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Tumor necrosis factor-like attenuated apoptosis inducer (TWEAK) is a new member of the tumor necrosis factor super family.TWEAK regulates cellular proliferation,differentiation,migration,apoptosis and inflammation by binding its receptor,fibroblast growth factor-inducible 14 (Fn14),through the interaction between cells or in a paracrine fashion.The role of TWEAK / Fn14 signaling pathway in the development of lung diseases such as lung injury,asthma and lung cancer has drawn increasing attention.
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Objective:To investigate effects of miR-503 on cisplatin sensitivity in BEL-7402 cells by targeting of bcl-2.Methods:MiR-503 and bcl-2 mRNA expression levels in hepatocellular carcinoma cells were measured by real-time quantitative (qRT)-PCR;Bcl-protein level was detected by Western blot;miR-503 mimics were transiently transfected to the BEL-7402 cells by liposome transfection;potential target genes of miR-503 were predicted by Bioinformatics software;miR-503 potential targets were validated by dual luciferase activity;and the cell viability was measured by MTT assay.Results:MiR-503 level was down-regulated and Bcl-2 protein expression level was up-regulated in BEL-7402 cells compared with HL-7702 cells.MiR-503 could interact with bcl-2 and inhibit its expression.Cell vitality with miR-503 transfection was significantly reduced compared to that in the negative control.Conclusion:MiR-503 may enhance the sensitivity of BEL-7402 cells to cisplatin and inhibit the cell proliferation by targeting bcl-2.
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Objective To study the effect of bel-2 siRNA on apoptosis of HL-60 cells.Methods bcl-2 siRNA was synthesized in vitro transcription with silencer siRNA construction kit.The synthesized siRNA was transfected into HL-60 cells with Amine siPORT transfection.We used MTT flow cytometer and hoechst 33258 flourescence stainning t0 evaluate cell proliferation and apoptosis. Results.Bcl-2 siRNA could partially inhibit the growth of HL-60 cells.After incubated with bcl-2 siRNAl for 48 hours,HL-60 cells exhibited morphologic characteristic of apoptosis including chromatin condensation,crescents formation and nuclear fragmentation.Conclusion Effective bcl-2 siRNA can induce apoptosis and inhibit cell proliferation.
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Aim To study the interference role of Bcl-2 siRNA on HL-60 cells. Methods Bcl-2 siRNA was synthesized in vitro transcription with silencer siRNA construction kit. The synthesized siRNA was transfected into HL-60 cells with lipid siPORT transfection. Forty-eight hours after the transfection, we used MTT and immunofluorescence to detect cell proliferation and apoptosis,and used RT-PCR and immunofluorescence to detect the level of Bcl-2 mRNA and Bcl-2 protein expression. Result Bcl-2 siRNA reduced the level of Bcl-2 mRNA and Bcl-2 protein expression in HL-60 cells and induced cell apoptosis. There was no difference on the effect of other groups compared with the control. Effective Bcl-2 siRNA specifically degraded Bcl-2 expression in the levels of mRNA and protein and induced HL-60 cells apoptosis.Conclusion These results indicate that siRNA is a highly specific tool for targeted gene knockdown. siRNA-mediated gene silencing is a reliable approach for large-scale screening of gene function and drug target validation.
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AimTo explore the effects of bcl-2 antisense oligonucleotide targeting the coding region of the bcl-2 mRNA on apoptosis induced by Vp16 in HL60 and K562 cells. Methods Drug sensitivity was compared by MTT cytotoxicity assay, and expression of bcl-2 protein and apototic cells were assayed by flow cytometry. Results The bcl-2 antisense oligonucleotide of 10 μmol · L- 1 combined with etoposide inhibited expression of bcl-2 protein,increased apoptosis in HL60 and K562 cells and decreased IC50 of etoposide; The antisense oligonucleotide targeting the coding region of the bcl-2 mRNA had stronger effection than the antisense oligonucleotide targeting the translation initiation. ConclusionThe bcl-2 antisense oligonucleotide targeting the coding region of the bcl-2 mRNA can enhances etoposide induced apoptosis of HL60 and K562 cell.
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AIM:To explore the production and cytotoxicity of the reactive oxygen species(ROS)induced by diallyl trisulfide(DATS)in HL-60 cells.METHODS:HL-60 cells were either treated with various doses of DATS alone,or DATS combination with apocynin,a specific NADPH oxidase inhibitor,or with antioxidant N-acetyl-L-cysteine(NAC)for 0,1,3,6,12 and 24 h,respectively.The intracellular ROS level was measured by flow cytometry.The activity of NADPH oxidase was evaluated by NBT reduction experiment.The content of both malondialdehyde(MDA)and the protein carbonyl were analyzed by spectrophotometer.RESULTS:The results from flow cytometry indicated that DATS significantly increased the intracellular ROS level in HL-60 cells(P
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AIM The antisense drug design will be optimized based on bcl 2 mRNA secondary structure simulated with computer. METHODS bcl 2 mRNA second structures were simulated with computer and Mfold software, and the unstable zones on the second structure, as designing antisense zones, were selected. RESULTS Five antisense deoxynucletides were studied and evaluated with experiments of HL 60 and K562 leukemic cells. Two of them exist significant effect of inhibiting grow of HL 60 and K562 leukemic cells with dose of 10 ?mol?L -1 or more. CONCLUSION The designs with computer and corresponding software will be usefully efficient way to look for antisense drugs.
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AIM: To investigate whether the bcl-2 antisense oligonucleotide increases the sensitivity of HL60 and K562 cell lines to daunorubicin. METHODS: IC50 for HL60 and K562 was determined with MTT method, the expression levels of Bcl-2 protein were assayed by immunofluorescence using fluoresce isothiocyanate labeling. In addition, apoptosis was detected by morphological observation and flow cytometric analysis of DNA fragmentation. RESULTS: It was found that the two oligonucleotides directed against the coding region and the translation initiation of bcl-2 mRNA, combined respectively with daunorubicin, inhibited expression of bcl-2 protein, increased apoptosis in HL60 and K562 cells, and decreased IC50 of daunorubicin significantly ( P