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RNAi Down-regulated Thioredoxin Expression Sensitizes U251 Cells to Arsenic Trioxide-induced Apoptosis

Author: WangRun
Tutor: SunMaoMin
School: Suzhou University
Course: Human Anatomy and Embryology
Keywords: RNA interference thioredoxin glioma arsenic trioxide apoptosis
CLC: R739.4
Type: Master's thesis
Year: 2011
Downloads: 12
Quote: 0
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Abstract


Objective: To explore correlation between thioredoxin and the sensitivity of U251 cells to arsenic trioxide-induced apoptosis.Methods: （1） U251 cells were treated with 5μmol/L As2O3 for 0 h, 6 h, 12 h, 24 h and 48 h. U251 cells’viability and apoptosis were determined by MTT and stained with Hoechst 33324. Trx and Caspase-9 expression were detected by immunocytochemistry. Trx and Bcl-2 expression in As2O3-induced U251 cells apoptosis were determined by Western blotting assays. （2） RNA interference plasmid: TXN-69, TXN-107, TXN-130, TXN-213 and negative control plasmid were transfected transiently into U251 cells. Trx mRNA and protein expression were detected by RT-PCR and Western blotting and determined the effective plasmid. （3） U251 cells were transfected transiently with the effective plasmid or negative control plasmid and then treated or untreated with 5μmol/L As2O3 for definite time. According to different treatment, cells were divided to four groups: cells transfected with negative control plasmid, cells transfected with effective plasmid, cells transfected with negative control plasmid and treated with 5μmol/L As2O3 and cells transfected with effctive plasmid transfected and treated with 5μmol/L As2O3. Each group cells were stained with Annexin V-PE/7-AAD and the As2O3-induced apoptosis and the down-regulation of Trx effects on the As2O3-induced apoptosis were detected by flow cytometer.Results: （1） MTT assays showed that the cell viability significantly depressed after As2O3 treatment and in a time-dependent manner. The cell-cytotoxic reaction after treatment with As2O3 for 12 h is significantly enhanced. The assays of cells stained with Hoechst 33242 show that cells treated with As2O3 appeared apoptosis features such as chromatin condensation and nuecleus devided into fragments. And the number of apoptosis cells significantly increased in a time-dependent manner. Immunocytochemistry results show that both percentage of Trx and Caspase-9 positive cells increased. Western blotting results showed that expression of Trx increased and Bcl-2 wasn’t significantly changed in the apoptosis process. （2） U251 cells were transiently transfected with plasmid, which was detected by Fluorescence microscope. TXN-107 was able to effectively down-regulate the Trx expression according to the RT-PCR and Western blotting results. （3） Flow cytometer assays show that there was no significant correlation between cells transfected with negative control plamid and cells transfected with TXN-107, but apoptosis of cells transfected with TXN-107 and treated with 5μmol/L As2O3 （16h） significantly increased comparing with cells transfected with negative control plamid and treated with 5μmol/L As2O3 （16h） cells.Conclusion: （1） 5μmol/L As2O3 inhibited the U251 cell viability and induced the apoptosis in a manner of time-dependent. Both Trx and Caspase-9 are involved in As2O3-induced apoptosis. Bcl-2 could not involve in As2O3-induced apoptosis. （2） The effective interference plasmid was seized. （3） Down-regulation of Trx by RNAi was not affect U251 cell apoptosis, but it sensitized the U251 cells to As2O3-induced apoptosis.

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