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Updated: Jul 10, 2026
Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages
Published on: November 3, 2014
[Effects of RNA interference on expression of tumor necrosis factor-alpha in lipopolysaccharide-activated mouse
Bing Tan1, Yu-yuan Li, Yu-qiang Nie
1Department of Gastroenterology, First Municipal People's Hospital of Guangzhou, Guangzhou Medical College, Guangzhou 510180, China.
Objective:
To investigate the effects of siRNAs targeting mouse tumor necrosis factor (TNF)-alpha gene on the expression of TNF-alpha mRNA and protein in the LPS activated macrophages.
Methods:
Mouse macrophages of the line RAW264.7 were cultured. Three siRNA sequences targeting different sites of mouse TNF-alpha gene (siRNA 1 - 3) and a fluorescein-labeled double-stranded RNA (dsRNA) oligomer (siRNA 4), as negative control, were designed and chemically synthesized. All siRNAs were transfected into the mouse macrophages. 0, 3, 6, 9, 12, 15, and 18 hours after the transfection samples of supernatant were collected. Inverse microscopy was used to observe the efficacy of transfection. Then the macrophages were co-incubated with LPS for 9 hours. Macrophages stimulated by LPS only and macrophages without transfection and LPS stimulation were used as controls. Then samples of supernatant were collected. TNF-alpha protein expression was detected by ELISA. Real time PCR was used to detect the TNF-alpha mRNA expression.
Results:
The transfection rate was 72% - 80%. The TNF-alpha mRNA expression levels of the LPS + siRNA2 and LPS + siRNA3 groups were 0.158 +/- 0.031 and 0.114 +/- 0.028 respectively, both significantly lower than that of the LPS control group (0.294 +/- 0.147, P < 0.05 and P < 0.01 respectively), however, the TNF-alpha mRNA expression level of the LPS + siRNA1 and LPS + siRNA4 groups were not significantly different from that of the LPS control group. The TNF-alpha mRNA expression inhibition rates of the LPS + siRNA and LPS + siRNA3 groups were 46.0% and 61.2% respectively. The TNF-alpha protein expression levels of the LPS + siRNA2 and LPS + siRNA3 group were (1358 +/- 348) pg/ml and (817 +/- 138) pg/ml, both significantly lower than that of the LPS control group [(2104 +/- 32) pg/ml, P < 0.05 and P < 0.01], and the TNF-alpha mRNA expression levels of the LPS + siRNA1 and LPS + siRNA4 groups were not significantly different from that of the LPS control group.
Conclusion:
LPS time-dependently increases the expression of TNF-alpha in macrophages. SiRNAs targeting TNF-alpha inhibit the expression levels of TNF-alpha mRNA and protein in macrophages treated with LPS.
Insights
Small interfering RNAs (siRNAs) targeting the tumor necrosis factor (TNF)-alpha gene effectively reduced TNF-alpha mRNA and protein expression in lipopolysaccharide (LPS)-activated macrophages. These findings highlight siRNA's potential in modulating inflammatory responses.
Area of Science:
- Immunology
- Molecular Biology
- Gene Silencing
Context:
- Macrophages play a crucial role in the immune response, with tumor necrosis factor-alpha (TNF-alpha) being a key pro-inflammatory cytokine.
- Lipopolysaccharide (LPS) is a potent activator of macrophages, leading to increased TNF-alpha production.
- Small interfering RNAs (siRNAs) offer a targeted approach to gene expression modulation.
Purpose:
- To evaluate the efficacy of siRNAs designed to target the mouse TNF-alpha gene.
- To assess the impact of these siRNAs on TNF-alpha mRNA and protein expression in LPS-stimulated RAW264.7 macrophages.
- To determine the optimal siRNA sequences for inhibiting TNF-alpha expression.
Summary:
- Three distinct siRNA sequences targeting mouse TNF-alpha were synthesized and transfected into RAW264.7 macrophages.
- Macrophages were subsequently stimulated with LPS, and TNF-alpha mRNA and protein levels were quantified using real-time PCR and ELISA, respectively.
- siRNA sequences 2 and 3 demonstrated significant inhibition of both TNF-alpha mRNA (46.0% and 61.2% reduction) and protein expression compared to controls.
Impact:
- This study demonstrates the successful application of siRNA technology to suppress TNF-alpha expression in activated macrophages.
- The findings suggest that targeted inhibition of TNF-alpha via siRNA could be a therapeutic strategy for inflammatory conditions.
- Optimized siRNA sequences provide valuable tools for further research into TNF-alpha-mediated inflammatory pathways.
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