[Inhibition of survivin gene expression in PC-3 cells by RNAi].
OBJECTIVE
To construct eukaryotic expression vectors by using the pSilencer3. 1-H1 neo vector for inhibiting human survivin gene by RNA interference, and to detect the effect of the silenced survivin gene on PC-3 cells.


METHODS
Three target gene segments were synthesized and cloned into the pSilencer3. 1-H1 neo vector respectively to construct three recombinant eukaryotic expression vectors: pSilencer3. 1-SVV1, pSilencer3. 1-SVV2 and pSilencer3. 1-SVV3, which were identified by enzyme digestion analysis and DNA sequencing. Then the PC-3 cells were transfected with the recombinant vectors and the interference effect detected by RT-PCR, Western blot and immunohistochemical staining. The apoptosis index of the PC-3 cells was detected by flow cytometry and their proliferation detected by MTT method.


RESULTS
Enzyme digestion analysis and DNA sequencing showed that three target segments were cloned into pSilencer3. 1-H1-neo vectors. The results of RT-PCR, Western blot and immunohistochemical staining indicated that pSilencer3. 1-SVV2 and pSilencer3. 1-SVV3 vectors could knock down the transcription and expression of survivin gene. After transfected with pSilencer3. 1-SVV2 and pSilencer3. 1-SVV3 vectors, the apoptosis index of the PC-3 cells was increased by 10% - 15% and their growth obviously slowly down.


CONCLUSIONS
The transcription and expression of survivin gene were inhibited effectively by the recombinant eukaryotic expression vectors (pSilencer3. 1-SVV2 and pSilencer3. 1-SVV3) in the prostate cancer cell line PC-3. After transfected with pSilencer3. 1-SVV2 and pSilencer3. 1-SVV3 vectors, the apoptosis index of the PC-3 cells was increased and their growth inhibited.
Shen
Jian-Jun
JJ
Department of Clinical Laboratory, Xijing Hospital, Fourth Military Medical University, Xi'an, Shaanxi 710032, China.
Hao
Xiao-Ke
XK
Liu
Jia-Yun
JY
Su
Ming-Quan
MQ
Miao
Ying-Ye
YY
Zhang
Qing
Q
D017209:Apoptosis; D045744:Cell Line, Tumor; D015536:Down-Regulation; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D005822:Genetic Vectors; D006801:Humans; D051034:Inhibitor of Apoptosis Proteins; D008297:Male; D008869:Microtubule-Associated Proteins / Q000096:biosynthesis* / Q000235:genetics; D009363:Neoplasm Proteins / Q000096:biosynthesis* / Q000235:genetics; D011471:Prostatic Neoplasms / Q000378:metabolism* / Q000473:pathology; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000494:pharmacology*; D000077022:Survivin; D014162:Transfection
C107489:BIRC5 protein, human; D051034:Inhibitor of Apoptosis Proteins; D008869:Microtubule-Associated Proteins; D009363:Neoplasm Proteins; D034741:RNA, Small Interfering; D000077022:Survivin