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Updated: Jul 3, 2026
DNA Vector-based RNA Interference to Study Gene Function in Cancer
Published on: June 4, 2012
[Selection and construction of cell line stably expressing survivin gene in lower level through eukaryotic plasmid
Wen-Xia Wang1, Shan-Zhen Sun, Ying Song
1Department of Periodontology, College of Stomatology, Shandong University. Jinan 250012, Shandong Province, China. wangwx@sdu.edu.cn
Purpose:
To construct a short hairpin RNA(shRNA) interference expression plasmid vector of survivin gene, transfect tongue squamous cell carcinoma line Tca8113 which expressed survivin gene in a high level, and choose the cells whose survivin gene were suppressed significantly.
Methods:
Two pairs of oligonucleotide sequences specific for survivin gene were designed and synthesized, and cloned into pSilencer-2.1U6-neo plasmid. The recombinant plasmids (named PS1 and PS2) were amplified in Ecoli. DH5alpha was identified by restriction digestion, PCR and sequencing. The vectors were transfected into Tca8113 cells with lipofectamine 2000. After selection with G418, the stable cell clones were attained. Survivn expression was assayed with real-time quantitative PCR and Western blotting. SAS8.0 software package was used for Student t test.
Results:
Two vectors were constructed successfully and stable cell clones with PS1 or PS2 plasmid were obtained. As compared with those of control, survivin expression of transfected cell with PS1 or PS2 in mRNA level was significantly suppressed (P<0.05). In protein level, only those of transfected cell with PS2 was significantly suppressed (P<0.01).
Conclusions:
The shRNA interference expression plasmid vectors of survivin gene are successfully constructed, and Tca8113 cells which express survivin gene in a stable lower level are attained, which enable us to carry out further research on gene therapy of oral squamous cell carcinoma. Supported by National Natural Science Foundation of China (Grant No.30572056).
Insights
Researchers successfully created short hairpin RNA (shRNA) vectors to suppress survivin gene expression in tongue squamous cell carcinoma cells. This significant reduction in survivin at both mRNA and protein levels offers potential for oral cancer gene therapy.
Area of Science:
- Molecular Biology
- Cancer Research
- Gene Therapy
Background:
- Survivin is a key protein overexpressed in many cancers, including oral squamous cell carcinoma.
- Targeting survivin offers a potential therapeutic strategy for oral cancer.
Purpose of the Study:
- To construct short hairpin RNA (shRNA) interference expression plasmid vectors targeting the survivin gene.
- To transfect Tca8113 tongue squamous cell carcinoma cells with these vectors.
- To select and validate cell lines with significantly suppressed survivin expression.
Main Methods:
- Designed and synthesized survivin-specific oligonucleotide sequences.
- Cloned sequences into the pSilencer-2.1U6-neo plasmid, creating PS1 and PS2 vectors.
- Transfected Tca8113 cells using lipofectamine 2000 and selected stable clones with G418.
- Assessed survivin expression via real-time quantitative PCR and Western blotting.
Main Results:
- Successfully constructed PS1 and PS2 shRNA vectors and obtained stable transfected cell clones.
- Significantly suppressed survivin mRNA levels in cells transfected with both PS1 and PS2 (P<0.05).
- Significantly suppressed survivin protein levels in cells transfected with PS2 (P<0.01).
Conclusions:
- Successfully developed shRNA expression vectors for survivin gene suppression.
- Established Tca8113 cell lines with stably reduced survivin expression.
- These cell lines provide a valuable model for further research into oral squamous cell carcinoma gene therapy.
