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BACKGROUND: MicroRNAs (miRNAs) can function as either oncogenes or tumor suppressor genes via regulation of cell proliferation and/or apoptosis. MiR-221 and miR-222 were discovered to induce cell growth and cell cycle progression via direct targeting of p27 and p57 in various human malignancies. However, the roles of miR-221 and miR-222 have not been reported in human gastric cancer. In this study, we examined the impact of miR-221 and miR-222 on human gastric cancer cells, and identified target genes for miR-221 and miR-222 that might mediate their biology. METHODS: The human gastric cancer cell line SGC7901 was transfected with AS-miR-221/222 or transduced with pMSCV-miR-221/222 to knockdown or restore expression of miR-221 and miR-222, respectively. The effects of miR-221 and miR-222 were then assessed by cell viability, cell cycle analysis, apoptosis, transwell, and clonogenic assay. Potential target genes were identified by Western blot and luciferase reporter assay. RESULTS: Upregulation of miR-221 and miR-222 induced the malignant phenotype of SGC7901 cells, whereas knockdown of miR-221 and miR-222 reversed this phenotype via induction of PTEN expression. In addition, knockdonwn of miR-221 and miR-222 inhibited cell growth and invasion and increased the radiosensitivity of SGC7901 cells. Notably, the seed sequence of miR-221 and miR-222 matched the 3'UTR of PTEN, and introducing a PTEN cDNA without the 3'UTR into SGC7901 cells abrogated the miR-221 and miR-222-induced malignant phenotype. PTEN-3'UTR luciferase reporter assay confirmed PTEN as a direct target of miR-221 and miR-222. CONCLUSION: These results demonstrate that miR-221 and miR-222 regulate radiosensitivity, and cell growth and invasion of SGC7901 cells, possibly via direct modulation of PTEN expression. Our study suggests that inhibition of miR-221 and miR-222 might form a novel therapeutic strategy for human gastric cancer.
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Proliferação de Células , Regulação Neoplásica da Expressão Gênica , MicroRNAs/fisiologia , PTEN Fosfo-Hidrolase/metabolismo , Tolerância a Radiação/genética , Neoplasias Gástricas/patologia , Animais , Apoptose , Northern Blotting , Western Blotting , Adesão Celular , Ciclo Celular , Movimento Celular , Células Cultivadas , Fibroblastos/citologia , Fibroblastos/metabolismo , Raios gama , Humanos , Técnicas Imunoenzimáticas , Rim/citologia , Rim/metabolismo , Camundongos , Células NIH 3T3 , PTEN Fosfo-Hidrolase/antagonistas & inibidores , PTEN Fosfo-Hidrolase/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Gástricas/genética , Neoplasias Gástricas/radioterapiaRESUMO
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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<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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Humanos , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , RNA Helicases DEAD-box , Genética , Metabolismo , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Glioblastoma , Genética , Metabolismo , RNA Interferente Pequeno , Genética , Metabolismo , Ribonuclease III , Genética , MetabolismoRESUMO
<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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Animais , Humanos , Camundongos , Apoptose , Sequência de Bases , Caspase 3 , Metabolismo , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Terapia Genética , Glioma , Metabolismo , Patologia , Antígeno Ki-67 , Metabolismo , Camundongos Endogâmicos BALB C , Camundongos Nus , MicroRNAs , Genética , Dados de Sequência Molecular , Transplante de Neoplasias , Oligonucleotídeos Antissenso , Farmacologia , PTEN Fosfo-Hidrolase , Metabolismo , Proteínas Proto-Oncogênicas c-bcl-2 , Metabolismo , RNA Mensageiro , Metabolismo , Inibidor Tecidual de Metaloproteinase-3 , Metabolismo , TransfecçãoRESUMO
<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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Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem , Astrocitoma , Metabolismo , Biomarcadores Tumorais , Metabolismo , Encéfalo , Metabolismo , Neoplasias Encefálicas , Metabolismo , Regulação Neoplásica da Expressão Gênica , Meduloblastoma , Metabolismo , Receptor Notch1 , Metabolismo , Fisiologia , Receptor Notch2 , Metabolismo , FisiologiaRESUMO
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 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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Humanos , Adenoviridae , Genética , Western Blotting , Proteínas de Ciclo Celular , Genética , Fisiologia , Linhagem Celular Tumoral , Movimento Celular , Genética , Vetores Genéticos , Genética , Glioma , Metabolismo , Patologia , Integrina alfaVbeta3 , Metabolismo , Metaloproteinase 14 da Matriz , Metabolismo , Metaloproteinase 2 da Matriz , Metabolismo , Metaloproteinase 9 da Matriz , Metabolismo , Microscopia Confocal , Invasividade Neoplásica , Genética , Septinas , Inibidor Tecidual de Metaloproteinase-1 , Metabolismo , Inibidor Tecidual de Metaloproteinase-2 , MetabolismoRESUMO
<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>