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Published on: March 30, 2019
Construction of survivin siRNA expression vector and its regulation on cell cycle and proliferation in MCF-7 cells
Li-Ping Li1, Nian-Ci Liang, Chao-Quan Luo
1Department of Biochemistry, Zhongshan Medical College, Sun Yat-sen University, Guangzhou, Guangdong, 510089, P.R.China.
Background & Objective:
Survivin is specifically overexpressed in tumor tissues. Many reports have shown that the abrogation of its functions is useful for tumor therapy. RNA interference is a new technique that proved to be effective for suppressing gene expression. The aim of this study was to construct a siRNA expression plasmid against gene survivin, and then to assess its functions on breast cancer cells MCF-7.
Methods:
A survivin siRNA plasmid was constructed using a mU6pro vector contained U6 promotor, the expression change of survivin was examined by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis after the plasmid had been transfected into MCF-7 cells, and the effect of the plasmid on the cell cycle and cell proliferation was analyzed by flow cytometry and MTT method,respectively.
Results:
Survivin siRNA plasmid knocked down survivin expression in MCF-7 cells obviously, arrested the cell cycle in G1 phase, inhibited the cell proliferation significantly, and promoted cell apoptosis in a tendency.
Conclusion:
Survivin expression was knocked down at mRNA and protein levels by RNAi.
Insights
This study demonstrates that survivin siRNA effectively suppresses survivin gene expression in breast cancer cells. This inhibition leads to cell cycle arrest and reduced proliferation, suggesting a therapeutic potential for survivin in cancer treatment.
Area of Science:
- Molecular Biology
- Cancer Research
- Gene Therapy
Background:
- Survivin is overexpressed in tumors and is a target for cancer therapy.
- RNA interference (RNAi) is a potent method for gene expression suppression.
Purpose of the Study:
- To create a survivin siRNA expression plasmid.
- To evaluate the plasmid's efficacy on MCF-7 breast cancer cells.
Main Methods:
- Constructed a survivin siRNA plasmid using mU6pro vector.
- Transfected MCF-7 cells and analyzed survivin expression via RT-PCR and Western blot.
- Assessed cell cycle and proliferation using flow cytometry and MTT assay.
Main Results:
- Survivin siRNA plasmid significantly reduced survivin expression at mRNA and protein levels.
- Induced G1 phase cell cycle arrest.
- Inhibited cell proliferation and showed a trend towards promoting apoptosis.
Conclusions:
- RNAi effectively knocked down survivin expression in breast cancer cells.
- Survivin suppression impacts cell cycle progression and proliferation.
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