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[Effect of shRNA Survivin-mediated inhibition of proliferation in A549 cells]
Lihong Wu1, Jinhe Wang, Qian Wang
1Tuberculosis Research Institute, the 309th Hospital of Chinese PLA, Beijing 100091, China.
Background And Objective:
Survivin is a member of the inhibitor of apoptosis family of proteins. The Survivin protein is highly expressed in most human tumors, but it is completely absent in terminally differentiated cells. Consequently, Survivin is an ideal target for cancer therapy because cancer cells are targeted and normal cells are left alone. The aim of this study is to construct a lentivirus-shRNA vector, and to disrupt the expression of Survivin in A549 cells. The effect of sh-RNA Survivin on A549 cells was analyzed.
Methods:
Target DNA sequences of Survivin shRNA were designed to obtain recombinant plasmids. After the plasmids were transfected into 293T cells, the virus was collected. Hela cells were used to detect the virus titer. Survivin mRNA and protein expression in the infected A549 cells were detected via reverse transcription polymerase chain reaction and Western blot analysis. The proliferation of A549 cells were detected via 3-(4,5-dimethylthiazolyl)-2,5-diphenyltetrazolium bromide and flow cytometry assays.
Results:
The recombinants were successfully constructed, and Survivin expression was inhibited. The cells were blocked at the G2/M phase.
Conclusions:
Recombinant lentivirus with shRNA targeting Survivin was successfully constructed. The lentivirus can down-regulate Survivin expression in A549 cells as well as inhibit proliferation, and is hence a potential gene therapy for lung cancer.
Insights
This study successfully created a lentivirus-shRNA to inhibit Survivin expression in lung cancer cells. This gene therapy approach effectively reduced cancer cell proliferation, offering a promising avenue for lung cancer treatment.
Area of Science:
- Molecular biology
- Cancer research
- Gene therapy
Background:
- Survivin, an inhibitor of apoptosis protein, is highly expressed in human tumors but absent in normal cells.
- This differential expression makes Survivin an attractive target for cancer therapy, aiming to selectively target cancer cells.
- The study focuses on Survivin's role in A549 lung cancer cells.
Purpose of the Study:
- To construct a lentivirus-short hairpin RNA (shRNA) vector targeting Survivin.
- To disrupt Survivin expression in A549 lung cancer cells.
- To analyze the effects of Survivin shRNA on A549 cell proliferation and cell cycle progression.
Main Methods:
- Design and construction of recombinant lentivirus plasmids expressing Survivin shRNA.
- Transfection into 293T cells, virus collection, and titer detection using Hela cells.
- Assessment of Survivin mRNA and protein levels via RT-PCR and Western blot.
- Evaluation of A549 cell proliferation using MTT assay and flow cytometry.
Main Results:
- Successful construction of recombinant lentivirus vectors.
- Significant inhibition of Survivin expression at both mRNA and protein levels in A549 cells.
- A549 cells infected with the lentivirus were arrested at the G2/M phase of the cell cycle.
- Inhibition of A549 cell proliferation was observed.
Conclusions:
- A lentivirus carrying shRNA targeting Survivin was successfully constructed.
- This lentivirus effectively down-regulates Survivin expression in A549 lung cancer cells.
- The developed lentivirus demonstrates potential as a gene therapy agent for lung cancer by inhibiting proliferation.
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