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Abstract
Objective:
Small interfering RNA (siRNA) triggers RNA interference in mammalian somatic cells. TNF-α is a proinflammatory cytokine implicated in the pathogenesis of inflammatory arthritis including rheumatoid arthritis (RA). This study was to use TNF receptor 1 (TNFRI)- specific siRNA to inhibit the TNF-α mediated signaling in RA fibroblast like synoviocytes (FLS) and chondrocytes.
Methods:
TNFRI specific siRNA was produced by targeting 3 nucleotide sequences at 474 ~ 494, 562~582 and 668~688. Reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot were performed to optimize the silencing effects of TNFRI siRNA in cultured FLS and chondrocytes. The inhibition of TNF-α mediated signaling was determined by ELISA assay of metalloproteinase 1 secretion induced by TNF-α.
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Fig. 1.
Inhibition of TNFRI mRNA by TNFRI specific siRNA in articular chondrocytes. (A) Expression of TNFRI mRNA in articular chondrocytes were detected by RT-PCR. Data represent samples from 3 different donors. (B) The band densities were quantified, the percent GAPDH density was calculated for TNFRI and the value for control was set at 100. ∗p<0.05 compared with control by Mann-Whitney U test. s: scrambled siRNA(negative control).
Fig. 2.
Inhibition of TNFRI protein by TNFRI specific siRNA in articular chondrocytes. Expression of TNFRI protein in articular chondrocytes was detected by Western blot. Data represent samples from 3 different donors. s: scrambled siRNA.
Fig. 3.
Inhibition of TNFRI mRNA and protein by TNFRI specific siRNA in RA fibroblast like synoviocytes(FLS). (A) Expression of TNFRI mRNA in RA FLS were detected by RT-PCR. Data represent samples from 5 different donors. (B) Expression of TNFRI protein in articular chondrocytes was detected by Western blot. Data represent samples from 4 different donors. s: scrambled siRNA.
Fig. 4.
Inhibition of MMP-1 secretion induced with TNF- α by TNFRI siRNA. MMP-1 production was measured 24 hours after TNF- α treatment from the conditioned media by ELISA. Results are given as the mean and standard deviation of the mean percentage of TNF- α treatment alone (control, representative of duplicate experiments from 4 donors). ∗p<0.05 compared with control by Mann-Whitney U test. s: scrambled siRNA (negative control)
Table 1.
The sequences of siRNA used in the experiment
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