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[Effect of RNA interference targeting brain-derived neurotrophic factor gene on HeLa cell proliferation and
Jing Huang1, Chun-Yan Sun, Tao Guo
1Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China.
Objective:
To screen effective sequences of short hairpin RNA on brain-derived neurotrophic factor (BDNF) gene and the effect of RNA interference on the proliferation and apoptosis of HeLa cells, a cervix carcinoma cell line with high expression of BDNF.
Methods:
Two recombinant eukaryotic human-BDNF siRNA expression vectors were designed and constructed. Sequences were confirmed by restrictive endonuclease digestion and DNA sequencing. The empty vector pGenesil-1 and two recombinant plasmids, pGenesil-shRNA-BDNF1 and pGenesil-shRNA-BDNF2, were transfected into HeLa cells using Lipofectamine 2000 (groups: P(0), P(1) and P(2), respectively). The mRNA and protein levels of BDNF in HeLa cells were detected by RT-PCR and Western blot, respectively. The cellular proliferation rates were determined by MTT assay and the apoptotic rates were measured by flow cytometry and Hoechest 33258.
Results:
The recombinant eukaryotic BDNF siRNA expression vectors were successfully constructed. The expression of mRNA and protein of BDNF in P(1) group were significantly decreased, comparing with non-transfected group, P(0) and P(2) groups (F = 48.19, P < 0.01). P(2) group failed to meet the expected results (P > 0.05). In addition, the proliferation activity was reduced in P(1) group and the peak point of proliferation curve was prolonged. Moreover, the early cell apoptotic rates were statistically increased in P(1)[(53.4 +/- 4.2)%] VS. non-transfected [(0.8 +/- 0.4)%], P(0) [(5.1 +/- 1.8)%] and P(2)[(7.9 +/- 2.4)%] groups (F = 269.77, P < 0.01).
Conclusion:
HeLa cells express a high level of BDNF. BDNF gene silencing by RNA interference increases the apoptosis of HeLa cells and inhibits cell proliferation, offering a possible target for efficient tumor therapy.
Insights
Short hairpin RNA targeting brain-derived neurotrophic factor (BDNF) effectively silenced the gene in HeLa cells. This gene silencing inhibited cell proliferation and increased apoptosis, suggesting BDNF as a potential cancer therapy target.
Area of Science:
- Molecular Biology
- Gene Expression Regulation
- Cancer Biology
Context:
- HeLa cells, a human cervix carcinoma line, exhibit high expression of brain-derived neurotrophic factor (BDNF).
- Understanding BDNF's role in cervical cancer progression is crucial for developing targeted therapies.
Purpose:
- To design and validate effective short hairpin RNA (shRNA) sequences for silencing the BDNF gene.
- To investigate the impact of BDNF gene silencing on HeLa cell proliferation and apoptosis.
Summary:
- Two shRNA expression vectors targeting BDNF were constructed and confirmed. Transfection into HeLa cells demonstrated significant reduction in BDNF mRNA and protein levels with one vector (pGenesil-shRNA-BDNF1).
- This effective BDNF silencing led to reduced cell proliferation and a statistically significant increase in early apoptosis rates compared to control groups.
- The other vector (pGenesil-shRNA-BDNF2) did not yield significant results.
Impact:
- BDNF gene silencing via RNA interference effectively inhibits HeLa cell proliferation and induces apoptosis.
- This study identifies BDNF as a potential therapeutic target for cervical cancer treatment.
- The findings provide a basis for developing novel RNA interference-based cancer therapies.
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