[Vector construction and silencing effect of Edg4 gene targeted small interfering RNA in ovarian cancer cell line].
OBJECTIVE
To construct the recombinant eukaryotic expression vector pRNAT-U6.1-siEdg4 which carries small interfering RNA (siRNA) of Edg4 and observe the silencing effect of Edg4 gene targeted siRNA in ovarian cancer cell line SKOV3.


METHODS
The Edg4 gene-targeted hairpin siRNA sequence was designed according to the Edg4 sequence in Genbank, and the two complementary oligo nucleotide strands were synthesized and annealed and inserted into the pRNAT-U6.1 plasmid to build a recombinant Edg4 siRNA eukaryotic expression vector, which was sequenced and identified to contain the correct Edg4 siRNA sequence. The human ovarian carcinoma cell lines SKOV3 were transfected with the vector using lipofectamine method. The efficiency of transfecting cells was observed with fluorescent microscope and the mRNA expression level of Edg4 gene was detected by real time quantitative PCR. The LPA levels in cell supernatants were detected using a biochemical method. And the apoptosis of SKOV3 cells induced by the vector was evaluated by flow cytometry.


RESULTS
The recombinant eukaryotic expression vector was confirmed to contain correct Edg4 siRNA sequence by PCR and sequencing. After transfection large amounts of green fluorescence were seen in plasma and nuclei of SKOV3 cells and the positive cell rates were 64%. The expression level of Edg4 mRNA in transfected SKOV3 cell line was significantly decreased (0.05 +/- 0.01vs 0.29 +/- 0.04, P < 0.05). The decrease in LPA level in the cell supernatants was revealed [(3.0 +/- 1.0) vs (7.5 +/- 2.2)micromol/L, P < 0.05]. The apoptosis rate of transfected SKOV3 was increased obviously (53.38% vs 0.51%, P < 0.05).


CONCLUSIONS
We have successfully constructed the recombinant eukaryotic expression vector containing Edg4 gene targeted siRNA (pRNAT-U6.1-siEdg4). The vector could effectively transfect SKOV3 cell line, and obviously suppress the Edg4 mRNA expression and induce cell apoptosis in ovarian cancer cell line SKOV3.
Qiao
Yu-huan
YH
Department of Obstetrics and Gynecology, the First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China. yhqiao@zzu.edu.cn
Li
Liu-xia
LX
Guo
Rui-xia
RX
Zhou
Wei
W
Wang
Miao
M
Zhang
Xiao-yan
XY
Zhang
Jian-hao
JH
Zhao
Xian-lan
XL
Zhang
Meng-zhen
MZ
Zhao
Guo-qiang
GQ
D017209:Apoptosis / Q000235:genetics / Q000502:physiology*; D045744:Cell Line, Tumor; D005260:Female; D005434:Flow Cytometry; D015972:Gene Expression Regulation, Neoplastic; D020868:Gene Silencing; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D008246:Lysophospholipids / Q000378:metabolism; D010051:Ovarian Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D010957:Plasmids / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics*; D049368:Receptors, Lysophosphatidic Acid / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection / Q000379:methods
D008246:Lysophospholipids; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D049368:Receptors, Lysophosphatidic Acid; C032881:lysophosphatidic acid