[Construction of PTTGas antisense expression vector and its inhibitory effects on human ovarian carcinoma cell line SK-OV-3].
OBJECTIVE
Pituitary tumor transforming gene (PTTG) is a new proto-oncogene and shows multiple actions of promoting tumorigenesis and metastasis. Most researches mainly focus on the problems of PTTG expression in different tumor tissues and its relative regulatory mechanisms, but no research of exploring the possibility of antisense blocking of PTTG gene has been reported by now. In present study, the authors constructed eukaryotic expression vector expressing full-length anti-sense PTTG mRNA and observed its blocking effect on the potential invasion of human ovarian carcinoma cell line SK-OV-3.


METHODS
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 analysis. The changes of cell proliferation were analyzed by MTT method. The biological behavior change of transfection positive cells was observed by colony formation in soft agar assay.


RESULTS
SK-OV-3 clones stably expressing full-length recombinant pcDNA3.1- PTTGas were obtained.The expression of PTTG and bFGF proteins in transfected cells were decreased by 61.5% and 52.3% respectively as compared with non-transfected ones. The cell proliferation was accelerated in transfected cells. The number of colony formation is reduced significantly in transfected cells (2.4+/-0.8) as compared with non-transfected and empty vector transfected cells (23.3+/-5.7 and 21.5+/-7.9, respectively, P< 0.01).


CONCLUSIONS
The recombinant vector pcDNA3.1-PTTGas is a novel tool and brings us a new possibility of anti-sense gene therapy targeted at PTTG in human carcinoma.
Chen
Gang
G
Cancer Biological Medicine Center of Tongji Hospital,Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, 430030, PR China.
Li
Jing
J
Li
Fu-Jun
FJ
Zhou
Jian-Feng
JF
Lu
Yun-Ping
YP
Ma
Ding
D
D045744:Cell Line, Tumor; D049109:Cell Proliferation; D005260:Female; D016222:Fibroblast Growth Factor 2 / Q000235:genetics; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D009363:Neoplasm Proteins / Q000235:genetics*; D010051:Ovarian Neoplasms / Q000473:pathology / Q000628:therapy*; D000090063:Proto-Oncogene Mas; D016372:RNA, Antisense / Q000235:genetics*; D064249:Securin; D014162:Transfection
C000715470:MAS1 protein, human; D009363:Neoplasm Proteins; D000090063:Proto-Oncogene Mas; D016372:RNA, Antisense; D064249:Securin; C578002:pituitary tumor-transforming protein 1, human; D016222:Fibroblast Growth Factor 2