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[Apoptosis of glioma cell line U251 induced by small interfering RNA targeting survivin]
Ru-xiang Xu1, Yan-yang Tu, Xiao-dan Jiang
1Department of Neurosurgery and Institute of Neuroscience of Guangdong Province, Zhujiang Hospital, Southern Medical University, Guangzhou 510282, China.
Objective:
To construct recombinant expression vectors of small interfering RNA (siRNA) targeting survivin and investigate apoptosis of glioma cell line U251 mediated by the survivin-targeting siRNA.
Methods:
According to the sequence of the coding region of survivin gene, two strings of 19 nucleotides of inverted sequence flanking the loop sequence of two complementary 9-base oligonucleotides were designed and synthesized to form hairpin construct as the DNA templates for the target siRNA. The siRNA templates were cloned into siRNA expression vector pGenesil-1, and the resulted vector pGenesil-1/survivin was transfected into U251 cells using Metafectene following the standard protocols. Real-time PCR and Western blotting were performed to evaluate survivin gene silencing induced by siRNA transfection at the RNA and protein levels, respectively. Flow cytometry analysis with Annexin-V/PI double staining was used to determine the cell apoptosis.
Results:
Real-time RT-PCR and Western blotting revealed significantly lowered survivin expression at both RNA and protein levels in transfected U251 cells, which exhibited a significantly higher apoptosis rate after transfection as shown by flow cytometry analysis.
Conclusion:
RNA interference mediated by the siRNA expression vector pGenesi-l/survivin can significantly reduce survivin expression and induce remarkable apoptosis in U251 cells.
Insights
Small interfering RNA (siRNA) targeting survivin significantly reduces survivin expression in glioma cells. This RNA interference approach effectively induces apoptosis in U251 glioma cells.
Area of Science:
- Molecular Biology
- Cancer Research
- Gene Silencing
Background:
- Glioma is a common type of brain tumor.
- Survivin is an oncoprotein that inhibits apoptosis and promotes cell proliferation.
- Targeting survivin offers a potential therapeutic strategy for glioma treatment.
Purpose of the Study:
- To construct recombinant expression vectors for small interfering RNA (siRNA) targeting survivin.
- To investigate the induction of apoptosis in the U251 glioma cell line using survivin-targeting siRNA.
Main Methods:
- Designed and synthesized 19-nucleotide hairpin constructs for survivin-targeting siRNA.
- Cloned siRNA templates into the pGenesil-1 vector, creating pGenesil-1/survivin.
- Transfected U251 cells with pGenesil-1/survivin and analyzed survivin expression and apoptosis rates.
Main Results:
- Real-time RT-PCR and Western blotting confirmed significantly reduced survivin expression at both RNA and protein levels in transfected cells.
- Flow cytometry analysis showed a significantly higher apoptosis rate in U251 cells transfected with survivin-targeting siRNA.
Conclusions:
- RNA interference mediated by the pGenesil-1/survivin vector effectively reduces survivin expression in U251 glioma cells.
- Survivin-targeting siRNA induces significant apoptosis in the U251 glioma cell line, suggesting therapeutic potential.
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