Apoptosis of glioma cell line U251 induced by small interfering RNA targeting survivin / 南方医科大学学报

ABSTRACT

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>RNA interference mediated by the siRNA expression vector pGenesi-l/survivin can significantly reduce survivin expression and induce remarkable apoptosis in U251 cells.</p>