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Objective To explore the effect of acetyl-CoA carboxylase 1(ACC1) on cell proliferation, migration and invasion of human glioma cell line U87. Methods Western blotting was performed to examine endogenous ACC1 expression in human glioma cell lines U87, U251 and U373. ACC1 overexpression plasmid and the plasmid vector were transiently transfected into U87 cells. The level of ACC1 in control and ACC1 overexpression cells was examined by Western blotting. The effect of ACC1 on U87 cells migration and invasion was detected by Transwell assay. The effect of ACC1 on U87 cells scratch healing ability was detected by scratch test. The effect of ACC1 on U87 cells proliferation was investigated by MTT assay. Western blotting was conducted to detect the level changes of proteins. Results Among three human glioma cell lines U87, U251 and U373, endogenous ACC1 level in U87 cells was lower than that in other two cell lines. ACC1 overexpression inhibited U87 cell proliferation, as well as cell migration, invasion and scratch healing ability (P < 0.05). Vimentin, fibronectin, urokinase type plasminogen activator (uPA), Bcl-2, cyclin B, cyclin D and p-STAT3 were down-regulated (P< 0.05), P21 was up-regulated (P < 0.05) after ACC1 overexpression. Conclusion These results suggest that ACC1 suppresses the proliferation, migration and invasion of human glioma cells, probably by inhibiting STAT3 activity.
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@#[Abstract] Objective: To investigate the miR-423-5p expression in brain glioma tissues and cell lines, and its promotive effect on temozolomide (TMZ) chemoresistance by targeting PDCD5 (programmed cell death protein 5). Methods: Tumor tissues and matched peritumoral tissues were collected from 20 brain glioma patients who were surgically treated in the Department of Neurosurgery, Affiliated Hospital of Beihua University between January 2017 and December 2018. Glioblastoma cell lines (U251, U87, SHG-44) and human normal glial cell line HMC-3 were also used in the study. The relative expression of miR-423-5p and PDCD5 in brain glioma and peritumoral tissues and cell lines was detected by qPCR. The synthesized miR-423-5p mimics and miR-NC were respectively transfected into U251 and U87 cells; meanwhile, TMZ at different concentrations (50, 100, 150 and 200 μmol/L) were also used to treat the cells. Then, the chemoresistance of cells to TMZ were determined. MTT assay and colony formation assay were used to examine the proliferation of U251 and U87 cells, andWestern blotting was used to detect the expression of c-caspase 3, Bcl-2 and PDCD5 proteins in U251 and U87 cells. The targeting relationship between PDCD5 and miR-423-5p was validated through Dual luciferase reporter gene assay. Results: miR-423-5p was highly expressed in glioma tissues and glioma cell lines (all P<0.01). As compared with the miR-NC group, the proliferation and TMZ-chemoresistance of U251 and U87 cells in miR-423-5p mimics group significantly increased (all P<0.01). Dual luciferase reporter gene assay validated that miR-423-5p could bind with PDCD5 3' UTR to suppress the expression of PDCD5. Conclusion: High expression of miR-423-5p enhances the chemoresistance of glioma cells to TMZ, and miR-423-5p may serve as a potential therapeutic target in the treatment of brain glioma.
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Aim To explore the inhibitory effect of hawthorn leaves flavonoids (HLF) on human glioblastoma U87 cells. Methods U87 cells were cultured in vitro. The concentration of HLF was 25, 50, 100 mg . L¹ as drug concentration. The effects of HLF on the proliferation, migration, invasion and adhesion of U87 cells were detected by CCK-8 assay, scratch assay, Transwell assay and adhesion assay, and clone forming ability was detected by cell clonogenic assay. Results HLF inhibited the proliferation, migration, invasion and adhesion of U87 cells in a concentration-dependent manner, and the difference was statistically significant compared with control group (P < 0. 05). When HLF reached 50 mg . L¹ the inhibition of tumor growth was the most significant. Conclusion HLF has a certain inhibitory effect on glioma U87 cells.
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@# Objective: To explore the role of tumor suppressor gene programmed cell death 5 gene (PDCD5) in the growth and temozolomide (TMZ) sensitivity of brain glioma cells. Methods:Atotal of 116 patients with cerebral glioma admitted to the Department of Neurosurgery, First Clinical Hospital of Jilin University from January 2009 to December 2014 were enrolled in this study. QPCR, WB and immunohistochemistry method were used to detect the mRNAand protein expressions of PDCD5 in glioma cell lines (U87, U251), U87 cell line with stable PDCD5 expression (U87-PDCD5), glioma cells with si-PDCD5 transfection and primary cerebral glioma tissues, respectively. MTT assay was used to detect the effect of over-expression or knockdown of PDCD5 on the growth and TMZ-sensitivity of glioma cells. The subcutaneous tumor-bearing model of glioma cell line U87 was established in nude mice, and then the experimental mice were randomly divided into control group, TMZ group, PDCD5 group and TMZ+exogenous PDCD5 recombinant expression vector group.After 20 days, the animals were sacrificed by cervical dislocation and the tumor tissue was excised to measure the tumor volume and weigh. The expression of PDCD5 in tumor tissues was detected by qPCR and WB methods, and the effects of PDCD5 combined with TMZ on the growth of gliomas were also analyzed. Results: The relative mRNA and protein expressions of PDCD5 in U87 cells were significantly lower than those in U251 cells (both P<0.05), and the mRNA and protein expressions of PDCD5 in high level glioma tissues were significantly lower than those in low level tissues (all P<0.05). The sensitivity of U87-PDCD5 cells and U251 cells to TMZ was higher than that of U87 cells (all P<0.05). The sensitivity of cells to TMZ in U87-PDCD5-siRNA group and U251siRNA group was significantly lower than that of the control group (both P<0.05). The tumor volume and weigh to fnudemicexenografts were compared,and the results showed control group>TMZ group>PDCD 5group>combined group(allP<0.05);however, the mRNA and protein expressions of PDCD5 in the transplanted tumor tissues of each group showed the opposite trend (all P<0.05). Conclusion: PDCD5 over-expression can enhance the chemosensitivity of braingliomato the chemotherapy drug TMZ, while silencing of PDCD5 expression exertsthe opposite effect.The combination of PDCD5 and TMZ can better inhibit the growth of xenografts in nude mice.
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Objective: To investigate the effects of silencing zinc finger E-box binding homeoboxl (ZEB1) gene on the expressions of mesenchymal markers and cell migration in the glioma U87 cells, and to clarify the effect of ZEB1 on the epithelial to mesenchymal transition (EMT) in the glioma cells. Methods: The constructed ZEB1 shRNA interfering plasmid and control plasmid (shCtrl) were transfected into the glioma U87 cells and the interfering effects were detected by Western blotting method. The glioma U87 cells were divided into control group (the glioma U87 cells were transfected with shCtrl), EMT group (EMT was induced by TGF-fil in the glioma U87 cells transfected with shCtrl) and ZEB1 silence group (EMT was induced by TGF-fil in the glioma U87 cells transfected with ZEB1 shRNAs plasmid). The protein expression levels of mesenchymal markers (N-cadherin, Vimentin), and matrix metalloproteinase-9 (MMP-9) in the glioma U87 cells were measured by Western blotting method. The scratch-healing assay was performed to examine the migration ability of glioma cells. Results: The Western blotting results showed that the expression levels of ZEB1 in the glioma U87 cells transfected with shZEBl # 1 and shZEBl # 2 were significantly lower than that in the cells transfected with shCtrl (P<0. 05 or P< 0. 01), and the inhibitory effect of shZEBl #2 on the ZEB1 expression was more obvious, indicating that ZEB1 was stably transfected into the U87 cells. Compared with control group, the expression levels of mesenchymal markers N-cadherin, Vimentin, and MMP-9 in EMT group were significantly increased (P<0. 05 or P<0. 01). Compared with EMT group, the expression levels of the above proteins in ZEB1 silencing group were markedly reduced (P< 0. 05 or P<0. 01). The cell migration rate in EMT group was obviously elevated compared with control group (P< 0. 01), and the cell migration rate of the glioma U87 cells in ZEB1 silence group was significantly lower than that in EMT group (P<0. 01). Conclusion: Silencing ZEB1 gene expression can inhibit the EMT in the glioma U87 cells and reduce the cell migration abilities, suggesting ZEB1 as an important therapeutic target of invasive glioma.
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@#[Abstract] Objective: To detect the expression of zinc transporter 1 (ZnT1) gene in glioma tissue, and to explore its effect on the proliferation, migration and invasion of U87 cells. Methods: From October 2015 to January 2017, 20 patients with glioma, who received no chemoradiotherapy before operation, were collected from Department of Neurosurgery, the First Affiliated Hospital of China Medical University. The protein and mRNA content of ZnT1 in glioma tissues and adjacent tissues were detected by Western blotting and Realtime PCR, respectively. ZnT1 and si-ZnT1 plasmids were transfected into glioma U87 cell line respectively to construct ZnT1 over-expression U87 cell line and ZnT1 knockdown U87 cell line. The effects of ZnT1 on proliferation, migration and invasion of U87 cells were detected by MTT and transwell assay. Results: Both mRNA and protein expressions of ZnT1 in glioma tissues was significantly higher than those in adjacent tissues (all P<0.05). U87 cell lines with ZnT1 over-expression and knockdown were successfully constructed. Compared with the control group and empty plasmid control group, the proliferation (0.54±0.01 vs 0.45±0.04, 0.43±0.03, P<0.01), invasion and migration (all P<0.05) of U87 cells with ZnT1 over-expression were significantly increased at 12 h after transfection; however, the proliferation (0.37±0.03 vs 0.45±0.01, 0.44±0.03, P<0.01), invasion and migration (all P<0.05) of U87 cells with ZnT1 knockdown were decreased significantly. Conclusion: ZnT1 was highly expressed in glioma tissues, and promoted the proliferation, migration and invasion of glioma U87 cells.
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@#[Abstract] Objective:To prepare the third generation CAR-T cells targeting EGFRvⅢ (EGFRvⅢCAR-T) and to detect its specific killing effect against EGFRvⅢ+ U87 cells in vitro and in vivo. Methods: Human CD3+ T cells were transfected with lentiviral EGFRv Ⅲ/3CAR, which was generated by calcium phosphate co-precipitation of three plasmids. The expression of EGFRvⅢ/3CAR in T cells was detected by Western blotting and flow cytometry. In vitro killing effect of EGFRvⅢ/3CAR-T cells on EGFRvⅢ+ U87 cells was detected by 51Cr release assay. The secretion of cytokine IFN-γ of EGFRvⅢ/3CAR-T cells was detected by ELISA. Nude mouse xenograft model was constructed to detect the in vivo cytotoxicity of EGFRvⅢ/3CAR-T cells on xenograft tumor. Results: The EGFRvⅢ/3CAR lentivirus was successfully packaged with an average titer of 5×106 TU/ml. Western blotting showed that a protein band of approximate 58 000 molecular weight was observed in EGFRvⅢ/3CAR-T cells but absent in untransfected T cells. Flow cytometry indicated the average transduction efficiency of EGFRvⅢ/3CAR was 52.3%. 51Cr release assay showed that the specific killing effect of EGFRvⅢ/ 3CAR-T cells was positively correlated with E/T ratio (E∶T=4∶1, 8∶1, 16∶1, 32∶1). ELISA showed that cytokine IFN-γ secretion was (1 836±148.2) pg/ml, which was significantly different from that of NTT and GFP+ T cells (P<0.01). The specific killing activity of EGFRvⅢ/3CAR-T cells and IFN-γ secretion were both dependent on the expression level of EGFRvⅢ in U87 cells. The tumor growth monitoring results showed that the tumor volume of EGFRvⅢ/3CAR-T cell group was significantly different from that of GFP+ T cell group and PBS group around 3 weeks after injection (P<0.01). Conclusion: EGFRvⅢ/3CAR-T cells demonstrated specific antitumor effectagainstEGFRvⅢ+U87cellsbothinvitro and in vivo, providing basis for immunotherapyofgliomainfuture clinical use.
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Objective: To study the effects of celastrol on the proliferation, apoptosis and migration of human Glioma U87 cells and investigate its preliminary action mechanism. Methods: MTT assay were used to evaluate the effects of celastrol on U87 cells proliferation, and flow cytometry were performed to detect U87 cells apoptosis and mitochondrial membrane potential. Expression of apoptosis related proteins in mitochondria pathway were detected by western blotting. Transwell migration assay and wound healing assay were used to study the migration ability of U87 cells. Results: After treatment with different concentrations of celastrol, the proliferation of U87 cells was significantly inhibited. The flow cytometry assay showed that celastrol decreased the mitochondrial membrane potential and induced the apoptosis of U87 cells. The mechanism of apoptosis showed that celastrol could significantly regulate the expression levels of Bcl-2, Bax, cytochrome C, caspase-9, and caspase-3 in mitochondria pathway. Additionally, celastrol significantly inhibited the migration of U87 cells. Conclusion: Celastrol significantly inhibited the proliferation and migration, and induced apoptosis of U87 cells, which may be mediated by the regulation of mitochondrial pathway related apoptosis proteins.
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Objective To evaluate the diagnostic value of TSSC1 in glioma patients and its influence on cell biologi-cal behavior of glioma U87 cells. Methods RT-PCR and Western blot were used to examine the expression of TSSC1 in glioma samples, including 80 normal paraneoplastic tissues and 80 primary tumors. MTT and transwell were used to analyze the effect of TSSC1 knockout on proliferation, migration, and invasion in U87 cells. Results TSSC1 is down-regulated in glioma compared to its paraneoplastic counterparts and negatively related to higher grade. Furthermore, knockdown of TSSC1 expression results in increased proliferation, migration and invasion in U87 cells in vitro. Conclusion Our results may worked as a marker for early diagnosis and prognosis of glioma.