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Objective:To investigate the silencing effect of gene encoding peroxisome proliferator-activated receptor γ (PPARγ) on the expression of tumor necrosis factor alpha (TNFα) by constructing vectors for RNA interference in RAW264.7 cells. Methods: The pSUPER-EGFP vectors were used to transcribe functional small interfering RNA (siRNA). Four pairs of oligonucleotides (64 nt) targeting PPARγ gene were inserted into the downstream of the H1 promotor, with their veracity confirmed by double digestion and sequencing. Western blotting and immunofluorescence assay were used to examine the silencing effect of PPARγ gene in RAW264.7 cells. Meanwhile, the TNFα level was determined by Sandwich ELISA. Results: Compared with other recombinant pSUPER-EGFP vectors (R-pSUPER.EGFP), R-pSUPER.EGFP2 induced the best silencing effect on the expression of PPARγ in RAW264.7 cells, which played an obvious inhibitory role in down-regulating the TNFα expression after the curcumin and lipopolysaccharide (LPS) stimulation. Conclusions: PPARγ-pSUPER-EGFP inducing a silencing effect on the expression of PPARγ can efficiently play a negative role in controlling the inflammatory responses of RAW264.7 cells.
Objective: To investigate the silencing effect of gene encoding peroxisome proliferator-activated receptor γ (PPARγ) on the expression of tumor necrosis factor alpha (TNFα) by constructing vectors for RNA interference in RAW264.7 cells. Methods: The pSUPER-EGFP vectors were used to transcribe functional small interfering RNA (siRNA). Four pairs of oligonucleotides (64 nt) targeting PPARγ gene were inserted into the downstream of the H1 promotor, with their veracity confirmed by double digestion and sequencing. Western blotting and immunofluorescence assay were used to examine the silencing effect of PPARγ gene in RAW264.7 cells. Meanwhile, the TNFα level was determined by Sandwich ELISA. Results: Compared with other recombinant pSUPER-EGFP vectors (R-pSUPER.EGFP), R-pSUPER.EGFP2 induced the best silencing effect on the expression of PPARγ in RAW264.7 cells, which played an obvious inhibitory role in down-regulating the TNFα expression after the curcumin and lipopolysaccharide (L PS) stimulation. Conclusions: PPARγ-pSUPER-EGFP inducing a silencing effect on the expression of PPARγ can efficiently play a negative role in controlling the inflammatory responses of RAW264.7 cells.