[Inhibitory effect of short hairpin RNA targeting survivin gene on human ectopic endometrial cells].
OBJECTIVE
To construct a lentiviral expression vector for short hairpin RNA (shRNA) of human survivin gene, and assess its gene silencing effect in human ectopic endometrial cells.


METHODS
Human survivin gene shRNA sequence was designed using a software available on-line. The synthesized shRNA sequence was cloned into the pGCL-GFP vector to construct LV-survivin shRNA, which was confirmed by PCR and DNA sequence analysis. The packaging 293T cells were cotransfected with LV-survivin shRNA, pHelper 1.0 and pHelper 2.0, and the titer of the lentivirus was determined. The recombinant lentivirus was injected into human ectopic endometrial cells and the survivin mRNA expression was examined by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) in comparison with that in the non-transfected and blank vector-transfected human ectopic endometrial cells.


RESULTS
PCR analysis and DNA sequencing confirmed correct insertion of the shRNA sequence into the lentiviral vector. The titer of virus after packaging was 8x10(8) U/ml. Survivin mRNA expression in human ectopic endometrial cells transfected by LV-survivin shRNA was significantly inhibited compared with those in the non-transfected and empty vector transfected human ectopic endometrial cells (P<0.01), and no significant difference was found between the latter two groups.


CONCLUSIONS
The lentiviral shRNA vector of survivin gene constructed can effectively inhibit the expression of survivin gene in human ectopic endometrial cells in vitro. This vector provides a tool for investigating the role of survivin gene in the occurrence and progression of endometriosis and for searching new therapeutic targets.
Peng
Dong-xian
DX
Department of Gynecology and Obstetrics, Zhujiang Hospital, Southern Medical University, Guangzhou 510282, China. pdx@fimmu.com
He
Yuan-li
YL
Qiu
Li-wen
LW
D002478:Cells, Cultured; D004715:Endometriosis / Q000235:genetics* / Q000473:pathology; D005260:Female; D018390:Gene Targeting; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D051034:Inhibitor of Apoptosis Proteins / Q000096:biosynthesis / Q000235:genetics*; D016086:Lentivirus / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D000077022:Survivin; D014162:Transfection
C107489:BIRC5 protein, human; D051034:Inhibitor of Apoptosis Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D000077022:Survivin