Effects of RNA interference on expression of tumor necrosis factor-alpha in lipopolysaccharide-activated mouse macrophages

Abstract

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. 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. 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. 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.