OBJECTIVETo investigate the silencing effect of gene encoding peroxisome proliferator-activated receptor gamma (PPARgamma) on the expression of tumor necrosis factor alpha (TNFalpha) by constructing vectors for RNA interference in RAW264.7 cells.

METHODSThe pSUPER-EGFP vectors were used to transcribe functional small interfering RNA (siRNA). Four pairs of oligonucleotides (64 nt) targeting PPARgamma 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 PPARgamma gene in RAW264.7 cells. Meanwhile, the TNFalphalevel was determined by Sandwich ELISA.

RESULTSCompared with other recombinant pSUPER-EGFP vectors (R-pSUPER.EGFP), R-pSUPER.EGFP2 induced the best silencing effect on the expression of PPARgamma in RAW264.7 cells, which played an obvious inhibitory role in down-regulating the TNFalphaexpression after the curcumin and lipopolysaccharide (LPS) stimulation.

CONCLUSIONSPPARgamma-pSUPER-EGFP inducing a silencing effect on the expression of PPARgamma can efficiently play a negative role in controlling the inflammatory responses of RAW264.7 cells.