[Influence of short hairpin RNA on survivin mRNA expression and chemosensitivity to paclitaxel in ovarian cancer cells].
OBJECTIVE
To investigate the influence of short hairpin RNA (shRNA) expression plasmid against gene survivin (mU(6)/survivin) on survivin mRNA expression and chemosensitivity to paclitaxel in ovarian cancer cells OVCAR3.


METHODS
OVCAR3 cells were transfected with plasmid pEGFPC(2) formulated with lipofectamine 2000 at different concentrations. The transfection efficiency was examined by flow cytometry. The expression of survivin mRNA of OVCAR3 cells after transfection with the plasmid mU(6)/survivin with the high efficiency ratio was observed by RT-PCR. The effect of the plasmid on the cell cycle and apoptosis was analyzed by flow cytometry. The chemosensitivities of transfected cells to paclitaxel were determined by methyl thiazolyl tetrazolium (MTT).


RESULTS
The optimal transfection efficiency was obtained when pEGFPC(2): lipofectamine 2000 was 1: 2. Compared with the non-transfected groups, 0.81 +/- 0.05, the mRNA of survivin in OVCAR3 cells was reduced clearly after transfection with mU(6)/survivin, 0.26 +/- 0.04. shRNA reduced the expression level of survivin mRNA to 32% of non-transfected groups (P < 0.01). The apoptotic rate of survivin shRNA group reached (31.9 +/- 1.2)%, which was much higher than those of non-transfected (4.9 +/- 0.7)% and lip alone groups (5.6 +/- 0.5)% (P = 0.000). Cell cycle analysis showed that survivin shRNA induced accumulation of cells in G(0)/G(1) phase with a decrease of cells in G(2)/M phase after being cultured for 24 hours compared with non-transfected group (P < 0.01). MTT results showed that the 50% inhibiting concentration (IC(50)) of paclitaxel in non-transfected, lip alone and survivin shRNA transfected groups was (0.305 +/- 0.032), (0.157 +/- 0.031), and (0.019 +/- 0.001) micromol/L respectively. Compared with non-transfected group, shRNA increased the chemosensitivity of OVCAR3 cells to paclitaxel by 16 fold (P = 0.000).


CONCLUSIONS
Sequence specific shRNA targeting survivin can effectively suppress the expression of survivin mRNA and enhance the chemosensitivity to paclitaxel in ovarian cancer cells significantly.
Yan
Xiao-jian
XJ
Department of Gynecology, Cancer Center, Sun Yat-sen University, Guangzhou 510060, China.
Liang
Li-zhi
LZ
Zeng
Zong-yuan
ZY
Fu
Li-wu
LW
Shi
Zhi
Z
D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D005260:Female; D005434:Flow Cytometry; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D051034:Inhibitor of Apoptosis Proteins; D008869:Microtubule-Associated Proteins / Q000096:biosynthesis / Q000235:genetics*; D009363:Neoplasm Proteins / Q000096:biosynthesis / Q000235:genetics*; D010051:Ovarian Neoplasms / Q000235:genetics / Q000473:pathology; D017239:Paclitaxel / Q000494:pharmacology*; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000077022:Survivin; D014162:Transfection
D000972:Antineoplastic Agents, Phytogenic; C107489:BIRC5 protein, human; D051034:Inhibitor of Apoptosis Proteins; D008869:Microtubule-Associated Proteins; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D000077022:Survivin; D017239:Paclitaxel