Inhibitory effects of anti-sense PTTG on malignant phenotype of human ovarian carcinoma cell line SK-OV-3.
To construct eukaryotic expression vector expressing full length anti-sense pituitary tumor transforming gene (PTTG) mRNA and observe its blocking effect on the potential invasion of human ovarian carcinoma cell line SK-OV-3. PCR primers containing designed enzyme cut sites were used for cloning full-length PTTG gene fragment, and the resulting PCR product was inserted into the eukaryotic vector pcDNA3.1 in the antisense direction. The recombinant vector was then transfected into SK-OV-3 by Lipofectamine. The positive cell clone was screened by G418, PTTG and bFGF at protein level expression were detected by Western blot. The biological behavior change of transfection positive cells was observed by colony formation in soft agar assay. Our results showed that SK-OV-3 clones stably expressing full-length recombinant pcDNA3.1-PTTGas were obtained. The expressions of PTTG and bFGF protein in transfected cells were decreased by 61.5% and 52.3%, respectively as compared with non-transfected ones. The number of colony formation was reduced significantly in transfected cells as compared with empty vector transfected and non-transfected cells. It is concluded that the recombinant vector pcDNA3.1-PTTGas is a novel tool and provides an alternative anti-sense gene therapy targeted at PTTG in human carcinoma.
Chen
Gang
G
Molecular Cancer Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Li
Jing
J
Li
Fujun
F
Li
Xiao
X
Zhou
Jianfeng
J
Lu
Yunping
Y
Ma
Ding
D
D045744:Cell Line, Tumor; D005057:Eukaryotic Cells / Q000378:metabolism; D005260:Female; D016222:Fibroblast Growth Factor 2 / Q000096:biosynthesis / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D009363:Neoplasm Proteins / Q000096:biosynthesis* / Q000235:genetics; D010051:Ovarian Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D010641:Phenotype; D016372:RNA, Antisense / Q000096:biosynthesis* / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D011995:Recombination, Genetic; D064249:Securin; D014162:Transfection
D009363:Neoplasm Proteins; D016372:RNA, Antisense; D012333:RNA, Messenger; D064249:Securin; C578002:pituitary tumor-transforming protein 1, human; D016222:Fibroblast Growth Factor 2