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Construction of the antisense eukaryotic vector for proliferating cell nuclear antigen gene and its expression in
Qiangsong Tong1, Fuqing Zeng, Yipeng Qi
1Department of Urology, Xiehe Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022.
Abstract:
To explore a novel strategy for antisense gene therapy of cancer, the coding sequence of human proliferating cell nuclear antigen (PCNA) cDNA was reversely inserted into the eukaryotic vector pLXSN by molecular cloning techniques and transferred into bladder cancer EJ cells with liposome. The PCNA expression in transferred cells was dynamically detected by immunofluorescence and RT-PCR techniques. Changes of proliferation activities of cancer cells were assayed by MTT colorimetric and cloning formation methods. In the experiment, the antisense eukaryotic vector was successfully constructed and named as pLAPSN. After transfection with it for 1-7 days, PCNA protein and mRNA levels in cancer cells were blocked by 16.74%-84.21% (P < 0.05) and 23.27%-86.15% (P < 0.05) respectively. The proliferation activities of transferred cells were inhibited by 27.91%-62.07% (P < 0.01), with cloning formation abilities being decreased by 50.81% (P < 0.01). It was concluded that the in vitro proliferation activities of cancer cells could be effectively inhibited by blocking PCNA expression with antisense technique, which could serve as an ideal strategy for gene therapy of bladder cancer.
Insights
Antisense gene therapy effectively inhibited bladder cancer cell proliferation by blocking proliferating cell nuclear antigen (PCNA) expression. This novel strategy shows promise for future cancer treatment applications.
Area of Science:
- Oncology
- Molecular Biology
- Gene Therapy
Background:
- Cancer proliferation is often driven by specific proteins.
- Proliferating cell nuclear antigen (PCNA) is crucial for DNA replication and cell cycle progression.
- Targeting PCNA offers a potential strategy for cancer treatment.
Purpose of the Study:
- To investigate the efficacy of antisense gene therapy targeting PCNA in bladder cancer cells.
- To develop and validate a novel gene therapy approach for inhibiting cancer cell growth.
Main Methods:
- Constructed an antisense eukaryotic vector (pLAPSN) by inserting human PCNA cDNA into pLXSN.
- Transfected bladder cancer EJ cells using liposomes.
- Quantified PCNA expression (protein and mRNA) using immunofluorescence and RT-PCR.
- Assessed cell proliferation and cloning formation using MTT and colony formation assays.
Main Results:
- Successfully constructed the pLAPSN antisense vector.
- Demonstrated significant inhibition of PCNA protein (16.74%-84.21%) and mRNA (23.27%-86.15%) levels post-transfection (P < 0.05).
- Showed substantial reduction in cancer cell proliferation (27.91%-62.07%, P < 0.01) and cloning formation (50.81%, P < 0.01).
Conclusions:
- Antisense inhibition of PCNA expression effectively suppresses in vitro proliferation of bladder cancer cells.
- This approach represents a viable gene therapy strategy for bladder cancer.
- Targeting PCNA offers a promising avenue for developing novel cancer therapeutics.