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Updated: Jul 8, 2026
MISSION LentiPlex Pooled shRNA Library Screening in Mammalian Cells
Published on: December 21, 2011
[Construction of targeting-Skp2 shRNA plasmids and observation of their inhibitory effect on Tca8113 cells]
Liang Fang1, Qin-gang Hu, Zi-chun Hua
1Department of Oral and Maxillofacial Surgery, Nanjing Stomatological Hospital, The Affiliated Hospital of Medical School, Nanjing University, Nanjing 210008, China.
Objective:
To construct the recombinant plasmids expressing Skp2 short hairpin RNA (shRNA) by pRNAT-U6.1/Neo plasmid vector and observe the effects of RNAi-mediated Skp2 gene silencing on Tca8113 cells.
Methods:
Five recombinant eukaryotic expression vectors were successfully constructed using pRNAT-U6.1/Neo plasmid vector separately. After they were transfected into Tca8113 cells with PEI, the interference effects no Skp2 and p27 were detected by RT-PCR and Western blot. The cell cycle of Tca8113 cells were tested by flow cytometry. The proliferation of Tca8113 cells were examined by MTT.
Results:
In Skp2shRNA-2 and Skp2shRNA-3 vectors, the expression of Skp2 protein of Tca8113 cells was down-regulated and p27 protein up-regulated (P < 0.01). The cell number during G1/G 0 phases increased 22% (P < 0.01) and during G(2)/M and S phases the number decreased 10% and 12% (P < 0.01). The proliferation of Tca8113 cells slowed down and the cells number decreased (P < 0.01).
Conclusions:
Skp2shRNA-2 and Skp2shRNA-3 vectors of shRNA for Skp2 were successfully constructed. They could influence expression of Skp2 and p27 gene. Skp2 may be a promising target of gene therapy on human tongue squamous cell carcinoma.
Insights
Researchers constructed short hairpin RNA (shRNA) vectors targeting Skp2 (S-phase kinase-associated protein 2) to silence its gene in human tongue squamous cell carcinoma cells. This gene silencing effectively reduced Skp2 and increased p27 protein levels, inhibiting cell proliferation.
Area of Science:
- Molecular Biology
- Cancer Research
- Gene Therapy
Context:
- Human tongue squamous cell carcinoma (TSCC) is a significant health concern.
- Skp2 (S-phase kinase-associated protein 2) plays a crucial role in cell cycle regulation and cancer progression.
- Targeting oncogenic proteins like Skp2 is a key strategy in cancer therapy.
Purpose:
- To construct recombinant plasmids expressing Skp2 short hairpin RNA (shRNA) using the pRNAT-U6.1/Neo vector.
- To investigate the effects of RNA interference (RNAi)-mediated Skp2 gene silencing on Tca8113 cells, a human tongue squamous cell carcinoma cell line.
- To evaluate the impact of Skp2 silencing on cell cycle progression and proliferation.
Summary:
- Five recombinant eukaryotic expression vectors for Skp2 shRNA were successfully constructed.
- Transfection of Tca8113 cells with Skp2 shRNA vectors (specifically Skp2shRNA-2 and Skp2shRNA-3) led to significant down-regulation of Skp2 protein and up-regulation of p27 protein.
- RNAi-mediated Skp2 silencing resulted in cell cycle arrest at the G1/G0 phase, decreased proliferation, and reduced cell numbers.
Impact:
- Successful construction of functional Skp2 shRNA vectors provides a tool for further research into TSCC.
- Skp2 gene silencing demonstrates a therapeutic potential by inhibiting cancer cell growth.
- Skp2 emerges as a promising molecular target for gene therapy in human tongue squamous cell carcinoma.
