was inhibited significantly. The inhibitory effect was time-dependent. The strongest inhibition was observed in combined transfection group, at the 6th day, the survival rate was 37.6%, telomerase activity (only 28.8TPG) was inhibited significantly, hTERT protein expression was inhibited significantly too, which was 0.2106, but PTEN protein expression was increased significantly, which was 0.9630. In vivo, the growth of tumors was also effectively inhibited.</p><p><b>CONCLUSION</b>Growth of malignant glioma cells is effectively inhibited after transfection with combined antisense hTERT and PTEN in vitro and in vivo.</p>
Subject(s)
Animals , Humans , Mice , Adenoviridae , Genetics , Apoptosis , Blotting, Western , Brain Neoplasms , Pathology , Therapeutics , Cell Line, Tumor , Cell Proliferation , DNA, Antisense , Genetics , Metabolism , Flow Cytometry , Genetic Therapy , Methods , Glioma , Pathology , Therapeutics , Green Fluorescent Proteins , Genetics , Metabolism , Mice, Nude , Microscopy, Fluorescence , PTEN Phosphohydrolase , Genetics , Recombinant Fusion Proteins , Genetics , Metabolism , Telomerase , Genetics , Metabolism , Transfection , Tumor Burden , Xenograft Model Antitumor AssaysABSTRACT
<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>