[Apoptosis of glioma cell line U251 induced by small interfering RNA targeting survivin].
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.


CONCLUSIONS
RNA interference mediated by the siRNA expression vector pGenesi-l/survivin can significantly reduce survivin expression and induce remarkable apoptosis in U251 cells.
Xu
Ru-xiang
RX
Department of Neurosurgery and Institute of Neuroscience of Guangdong Province, Zhujiang Hospital, Southern Medical University, Guangzhou 510282, China.
Tu
Yan-yang
YY
Jiang
Xiao-dan
XD
Feng
Jiang-nan
JN
Huang
Jun
J
D017209:Apoptosis / Q000502:physiology*; D001932:Brain Neoplasms / Q000378:metabolism / Q000473:pathology*; D002455:Cell Division; D045744:Cell Line, Tumor; D015316:Genetic Therapy; D005910:Glioma / Q000378:metabolism / Q000473:pathology*; D006801:Humans; D051034:Inhibitor of Apoptosis Proteins; D008869:Microtubule-Associated Proteins / Q000096:biosynthesis / Q000235:genetics*; D009363:Neoplasm Proteins / Q000096:biosynthesis / Q000235:genetics*; D016372:RNA, Antisense / Q000235:genetics; D034741:RNA, Small Interfering; D000077022:Survivin; D014162:Transfection
C107489:BIRC5 protein, human; D051034:Inhibitor of Apoptosis Proteins; D008869:Microtubule-Associated Proteins; D009363:Neoplasm Proteins; D016372:RNA, Antisense; D034741:RNA, Small Interfering; D000077022:Survivin