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Published on: January 3, 2015
[Construction of PTTGas antisense expression vector and its inhibitory effects on human ovarian carcinoma cell line
Gang Chen1, Jing Li, Fu-Jun Li
1Cancer Biological Medicine Center of Tongji Hospital,Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, 430030, PR China.
Background & 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).
Conclusion:
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.
Insights
Researchers developed an antisense gene therapy targeting the Pituitary Tumor Transforming Gene (PTTG) in ovarian cancer cells. This approach successfully reduced PTTG expression and inhibited tumor cell invasion, offering a new therapeutic possibility.
Area of Science:
- Oncology
- Molecular Biology
- Gene Therapy
Background:
- Pituitary Tumor Transforming Gene (PTTG) is a proto-oncogene implicated in tumorigenesis and metastasis.
- Current research primarily focuses on PTTG expression and regulation, with limited exploration of therapeutic blocking strategies.
- Antisense gene therapy offers a potential method to inhibit oncogene expression.
Purpose of the Study:
- To construct a eukaryotic expression vector for antisense PTTG mRNA.
- To evaluate the efficacy of antisense PTTG gene therapy in blocking the invasion of human ovarian carcinoma SK-OV-3 cells.
Main Methods:
- Cloning of the full-length PTTG gene fragment using PCR.
- Construction of a recombinant eukaryotic expression vector (pcDNA3.1-PTTGas) with PTTG in antisense orientation.
- Transfection of SK-OV-3 cells with the recombinant vector and screening of positive clones.
- Assessment of PTTG and bFGF protein expression via Western blot.
- Analysis of cell proliferation using MTT assay and colony formation in soft agar.
Main Results:
- Stable SK-OV-3 clones expressing antisense PTTG were successfully generated.
- Transfected cells showed a significant decrease in PTTG (61.5%) and bFGF (52.3%) protein expression.
- Cell proliferation was accelerated in transfected cells.
- Colony formation in soft agar was significantly reduced in transfected cells compared to controls (2.4 vs. 23.3/21.5).
Conclusions:
- The recombinant vector pcDNA3.1-PTTGas is an effective tool for antisense gene therapy.
- This study presents a novel therapeutic strategy targeting PTTG in human carcinoma.
- Antisense PTTG gene therapy demonstrates potential in inhibiting ovarian cancer cell invasion.
